3. Biological Functions of PEDV Proteins
The viral proteins of PEDV perform different biological functions during viral entry, replication cycle and propagation (Table 1). PEDV S protein, a type I membrane glycoprotein protein located on the envelope of the virus, consists of an N-terminal signal peptide, a large extracellular region, a single transmembrane domain, as well as a short cytoplasmic tail [48,49]. The ectodomain of S protein comprises S1 and S2 subunits. The N-terminal S1 region, containing N- and C-terminal domains (S1-NTD and S1-CTD), is mainly responsible for receptor binding [50]. The C-terminal membrane-anchored S2 region is mainly involved in triggering the fusion of the viral envelope with host cell membranes [48,49]. The interaction of the CoV S protein with its host cell surface receptor is a key determinant for host tropism. The S1-CTD of most known members of α-CoV genus, including PEDV, interacts with aminopeptidase N (APN) to entry into the target cell [32,36,51,52,53,54]. In addition, S protein contains the epitopes that are the major targets of the neutralizing antibody. N protein is the most abundant viral protein during the early phase of infection in CoV-infected cells [55]. Similar to the N proteins of other CoVs, the PEDV N protein has multiple functions, such as acting as a structural protein that forms nucleocapsid with viral genomic RNA, playing important roles in viral replication, transcription, and assembly [56,57]. The expression of the N protein in intestinal epithelial cells extends the S-phase of cell cycle, causes endoplasmic reticulum (ER) stress, and upregulates interleukin-8 expression [44]. Moreover, the N proteins of several α-CoVs and β-CoVs, including PEDV, PDCoV, SARS-CoV, and mouse hepatitis virus (MHV), have been identified as innate immunity antagonists [58,59,60,61]. However, the involved antagonistic mechanisms are particularly different. PEDV M protein participates in virion assembly and virus budding through collaboration with other viral proteins, and engages in the induction of neutralizing antibodies against PEDV [62,63]. PEDV M protein is distributed throughout the cytoplasm. It induces the cell growth retardation in intestinal epithelial cells (IEC) and arrests the cells in S-phase [64]. In addition, PEDV M protein is identified as an interferon (IFN) antagonist with an unrecognized mechanism [65]. PEDV E protein is important for the virus packaging and budding [66]. It is predominantly localized in the ER, having no effect on cell growth, cell cycle and cyclin A expression in IEC. However, it causes ER stress and activates the nuclear factor-κB (NF-κB) pathway which is responsible for the up-regulation of IL-8 and the anti-apoptotic protein Bcl-2 expression [67]. ORF3 has been predicted to possess multiple transmembrane domains [68], while it is predominantly distributed in the cytoplasm [69]. ORF3 also detains cells at S-phase, facilitating vesicle formation, and thus promoting PEDV multiplication [69]. A recent study suggests that ORF3 interacts with S protein during PEDV assembly and consequently benefits viral replication [70].
CoV nsps play multiple roles in the synthesis or processing of viral RNA, or in virus-host interactions aiming to create an optimal environment for virus replication, such as facilitating viral entry, viral gene expression, RNA synthesis, and virion release. nsp1 is a N-terminal cleavage product of ORF1a polyprotein [71], a 9-kDa protein, that exists only in α-CoVs and β-CoVs [72]. The nsp1 of α-CoVs is not very similar to β-CoVs nsp1 with regard to sequence homology and size [73,74]. Based on the sequence alignment analysis of the genomes of different CoVs, the viral nsp1 can be regarded as a genus-specific marker [75]. Moreover, β-CoVs nsp1 has been widely reported to inhibit host protein expression. However, the biological functions of α-CoVs nsp1 remain largely unknown. Despite the lack of overall sequence similarity, the nsp1 of different CoVs shares a similar function to interfere with host protein expression [76]. These studies suggest the importance of nsp1 in the life cycle of different lineages of CoVs. It is shown that TGEV nsp1 inhibits host gene expression and is critical for viral virulence [77]. PEDV nsp1 induces the degradation of CBP and NF-κB to abate IFN response [78], but the detailed mechanisms remain unclear. The sizes and amino acid sequence identity of nsp 2 are variable among different CoVs [76]. Nsp2 of MHV and SARS-CoV are involved in viral RNA synthesis [79]. PEDV nsp2 has unknown functions in replication, and may implicate the virus-host interactions and virulence. Nsp3 is the largest nsp protein, containing two papain-like protease (PLP1 and PLP2) domains, of which PEDV PLP2 acts as a viral deubiquitinase (DUB), to negatively regulate type I IFN signaling [80]. CoVs PLPs domains exhibit multiple functions, serving as a viral protease, DUB, as well as an IFN antagonist [81].
