3. Structure, Components and Life Cycle of CoVs
CoVs are 60–140 nm in size and are enveloped (+) ssRNA viruses, which feature an RNA genome, directly available to function as mRNA and thus result in rapid infection. CoVs exhibit RNA genomes of 28–32 kb, comprised of two large overlapping open reading frames (ORFs), which encode the virus replicase (transcriptase) and structural proteins. The SARS-CoV-2 genome is 29,891 bp in size, which encodes 9860 amino acids. The ssRNA are capped and tailed with a 5′-capping structure and 3′-poly A tail at the termini. The genome is the same sense as virus mRNA indicating that the viral RNA is translated through its own (+) RNA to synthesize RNA dependent RNA polymerase (RdRp; PDB: 6M71).
Generally, viral families are determined by the genome structure and virion morphologies of an envelope or naked capsid. A virus with a naked capsid has a coat of nucleocapsid protein (N) coating the viral genome. Viruses with an envelope have lipid envelopes further surrounding the outmost protein layer. The 2019-nCoV (SARS-CoV-2) contains a spike (S) glycoprotein, E, dimeric HE enzyme, a membrane matrix glycoprotein (M), N and RNA [16]. The structural proteins are the S, N, M and E proteins, while the non-structural proteins are proteases such as Nsp3 and Nsp5 and RdRp such as Nsp12. Among the N, M and S glycoproteins, the S glycoprotein is a fusion protein that recognizes the host receptor and enters the host cells [17]. The S, M and E proteins anchored into the endoplasmic reticulum (ER) membrane are trafficked to the endoplasmic reticulum–Golgi intermediate compartment (ERGIC). The RNA genome linked with nucleoprotein buds into the ERGIC to form virus particles. Assembled virions transported to the vesicular surface are released to the extracellular milieu via exocytosis.
The RNA generates the replicase as two polyproteins, pp1a and pp1ab. The replicase-encoded viral proteases generate up to 16 nonstructural proteins (Nsps) in the cytosol to produce replicase enzyme and the replicase–transcriptase complex (RTC). These enzymes including RTC synthesize RNAs for replication and transcription to generate viral RNA genome. CoV genomes bear two or three protease genes and the coding enzymes cleave the replicases. Together with the replicases, nonstructural proteins, termed Nsps, assemble into the RTC complex. Nsp1 to Nsp16 are known to have multiple enzyme regions. For example, Nsp1 degrades cellular mRNAs and, consequently blocks protein translation in host cells and innate immune responses. Nsp2 recognizes the specific protein called prohibitin. Nsp3 is a multi-domain transmembrane (TM) protein with diverse activities. Ubiquitin-like 1 and acidic domains bind to N protein and ADP-ribose-1′-phosphatase (ADRP) activity induces cytokine expression. The papain-like protease (PLpro)(PDB:6WX4)/ deubiquitinase domain cleaves virus-produced polyprotein. Nsp4 is a TM scaffold protein for double-membrane vesicle structure. Nsp5 has a main protease domain which also cleaves virus-produced polyprotein and Nsp6 acts as a TM scaffold protein. The Nsp7 and Nsp8 proteins form the Nsp7-Nsp8 hexadecameric complex, which functions as an RNA polymerase-specific clamp and a primase enzyme. Nsp9 is an RNA-binding protein that activates ExoN and 2-O-methyltrnasferase (MTase) enzyme activity. Nsp10 binds to Nsp16 and Nsp14 to form a heterodimeric complex. Nsp12 is the RdRp and Nsps13 is the RNA helicase and 5′ triphosphatase. Nsps14 is a N7 MTase and 3′-5′ exoribonuclease. ExoN of Nsap14 acts as an N7 MTase and attaches the 5′ cap to viral RNAs. Viral exoribonuclease enzyme proofreads the viral RNA genome, where Nsp15 is a viral endoribonuclease (PDB: 6VWW). Nsp16 has 2-O-MTase enzyme activity, which shields viral RNA from melanoma differentiation associated protein-5 recognition.

