Alternative substitutions of N332 in HIV-1 AD8 gp120 differentially affect envelope glycoprotein function and viral sensitivity to broadly neutralizing antibodies targeting the V3-glycan.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332 but Asn at this position is not absolutely conserved or required for HIV-1 entry based on prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1 AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro . Our results suggest robust tolerability of HIV-1 AD8 Env N332 to changes with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIV AD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity even though cell surface expression levels are lower than most N332 variants. Thus, tolerance of HIV-1 AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and to evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs was in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states.