Post-translational modifications of coronavirus proteins: roles and function

Fung, To Sing; Liu, Ding Xiang

into the envelope and provides structural support to the virion. The E protein is a small, integral membrane protein present at a low amount in the virion, but it plays an essential role during virion assembly and release [11, 12] . Inside the envelope, the helically symmetric nucleocapsid is comprised of the RNA genome closely associated with the N protein in a beads-on-a-string fashion. The positive sense, nonsegmented, ssRNA genome, ranging from 27,000 to 32,000 nucleotides in size, is the largest RNA genome known to date. The replication cycle of coronavirus starts with the binding of the S protein to its cognate receptor(s) on the host cell surface (Figure 1 ), which triggers a conformational change in the S2 subunit and results in the fusion between the viral envelope and the cellular membrane, thereby delivering the nucleocapsid into the cytoplasm [9] . After uncoating, the genomic RNA containing a 5 -cap and a 3 -poly(A) tail is recognized by the host translation machinery to synthesize a polyprotein 1a (pp1a), as well as a larger polyprotein 1ab (pp1ab) in a process involving ribosomal frameshifting [13] . Autoproteolytic cleavage of pp1a and pp1ab produces 15-16 nonstructural proteins (nsps) with diverse functions. Among them, nsp3 and nsp5 encode the papain-like protease (PLPro) activity and the chymotrypsin-like main protease (M pro ) activity, respectively, whereas nsp12 encodes the critical RNAdependent RNA polymerase (RdRp) activity [14, 15] . In the replication/transcription complex closely associated with virus-induced double membrane vesicles (DMVs) or spherules, positive-sense progeny genomic RNA is synthesized from the negative-sense intermediate. On the other hand, a nested set of subgenomic RNA (sgRNA) species is synthesized by discontinuous transcription of the genome, from which structural and accessory proteins are translated. Transmembrane structural proteins (S, M and E) are synthesized, folded and modified in the N-linked glycosylation N-linked glycosylation of coronavirus S protein was first described for MHV in the 1980s [21] . MHV S protein in the rough ER was found to acquire high mannose oligosaccharides. Treatment of the Golgi transport blocker monensin inhibited the transport of MHV S protein from trans-Golgi network to the cell surface [21] . Later studies demonstrated that S proteins of IBV [24] , TGEV [25, 26] , bovine coronavirus (BCoV) [27] were also modified by N-linked glycosylation. Using pulse-chase experiments coupled with fractionation, it was found that high mannose glycans were acquired by monomer of the TGEV S protein, followed by the rate-limiting assembly of monomers into a trimeric structure and terminal glycosylation of the newly assembled trimers [28] . Similarly, SARS-CoV S protein was found to acquire high mannose oligosaccharides and trimerize as early as 30 min postentry into ER, prior to the acquisition of complex glycans in the Golgi complex [29] . The maturation status of SARS-CoV S protein can thus be monitored by its sensitivity to endoglycosidase H (endo H), which hydrolyzes high mannose glycans but not complex glycans [30] . Using mass spectrometry, the structure of N-linked glycans on SARS-CoV S protein was determined, which were composed of high mannose, hybrid and complex glycans with and without bisecting N-acetyl-galactosamine (GalNAc) and core fucose [31] . With the advent of molecular cloning technologies, the coding sequences of S proteins from numerous coronavirus