Regulation of epitope exposure in the gp41 membrane-proximal external region through interactions at the apex of HIV-1 Env

Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from EnvSF162 into the EnvHXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the EnvHXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312–315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits.Author summary: Surface glycoprotein Env is the main target for neutralizing antibodies elicited by HIV-1 vaccines. Env spontaneously fluctuates among different structures, limiting exposure of many attractive antibody-binding epitopes and, thereby, confounding vaccine development. To characterize these fluctuations, we examined how exposure of the MPER epitope found at the base of Env is regulated by interactions of the V3 loop located in the apex. Starting with an extremely flexible Env with a readily-exposed MPER, we identified two alterations that substantially restricted antibody access to the epitope. The first, a wholesale swap of V3 loops between HIV-1 strains, energetically stabilized Env in a closed structure that restricted access to antibodies throughout the protein. The second, a point mutation that altered V3-loop charge, specifically destabilized the MPER-exposed conformation but had minimal impact on antibody access to Env regions in between the apex and base. The results indicate that MPER exposure is not explicitly tied to the dynamics of Env regions between the apex and base and suggest a new structural fluctuation during Env activation.