Within the trimeric HIV-1 envelope (Env) spike, the first and second variable loops (V1V2 region) and the third variable loop (V3) of the gp120 subunit play dual roles in antibody recognition, because they contain neutralization
epitopes and also participate in
epitope masking. The spatial relationships between V1V2 and V3 and the associated mechanisms of
epitope masking remain unclear. Here we investigated interactions between these domains using two
monoclonal antibodies recognizing distinct conserved linear
epitopes that are subject to masking in the functional trimer, which limits their neutralizing activities. Using Env pseudotype
virus infection assays, we found that deleting the V1V2 region greatly enhanced neutralization by both
antibodies, leading us to consider two alternative models: V1V2 on one gp120
protomer masks V3 on the same
protomer (intraprotomer or cis masking) versus on an adjacent
protomer (interprotomer or trans masking). Our experimental approach exploited a previously described complementation system wherein two variant Envs harboring different inactivating mutations (one in gp120, the other in gp41) are coexpressed in the same cell; functional Env results only from cooperative interactions within mixed trimers, thereby enabling selective examination of mixed trimer activity. We introduced additional mutations that either promoted (V1V2 deletion, i.e., unmasking) or prevented (GPGR to GPGQ mutation, i.e.,
epitope destruction) interaction with the
antibodies. The observed neutralization sensitivities of mixed trimers produced from various combinations of constructs support the intraprotomer (cis) model of V1V2 masking of V3
epitopes.