3.1. Spike (S) Transmembrane Glycoprotein
In RNA viruses, the S glycoprotein (PDB: 6VSB) is the biggest protein, heavily glycosylated and its N-terminal domain (NTD) sequence binds to the host receptor to enter the ER of host cells. SARS-CoV-2 S-glycoprotein bears 22 N-glycan sequons in each protomer. Therefore, the trimeric S glycoprotein surface is dominated by 66 N-glycans. The S glycoprotein mediates direct and indirect interaction of virus with host cells in the infection cycle. All CoVs exhibit a surface S glycoprotein, which bears the receptor-binding domain (RBD). The S glycoprotein has a distinct spike structure. When S glycoprotein binds to its host receptor, a host furin-like protease cleaves the S glycoprotein, which liberates the spike fusion peptides, allowing entry of the virus into the host cell [18]. The furin-like protease-generated S1 and S2 exist as a S1/S2 complex, where S1 in a homotrimeric form interacts with the host cell membrane and S2 penetrates the cytosolic area. For SARS-CoV and MERS-CoV, the S1 C-terminal domains (CTDs) have a dual role in virus entry via attachment and fusion. The S1 NTD binds to carbohydrate receptors because the S1 domains act as the RBD. The CTD of S1 recognizes protein receptors via RBDs.

3.2. Nucleocapsid (N) Protein
In RNA viruses, the N protein recognizes the viral RNA genome. The N protein (PDB: 6M3M) binds to the RNA genome via the NTD and CTD. The N protein tethers to the viral RNA and replicase–transcriptase complex (RTC). NSP3 (PDB: 6VXS) of CoV blocks the innate immune responses of hosts. After entrance into the host cells, for CoV transcription and particle release, RNA chaperones such as nonspecific nucleic acid binding proteins potentiate ssRNA conformation shifts. Representatively, the N protein is known as the RNA chaperone protein. For example, glycogen synthase kinase 3 (GSK3) phosphorylates the SARS-CoV N-protein and thus, GSK3 inhibition contributes to reduced replication activity of SARS-CoV [19]. In addition, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) regulates the preformed mRNA splicing in the nucleus and continuous translation. hnRNPA1 interacts with SARS-CoV N protein to form a replication and transcription complex during ssRNA genome biosynthesis [20].

3.3. Envelope (E) Protein
E protein is the most abundant structural protein needed to assemble virus particles in the cytosol. As a TM protein, E protein is the smallest structural protein with a MW of 12 kDa. The E protein has an NTD in the extracellular region and a CTD in the cytosolic region. The E protein bears an ectodomain in the NTD and an endodomain in the CTD. It has also an ion channel domain. E protein is present in the cytosolic region of infected cells and only a limited amount is incorporated into the envelope of virions [21]. Most E proteins assemble and bud in new virus particles.

3.4. Membrane Glycoprotein (M)
The M protein contains three TM domains and is an abundant structural protein with a MW of 30 kDa. It consists of a small glycosylated NTD in the extracellular region and CTD in the cytoplasmic region. The M protein forms a scaffold for virus assembly in the cytosol via binding to S glycoprotein and N protein [22]. For example, E protein and N protein are co-expressed with M protein to form virus-like particles (VLPs) that are released from the cells, as the M and E protein are involved in CoV assembly. Then, CoVs bud into the ERGIC, trafficking by membrane vesicles and transported via the exocytosis-secretory pathway [23,24]. The dimeric M protein binds to the nucleocapsid.-M protein binds to S glycoprotein for S glycoprotein retention in the ERGIC/Golgi complex. The M-N protein complex keeps the N protein–RNA complex stable, for nucleocapsid and the viral assembly.-M and E proteins constitute the virus envelope for successful release of virus-like particles.

3.5. Hemagglutinin-Esterase (HE) Dimeric Protein
HE hemagglutinates and destroys receptors. As RNA viruses, CoVs bear RDEs, which are used in effective attachment to hosts and also reversely in detachment from the hosts. For example, enveloped RNA viruses evade the hosts via their RDEs. Currently, RDE-related functional enzymes such as neuraminidase (NA) and SA-O-acetyl-esterase are known. SA-O-acetyl-esterase was originally identified in influenza C virus and in nidoviruses (CoV and torovirus) as well as in salmon anemia virus (teleost orthomyxovirus). The origin and evolution of CoV SA-O-acetylesterases are correlated to other viruses. The fusion event of S glycoprotein and HE is specific for HCoV attachment to SA-associated receptors in the host [25]. The HE has acetylesterase activity [26]. In early SA-related biology, influenza A/B viruses were found to recognize chicken erythrocytes in 1942 [27]. They caused hemagglutination through clumping by virus-borne hemagglutinin. These phenomena were widely found in influenza viruses, paramyxoviruses, Newcastle disease (NDV) and mumps virus.