Quaternary structures of HIV Env immunogen exhibit conformational vicissitudes and interface diminution elicited by ligand binding

Abstract

The human immunodeficiency virus envelope protein is the key element mediating entry into host cells. Conformational rearrangement of Env upon binding to the host CD4 receptor and chemokine coreceptor drives membrane fusion. We elucidated the quaternary arrangement of the soluble Env trimeric immunogen o-gp140ΔV2TV1, in both its native (unliganded) and CD4-induced (liganded) states by cryoelectron microscopy and molecular modeling. The liganded conformation was elicited by binding gp140 to the synthetic CD4-mimicking miniprotein CD4m. Upon CD4m binding, an outward domain shift of the three gp120 subunits diminishes gp120-gp41 interactions, whereas a "flat open" concave trimer apex is observed consequent to gp120 tilting away from threefold axis, likely juxtaposing the fusion peptide with the host membrane. Additional features observed in the liganded conformation include rotations of individual gp120 subunits that may release gp41 for N-and C-helix refolding and also may lead to optimal exposure of the elicited coreceptor binding site. Such quaternary arrangements of gp140 lead to the metastable liganded conformation, with putative locations of exposed epitopes contributing to a description of sequential events occurring prior to membrane fusion. Our observations imply a mechanism whereby a soluble Env trimeric construct, as opposed to trimers extracted from virions, may better expose crucial epitopes such as the CD4 binding site and V3, as well as epitopes in the vicinity of gp41, subsequent to conjugation with CD4m. Structural features gleaned from our studies should aid the design of Env-based immunogens for inducement of potent broadly neutralizing antibodies against exposed conformational epitopes.T he human immunodeficiency virus envelope proteins gp120 and gp41 associate in a trimeric arrangement (1-3) and mediate membrane fusion through conformational rearrangement and fusion peptide insertion into the host membrane. The gp120 tertiary conformational change observed via X-ray crystallography has not been definitively characterized in the context of a quaternary structure. Several observations have been gleaned from tomography studies, though with inconclusive and sometimes contradictory results. Quaternary structural features reported previously include trimer morphology, with some groups reporting a mushroom-like shape and others a more rod-like arrangement, as well as the relative location of the primary epitope, hypervariable loops, and N-and C-termini, which have some variation among the different tomograms. Another point of conflict is the presence of a cavity at the threefold axis, capped by a density. One final feature is the arrangement of gp41, with some groups reporting a thin, stalk-like rod arrangement of gp41 and another group reporting a more splayed out, tripod-like conformation of gp41 proximal to the viral membrane. Taken together, these contrasting features portray a heterogeneous set of registers that seem to hinder a cohesive and thorough model of ligand-induced quaternary arrangement...

Kinetic mechanism for HIV-1 neutralization by antibody 2G12 entails reversible glycan binding that slows cell entry

“…4F, derives from the fact that N295 and N332 occur directly adjacent to the disulfide bond on opposite borders of the V3 loop. This is a critical fulcrum for gp120 function because CD4 weakens V3 constraints on gp41 (19) and induces V3 movement toward the apex of the gp120 trimer (16,17,19) where HIV-1 JRCSF -Ad that had been pretreated for 1 h at 37°C ± 4 μg/mL 2G12. Titers were normalized relative to maximum titers at 100 μM Nt (n = 3, error bars are ± SEM).…”

“…After CD4 binding, V3 loop regions of gp120 reduce their constraining hold on gp41 and move toward the trimer apex where they interact with coreceptors, thereby playing a pivotal role in controlling HIV-1 entry rates (16)(17)(18)(19). These and additional conformational changes in gp120 enable gp41 to refold by a multistep process that fuses the viral and cellular membranes.…”

Straightforward Selection of Broadly Neutralizing Single-Domain Antibodies Targeting the Conserved CD4 and Coreceptor Binding Sites of HIV-1 gp120

“…Interestingly, they possess CD4 functional properties, including the ability to unmask gp120 conserved neutralization epitopes that are cryptic on the unbound glycoprotein (15)(16)(17)(18)(19). In the present work, to increase the chance of selecting neutralizing sdAbs targeting the CD4 and coreceptor binding sites of gp120, we have immunized llamas with gp140 (trimeric version of gp120 bound to the gp41 ectodomain), either free or cross-linked to a CD4 mimic (16).…”

Phylogenetic study of SIVcpz MT145 virus based on proteome and genome analysis

“…Accordingly, our estimates of average evolutionary divergence over all sequence pairs confirmed the most diversity for Env with .57 followed by Gag and Pol with .30 and .21, respectively ( Table 2). The Env protein of HIV and SIV is the key element mediating entry into the host cells (Moscoso et al, 2011). The Env proteins contain considerable genetic variable sites that are very important for the agent viability.…”