CoVs, like other positive-stranded RNA viruses, induce membranous rearrangements of varying morphologies that are essential for RTCs anchoring [82,83]. The CoV-induced replicative structures consist of double-membrane vesicles (DMVs) and convoluted membranes (CMs), which form a large reticulovesicular network that are critical for viral replication and transcription [84,85,86,87,88,89,90,91,92,93]. Among the CoV nsps, nsp3, nsp4, and nsp6 include the hydrophobic transmembrane domains engaging in anchoring the viral RNA synthesis components to the membranes [94]. For MERS-CoV and SARS-CoV, co-expression of nsp3 and nsp4 is required to induce DMVs [95]. SARS-CoV nsp6 has membrane proliferation ability as well, which also contributes to DMVs formation [96]. The structure and functions of α-CoV nsp3 are largely unknown [97]. Nsp4 is also a marker for CoV-induced membrane structures; some results indicate that the nsp4–10 of pp1a act as a large complex through multidomain structure or scaffold during viral RNA replication progress, before its cleavage into individual products [98]. CoVs nsp5 encodes a 3C-like proteinase (3CLpro). The polyproteins pp1a and pp1ab are processed into individual elements of replicase by 3C-like protease and PLPs [99]. Moreover, PEDV nsp5 plays a crucial role in virus replication and also blocks host innate immune responses [100]. Crystallographic or nuclear magnetic resonance structures have shown that nsp3, nsp5, nsp7, nsp8, nsp9, and nsp10 have the PLprob and the ADP-ribose 1′′-phosphatase (ADRP) activity [101,102,103,104,105,106,107]. The crystal structure of SARS-CoV nsp9 suggests that nsp9 is dimeric and it is able to bind to single-stranded RNA [108]. The crystal structure of SARS CoV nsp10 protein suggests that nsp10 is a zinc-finger protein, which is existent exclusively in CoVs so far [107]. Moreover, nsp7–10 have RNA binding activity and nsp12 encodes a single RNA-dependent RNA polymerase (RdRp). The biochemical characterization and crystallization of SARS CoV nsp7 and nsp8 manifests that eight copies of nsp8 and eight copies of nsp7 form a supercomplex [106]. The complex is supportive for nucleic acid binding and may be associated with the processivity of viral RdRp [102,106]. Recently, structural studies have described that the SARS-CoV nsp12 polymerase binds to the nsp7 and nsp8 complex [109] that may increase the polymerase activity of nsp12 RdRp [110]. CoV nsp13, a NTPase/helicase, is also determined to play essential roles in viral replication [111]. CoV nsp14 is a multifunctional protein with 3′-5′ exoribonuclease activity and N-7-methyltransferase [MTase] activity [112,113]. Nsp14 catalyzes the N7-methylation of Gppp-RNA to form a cap-0 structure. CoV nsp15 encodes an endoribonuclease (EndoU), performing functions through a hexamer in many CoVs [114,115,116]. The endoribonuclease activity of nsp15 is not essential for CoV replication [117,118]. For CoVs, the 5′ end of the viral genomic RNA and subgenomic mRNA (sgmRNA) is supposed to have cap structures: an N-7 methylated guanosine nucleoside (m7GpppN) (cap 0) and a methyl group at the 2′-O-ribose position (cap 1) of the first nucleotide [119]. These cap structures enhance the initiation of translation of viral proteins, protect viral mRNAs against cellular 5′-3′-exoribonuclease and limit the recognition of viral RNA by host innate system [120,121]. Nsp13 is proposed to catalyze the first step of the 5′-capping reaction of viral RNAs [122]. The methylation of the two sites in the 5′ cap are catalyzed by three nsps; nsp14 (the N-7-MTase), nsp16 (the 2′-O-methyltransferase), and nsp10 [112,123,124,125,126]. In addition, the 3′-5′ exoribonuclease activity of nsp14 is involved in a replicative mismatch repair system during RNA synthesis, which improves the replication fidelity of CoV [42]. Although these nsps have been demonstrated to play essential roles in viral replication, transcription and/or post-translational polyprotein processing [127], the nsp12–16 of PEDV and other CoVs are poorly characterized to date, except for SARS-CoV.