For 185F, the 5-month Nab resistant EnvPB1.1 was chosen to determine which sequence adaptations were responsible for early Nab escape. Fig. 3A demonstrates that the chimeras containing either the region spanning the V1 loop through the end of the V5 domain (V1V5) or the V3 loop through the V5 domain (V3V5) from the 5-month Env displayed a level of resistance similar to the parental Env. However, the Nab sensitivity of the chimera containing the entire V1V2 domain (V1V2) was unchanged compared to the 0-month Env, despite a K192Q change in V2 (Fig. 3C) . We therefore surmised that Nab resistance was heavily dependent upon the V3V5 subregion, in which there were 3 residues that differed between 0-month EnvPB3.1 and 5-month EnvPB1.1. These changes were an E335A in the first position of the a2 helix, and two changes in the V5 region: I459T, which may also impact CD4 binding, and S463N ( Fig. 3C ; based on HXB2 numbering). None of these changes altered any of the predicted Nlinked glycosylation sites. To assess its individual contribution to Nab resistance, each amino acid change was introduced into the 0month EnvPB3.1. The V5 mutations I459T and S463N each independently produced a decrease in neutralization sensitivity, while these mutations combined recapitulated the Nab resistance level of the V3V5 chimera ( Fig. 3B ). In contrast, the E335A change in the a2 helix did not decrease neutralization sensitivity when introduced by itself into the 0-month Env (Fig. 3B ).