HIV-1 gp41 Residues Modulate CD4-Induced Conformational Changes in the Envelope Glycoprotein and Evolution of a Relaxed Conformation of gp120

Abstract

Entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by conformational changes in the envelope glycoprotein (Env) that are triggered by Env binding to cellular CD4 and chemokine receptors. These conformational changes involve the opening of the gp120 surface subunit, exposure of the fusion peptide in the gp41 transmembrane subunit, and refolding of the gp41 N- and C-terminal heptad repeat regions (HR1 and HR2) first into an extended prehairpin intermediate and then into a compact 6-helix bundle (6HB) that facilitates fusion between viral and host cell membranes. Previously, we reported that Envs resistant to HR1 peptide fusion inhibitors acquired key resistance mutations in either HR1 or HR2 that increased 6HB stability. Here, we identify residues in HR1 that contribute not only to fusion inhibitor resistance and 6HB stability but also to reduced reactivity to CD4-induced conformational changes that lead to 6HB formation. While all Envs show increased neutralization sensitivity to mimetic CD4 (mCD4), Envs with either the E560K or Q577R HR1 mutation reduced conformational reactivity to CD4 that resisted viral inactivation and triggering to the 6HB. Using a panel of monoclonal antibodies (mAbs), we further determined that Envs from both HR1 and HR2 resistance pathways exhibit a relaxed trimer conformation due to gp120 adaptive mutations in different regions of Env that segregate by resistance pathway. These findings highlight regions of cross talk between gp120 and gp41 and identify HR1 residues that play important roles in regulating CD4-induced conformational changes in Env. Binding of the HIV envelope glycoprotein (Env) to cellular CD4 and chemokine receptors triggers conformational changes in Env that mediate virus entry, but premature triggering of Env conformational changes leads to virus inactivation. Currently, we have a limited understanding of the network of residues that regulate Env conformational changes. Here, we identify residues in HR1 of gp41 that modulate conformational changes in response to gp120 binding to CD4 and show that the mutations in HR1 and HR2 that confer resistance to fusion inhibitors are associated with gp120 mutations in different regions of Env that confer a more open conformation. These findings contribute to our understanding of the regulation of Env conformational changes and efforts to design new entry inhibitors and stable Env vaccine immunogens.

Characterization of resistance to a potent D-peptide HIV entry inhibitor

“…Using multiple methods, Weiss, et al have investigated the effect of Q577R on Env structure and function. They have shown Q577R leads to: 1) a more stable 6-helix bundle structure, 2) greater neutralization sensitivity to CD4 mimetics, and 3) decreased susceptibility to CD4-induced gp41 conformational changes that lead to viral inactivation [34,35,37,41]. This wide influence of Q577 mutation on Env function supports the concept that both gp120 and gp41 modifications would be needed to restore viral fitness, as is seen in our PIE12-trimer resistant pools.…”

“…In previous entry inhibitor resistance studies that include Q577R and for which gp120 sequence is available [15,[34][35][36]41], compensatory mutations are found in CD4 binding residues (E426K), the V1/V2 loop (D167N), and in the V3 loop (R298K, S306G, I309M, T319I, and E322D), indicating that changes to gp120-based fusion determinants, such as CD4 and co-receptor binding, help restore gp41-based fitness defects. Our V1/V2 and V4 loop deletions may similarly affect Env function.…”

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

“…Receptor binding initiates formation of the pre-hairpin intermediate conformation, in which both the MPER epitopes, as well as the T-20 binding site in the NHR are exposed (31–33). Several recent publications have described Env mutations that promote unliganded trimer sampling of an intermediate conformation, termed “state 2”, between closed (state 1) and receptor bound (state 3) conformations (34–37). State 2 is characterized by a heightened intrinsic reactivity to ligand binding, increased capacity to infect cells expressing low levels of CD4 or CCR5 receptors, and increased exposure of MPER and NHR residues.…”

“…N/CHR mutations that enhanced usage of macaque receptors increased sensitivity to neutralization by MPER antibodies, and, in the BF520 background, to inhibition by the fusion inhibitor T-20. The MPER epitope (sites 671-683) and T-20 binding pocket (sites 547-556) are located immediately N, and C terminal, respectively, to the NHR and CHR mutants enriched in our DMS (36, 37). Our neutralization data thus indicate that these mutations cause Env conformation changes that are confined to regions of the trimer directly adjacent to the mutated residues.…”

“…This phenotype has been linked to Envs frequently sampling state 2 conformations, in which the thermodynamic barrier between unliganded and CD4-bound states is lowered (34, 35). Envs displaying a state 2 phenotype are also known to display heightened sensitivity to MPER antibodies (34–37), and in some cases, increased exposure of the NHR helix (34). Our observations that N/CHR mutations increase sensitivity to MPER antibodies, and in the BF520 background modestly increase T-20 sensitivity, are consistent with the hypothesis that these mutations promote a state 2 conformation.…”