HIV-1 evolved to encode an envelope protein (Env) that triggers fusion of viral and cellular membranes while minimizing the exposure of its key domains to the host immune response. Long variable domains protect HIV-1 Env from the host humoral response; however, they seem detrimental for the transmission or early expansion of HIV-1. In the scheme of opposite selective pressures exerted by the immune response and the bottleneck of transmission, V1/V2 domain of Env appears center stage. In our preliminary studies of the Lusaka cohort, the length of Env V1/V2 domains inversely correlated with viral entry and Env incorporation, suggesting that resistance to V1/V2-targeting antibody could be associated with a decrease in Env function. We also found that Env species encoding additional “twin cysteines” in V1—a genetic signature associated with transmission— packaged more Envs into viral particles. We now hypothesize that the length and cysteine composition of the V1/V2 domain influences Env function and stability in conditions relevant to in vivo transmission. In Aim 1, we will investigate the relationship between V1/V2 length, susceptibility neutralization, and Env function in the large Amsterdam cohort composed of subtype B isolates with variation in the length of V1/V2 domains and in sensitivity to neutralizing antibodies. In Aim 2, we will explore how twin cysteines in the V1/V2 domain impact Env ability to mediate fusion in biological fluids highly relevant to HIV transmission in vivo and in the presence of broadly neutralizing antibodies that target glycan structures in V1/V2. These aims will extend our initial observation on the impact of V1/V2 length on transmission and could unravel important functional consequences of acquiring resistance to neutralization or alternative disulfide bonds.