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

## TL;DR

This study shows that the OST-A complex plays a bigger role than OST-B in adding sugars to the HIV-1 envelope protein, which is important for vaccine design.

## Contribution

The study reveals the distinct roles of OST-A and OST-B in HIV-1 Env glycosylation and identifies specific glycosylation sites influenced by each complex.

## Key findings

- STT3A knockout significantly reduced HIV-1 production and infectivity, while STT3B knockout had no such effect.
- STT3A knockout increased susceptibility to some antibodies but conferred resistance to others in a strain-specific manner.
- OST-A primarily controls glycosylation of most sites, while OST-B mainly affects C-terminal glycosylation.

## Abstract

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 clade 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.

HIV-1 envelope glycoprotein (Env) complex is the sole target of broadly neutralizing antibodies (bNAbs), making it the primary focus of vaccine design efforts. The Env glycoprotein is one of the most heavily glycosylated proteins found in nature. However, the contributions of oligosaccharyltransferase (OST) isoforms STT3A and STT3B to Env glycosylation have not been fully characterized. Under-occupancy of potential N-linked glycosylation sites (PNGS) on recombinant Env glycoproteins can elicit off-target immune responses and pose challenges for HIV-1 vaccine development. Understanding and controlling the mechanisms behind PNGS occupancy is therefore critical for rational immunogen design. This study demonstrated that the viral Env glycosylation is mostly controlled by OST-A. Additionally, site-specific glycan analysis of recombinant Env proteins identified several STT3A-dependent sites and confirmed a dominant role of STT3B in C-terminal glycosylation. Our fundamental study contributes novel insights into host-cell-mediated glycosylation and informs the development of methods to regulate PNGS occupancy of Env-based immunogens.