Differential contributions of human oligosaccharyltransferase complexes OST-A and OST-B to HIV-1 envelope glycoprotein glycosylation.

N-linked glycosylation of glycoproteins during synthesis in the endoplasmic reticulum (ER) is mediated by oligosaccharyltransferase (OST) complexes OST-A and OST-B that have different catalytic subunits STT3A and STT3B, respectively. OST-A acts cotranslationally, while OST-B adds glycans posttranslationally. While there is redundancy between these two enzymes, it is unclear how they both contribute to glycosylation of the densely glycosylated HIV-1 envelope glycoprotein complex (Env). We found that knocking out STT3A had a profound negative impact on HIV-1 virus production and infectivity while STT3B ablation had no such effect suggesting that STT3A is more important than STT3B for Env glycosylation and preserved function. STT3A/3B knockout (KO) affected the neutralization sensitivity to broadly neutralizing antibodies (bNAbs) in a strain-specific manner with STT3A-KO increasing susceptibility to VRC01 bNAb for the tested HIV-1 strains. In contrast, for the BG505 strain A virus, it conferred increased resistance to glycan-dependent bNAbs 2G12 and PGT128. For other HIV-1 strains, STT3B-KO also led to resistance to glycan-dependent bNAb PGT151. Site-specific glycan analysis of recombinant Env proteins revealed that STT3A-KO reduced glycan occupancy of potential N-linked glycosylation sites (PNGS) more globally than STT3B-KO, with certain acceptor sites, including N234 and N386, showing STT3A dependence. In contrast, STT3B-KO appeared to have a more pronounced effect on gp41 glycosylation, suggesting that PNGS located near the C-terminus are more dependent on STT3B. Defining the roles of the OST-A and OST-B complexes in HIV-1 Env glycosylation may bring critical information for the development of methods to control PNGS glycan occupancy of recombinant glycoprotein immunogens.