Isoform-dependent glycosylation of respiratory viral class I fusion glycoproteins by oligosaccharyltransferases.

Viral fusion proteins decorate their antigenic surface with N-linked glycans which support processes such as protein folding, cell-specific interactions and shielding of vulnerable antibody epitopes. Asparagine-linked glycosylation is catalyzed by the oligosaccharyltransferase (OST) complexes containing the catalytic subunits STT3A or STT3B, which act predominantly co- and post-translationally, respectively. Here, we investigated the contributions of STT3A and STT3B to glycan attachment to recombinant SARS-CoV-2 Spike (S) and influenza A (H3N2) virus hemagglutinin (HA). Soluble proteins and pseudotyped viruses were produced in wild-type and STT3A or STT3B-knockout (KO) 293T cells. Site-specific glycan analysis of the recombinant proteins revealed significant changes at only a limited number of glycosylation sites upon deletion of either STT3A or STT3B, indicating partial redundancy in maintaining overall site occupancy. However, STT3A-KO reduced glycosylation at N717 and N1074 on the SARS-CoV-2 S protein, while STT3B KO reduced glycosylation at N483 on influenza HA, suggesting isoform-specific preferences for distinct glycosylation sequons. Infectivity assays further suggested that the glycosylation of both SARS-CoV-2 and influenza A (H3N2) viral glycoproteins are more dependent on STT3A, with STT3B contributing to a lesser but detectable extent. These findings highlight distinct, context-dependent and site-specific contributions of STT3A and STT3B to viral glycoprotein glycosylation, with implications for vaccine antigen design.