PMC:7352545 / 32537-69051 JSONTXT

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

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CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}

{"project":"LitCovid-PD-HP","denotations":[{"id":"T10","span":{"begin":389,"end":399},"obj":"Phenotype"},{"id":"T11","span":{"begin":12136,"end":12145},"obj":"Phenotype"},{"id":"T12","span":{"begin":12253,"end":12258},"obj":"Phenotype"},{"id":"T13","span":{"begin":14505,"end":14528},"obj":"Phenotype"},{"id":"T14","span":{"begin":20963,"end":20968},"obj":"Phenotype"},{"id":"T15","span":{"begin":25479,"end":25484},"obj":"Phenotype"},{"id":"T16","span":{"begin":27963,"end":27971},"obj":"Phenotype"},{"id":"T17","span":{"begin":28888,"end":28899},"obj":"Phenotype"},{"id":"T18","span":{"begin":29223,"end":29231},"obj":"Phenotype"},{"id":"T19","span":{"begin":29235,"end":29243},"obj":"Phenotype"},{"id":"T20","span":{"begin":31352,"end":31361},"obj":"Phenotype"},{"id":"T21","span":{"begin":31388,"end":31397},"obj":"Phenotype"},{"id":"T22","span":{"begin":31415,"end":31431},"obj":"Phenotype"},{"id":"T23","span":{"begin":31462,"end":31471},"obj":"Phenotype"}],"attributes":[{"id":"A10","pred":"hp_id","subj":"T10","obj":"http://purl.obolibrary.org/obo/HP_0012387"},{"id":"A11","pred":"hp_id","subj":"T11","obj":"http://purl.obolibrary.org/obo/HP_0012115"},{"id":"A12","pred":"hp_id","subj":"T12","obj":"http://purl.obolibrary.org/obo/HP_0031273"},{"id":"A13","pred":"hp_id","subj":"T13","obj":"http://purl.obolibrary.org/obo/HP_0001626"},{"id":"A14","pred":"hp_id","subj":"T14","obj":"http://purl.obolibrary.org/obo/HP_0012735"},{"id":"A15","pred":"hp_id","subj":"T15","obj":"http://purl.obolibrary.org/obo/HP_0031273"},{"id":"A16","pred":"hp_id","subj":"T16","obj":"http://purl.obolibrary.org/obo/HP_0002014"},{"id":"A17","pred":"hp_id","subj":"T17","obj":"http://purl.obolibrary.org/obo/HP_0002586"},{"id":"A18","pred":"hp_id","subj":"T18","obj":"http://purl.obolibrary.org/obo/HP_0001909"},{"id":"A19","pred":"hp_id","subj":"T19","obj":"http://purl.obolibrary.org/obo/HP_0002665"},{"id":"A20","pred":"hp_id","subj":"T20","obj":"http://purl.obolibrary.org/obo/HP_0012740"},{"id":"A21","pred":"hp_id","subj":"T21","obj":"http://purl.obolibrary.org/obo/HP_0012115"},{"id":"A22","pred":"hp_id","subj":"T22","obj":"http://purl.obolibrary.org/obo/HP_0100726"},{"id":"A23","pred":"hp_id","subj":"T23","obj":"http://purl.obolibrary.org/obo/HP_0012740"}],"text":"6. CoVs Infection of Human Hosts\n\n6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells\nSeveral β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].\n\n6.1.1. α-Coronavirus\nIn α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.\n\n6.1.2. β-Coronavirus\nIn β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].\n\n6.1.3. γ-Coronavirus\nIn γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.\n\n6.1.4. Torovirus\nIn torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].\n\n6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors\nThe S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities\nCoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].\nMERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.\n\n6.3. Host Receptors of CoVs\nCoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.\nBCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.\n\n6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor\n\nStructure and Role of the Host SARS-CoV Receptor ACE2\nSARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.\nACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.\nPulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.\n\nHost Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2\nA disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.\nTMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].\nThe fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.\n\n6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor\nThe Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].\n\n6.3.3. CEACAM Receptor\nEntry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.\nBoth raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.\n\n6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5\nMERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.\n\n6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E\nAmong the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].\n\n6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site\nFor glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.\n\n6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1\nAlthough HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].\n\n6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry\nSARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].\n\n6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases\nTetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].\n\n6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction\nSAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.\nOn the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136]."}