Membrane binding proteins of coronaviruses

J Alsaadi, Entedar A; Jones, Ian M

The 44 kDa CoV nsp4 protein is also a transmembrane protein, with four transmembrane helices and an internal C-terminal domain ( Figure 3 ) and with nsp3 is an indispensable component required to produce DMVs [73, 74] . All CoV-nsp4 molecules encode at least one predicted glycosylation site and in the case of MHV, it has been shown that mutation of the glycosylation site results in loss of virus fitness suggesting that nsp4 glycosylation is necessary for virus replication or the organization of the DMVs [75] . In an electron micrographic study, transfection of SARS-nsp3 and nsp4 alone caused considerable membrane deformation, producing a perinuclear double-walled maze-like body (MLB) [76] and the nsp3-nsp4 interaction was shown to be absolutely necessary for such membrane rearrangement. However the interaction of these two nsps was insufficient in itself to trigger membrane rearrangement and host factors such as EDEM1 and OS9 of the ER-associated degradation system have been shown to be co-factors [77, 78] . Despite them being a universal feature of CoVs the size and number of DMVs does not appear to correlate directly with viral fitness, at least when virus is grown at reduced temperatures [79] nor are they a determinant of pathogenicity [80] . For the γ CoV Infectious Bronchitis Virus (IBV), nsp4 was essential and sufficient to induce membrane pairing, recognized as extensive areas of membrane accumulation or small regions of paired membrane, but expression of nsp3, nsp4 and nsp6 was required for DMV production which, even then, was poor for strain BeauR and not seen at all for strain M41. DMVs formed by IBV nsp3, nsp4, and nsp6 alone were poorly efficient when compared with DMVs formed by Betacoronavirus infection so supplementation of nsp4 with nsp6 is not sufficient for authentic IBV DMV production [81] . CoV nsp6 is a membrane protein of approximately 34 kDa predicted molecular mass with six transmembrane helices (Figure 3) including, in almost all viruses, a highly conserved C-terminus [82] . Although nsp6-stimulated internal cellular membrane rearrangement is observed with the addition of nsp3 and 4, nsp6 also causes membrane proliferation alone, including the formation of Atg5 and LC3II-positive vesicles classically observed in autophagy [83] . The autophagosomes produced are somewhat different from those induced by starvation; however, as although their number is higher and their size is reduced [84] . As noted above, along with nsp3 and 4, nsp6 functions to produce the canonical DMVs as well as many other types of intracellular vesicles observed in CoV infected cells such as convoluted membranes, vesicle packages, tubular bodies, large virion-containing vacuoles (LVCVs), cubic membrane structures (CMSs) and zippered ER spherules in the case of IBV [85, 86] . An attenuated form of an IBV vaccine includes mutations in an nsp6 TM domain, confirming its role in virulence and replication [87] .