Post-translational modifications of coronavirus proteins: roles and function

Fung, To Sing; Liu, Ding Xiang

O-linked glycosylation O-linked glycosylation of the MHV M protein was first discovered in 1981 [90] . It was found that in the presence of tunicamycin, an inhibitor of N-linked glycosylation, synthesis of the S protein was completely inhibited, but M protein was still normally produced and glycosylated, resulting in the formation of noninfectious virions containing normal amounts of N and M protein, but lacking S completely [90] . When it was expressed from transfected future science group www.futuremedicine.com cDNA, M protein of MHV-A59 also underwent O-linked glycosylation and was localized in the Golgi region [92] . The structures of the O-linked glycans of MHV-A59 M protein were characterized [93] , and pulse-chase labeling experiments showed that the O-linked glycans were acquired in a two-step process: GalNAc was added before the addition of galactose and sialic acid [94] . After the sequential acquisition of GalNAc, galactose and sialic acid, the M protein of MHV-A59 was further modified in the trans-Golgi network [95] . Apart from MHV, Olinked glycosylation was also found in the M protein of two other lineage A Betacoronaviruses: BCoV [27, 96] and HCoV-OC43 [97] . Since its discovery, O-linked glycosylation has been used as a marker to study the maturation, membrane insertion and intracellular trafficking of MHV M protein [98, 99] . In fact, due to its high expression level in transfected or MHV-infected cells, the M protein of MHV has also been used as a model protein to study O-linked glycosylation and vesicular trafficking between ER and the Golgi compartments [100] . Initial studies proposed the four highly conserved hydroxyamino acids (S2, S3, T4 and T5) at the extreme N terminus of MHV M protein as the putative O-linked glycosylation sites [93] . Follow-up investigations further pinpointed T5 as the functional acceptor site, and the downstream P8 was also required for efficient O-linked glycosylation [101] . However, the hydroxylamino acid cluster per se was not sufficient, as downstream amino acids must also be included to introduce a functional O-linked glycosylation site into a foreign protein [101] . Interestingly, in the highly virulent strain MHV-2, the S-S-T-T sequence was mutated to N-S-T-T, and N-linked glycosylation was shown to be added to the N2 residue [102] . However, whether the presence of extra sugars would affect the function of MHV-2 M protein has not been fully understood.