Identification of novel structural determinants in MW965 Env that regulate the neutralization phenotype and conformational masking potential of primary HIV-1 isolates

The subtype C HIV-1 isolate MW965.26 is a highly neutralization-sensitive tier 1a primary isolate that is widely used in vaccine studies, but the basis for the sensitive neutralization phenotype of this isolate is not known. Substituting the MW965.26 V1/V2 domain into a neutralization-sensitive SF162 Env clone resulted in high resistance to standard anti-V3 monoclonal antibodies, demonstrating that this region possesses strong masking activity in a standard Env backbone and indicating that determinants elsewhere in MW965.26 Env are responsible for its unusual neutralization sensitivity. Key determinants for this phenotype were mapped by generating chimeric Envs between MW965.26 Env and a typical resistant Env clone, the consensus C (ConC) clone, and localized to two residues, Cys384 in the C3 domain and Asn502 in the C5 domain. Substituting the sensitizing mutations Y384C and K502N at these positions into several resistant primary Envs resulted in conversion to neutralization-sensitive phenotypes, demonstrating the generalizability of this effect. In contrast to the sensitizing effects of these substitutions on normally masked epitopes, these mutations reduced the sensitivity of VRC01-like epitopes overlapping the CD4-binding domain, while they had no effect on several other classes of broadly neutralizing epitopes, including members of several lineages of V2-dependent quaternary epitopes and representatives of N332 glycan-dependent epitopes (PGT121) and quaternary, cleavage-dependent epitopes centered at the gp41-gp120 interface on intact HIV-1 Env trimers (PGT151). These results identify novel substitutions in gp120 that regulate the expression of alternative conformations of Env and differentially affect the exposure of different classes of epitopes, thereby influencing the neutralization phenotype of primary HIV-1 isolates.