Post-translational modifications of coronavirus proteins: roles and function

Fung, To Sing; Liu, Ding Xiang

Among all the coronavirus nsps, three of them are known to contain TM domains that facilitate their insertion into ER membrane. Nsp3 and nsp4 have two and four TM domains respectively [141] , while nsp6 contains six TM domains with a hydrophobic C-terminal cytosolic tail [142] . These three nsps are proposed to reorganize ER membrane to form DMVs and to facilitate the assembly and anchorage of the replication/transcription complex to the DMVs. In fact, co-expression of SARS-CoV nsp3, nsp4 and nsp6 induced DMV formation in the transfected cells [143] . A more recent study showed that, for both MERS-CoV and SARS-CoV, co-expression of nsp3 and nsp4 was already sufficient to induce DMV formation [144] . On the other hand, overexpression of coronavirus nsp6 induced the formation of autophagosomes [145] , but at the same time restricted its expansion [146] . Therefore, nsp3, nsp4 and nsp6 are closely associated with cellular membrane dynamics in coronavirus-infected cells. Given their membrane multispanning nature, it is not surprising that some of the luminal domains undergo N-linked glycosylation in the ER (Figure 6 ). For example, MHV nsp3 is inserted into ER co-translationally and glycosylated at N1525 [147] . Glycosylation of nsp4 was first identified in IBV (Lim et al., 2000) . By glycosidase digestion and site-directed mutagenesis, the glycosylation site of IBV nsp4 was confirmed to be at N48 [148] . As for the nsp4 of MHV, two glycosylation sites were predicted at N176 and N237. In one early study using reverse genetics, it was found that whereas recombinant MHV harboring nsp4-N176A mutation replicated identically to the WT control, nsp4-N237A was lethal and no recombinant virus could be recovered [149] . In a later study using identical infectious clone system based on MHV-A59, Gadlage et al. successfully recovered recombinant MHV with N176A, N273A or N176A/N273A mutation in nsp4 [150] . Interestingly, all nsp4 glycosylation mutants exhibited aberrant morphology of DMVs and were defective in viral RNA synthesis and virus growth, supporting a critical role of N-linked glycosylation in the DMV formation activity of MHV nsp4 [150] . In a recent follow-up study, other mutations distinct from glycosylation sites were introduced in MHV nsp4. Similar to the glycosylation mutants, some of these mutants also exhibited altered DMV morphology. However, only mutations in the nsp4 glycosylation sites resulted in a loss of fitness in the recombinant MHV [151] . Therefore, apart from DMV formation, N-linked glycosylation of MHV nsp4 may serve other critical roles during viral replication. future science group www.futuremedicine.com