An Allosteric Rheostat in HIV-1 gp120 Reduces CCR5 Stoichiometry Required for Membrane Fusion and Overcomes Diverse Entry Limitations

Abstract

SUMMARYBinding of the HIV-1 envelope glycoprotein gp120 to the CCR5 coreceptor reduces constraints on the metastable transmembrane subunit gp41, thereby enabling gp41 refolding, fusion of viral and cellular membranes, and infection. We previously isolated adapted HIV-1 JRCSF variants that more efficiently use mutant CCR5s, including CCR5(Δ18) lacking the important tyrosine sulfatecontaining amino terminus. Effects of mutant CCR5 concentrations on HIV-1 infectivities were highly cooperative, implying that several may be required. However, because wild-type CCR5 efficiently mediates infections at trace concentrations that were difficult to accurately measure, analyses of its cooperativity were not feasible. New HIV-1 JRCSF variants efficiently use CCR5 (HHMH), a chimera containing murine extracellular loop 2. The adapted virus induces large syncytia in cells containing either wild-type or mutant CCR5s and has multiple gp120 mutations that occurred independently in CCR5(Δ18)-adapted virus. Accordingly, these variants interchangeably use CCR5 (HHMH) or CCR5(Δ18). Additional analyses strongly support a novel energetic model for allosteric proteins, implying that the adaptive mutations reduce quaternary constraints holding gp41, thus lowering the activation energy barrier for membrane fusion without affecting bonds to specific CCR5 sites. In accordance with this mechanism, highly adapted HIV-1s require only one associated CCR5 (HHMH), whereas poorly adapted viruses require several. However, because they are allosteric ensembles, complexes with additional coreceptors fuse more rapidly and efficiently than minimal ones. Similarly, wild-type HIV-1 JRCSF is highly adapted to wild-type CCR5 and minimally requires one. The adaptive mutations cause resistances to diverse entry inhibitors and cluster appropriately in the gp120 trimer interface overlying gp41. We conclude that membrane fusion complexes are allosteric machines with an ensemble of compositions, and that HIV-1 adapts to entry limitations by gp120 mutations that reduce its allosteric hold on gp41. These results provide an important foundation for understanding the mechanisms that control membrane fusion and HIV-1's facile adaptability.

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

“…These similarities suggest that 2G12 inhibits assembly and lowers the steady-state concentration of FCs. Consistent with this interpretation and evidence that FCs mediate gp41 refolding (18,25,35), 2G12 and these other factors increase enfuvirtide (T-20) sensitivities (Fig. 2 E-H), suggesting that they HIV-1 JRCSF was incubated in culture media in the presence (+) or absence (−) of NMAbs 2G12 (100 μg/mL), b12 (15 μg/mL), or 2F5 (100 μg/mL) for 1 h at 37°C before adding onto JC.53 cultures and incubating an additional 30 min at 37°C, after which cell-attached virions were fixed, permeabilized, and stained for Gag p24.…”

“…To learn how 2G12 inhibits FC assembly, we used HeLa-CD4/CCR5(Δ18) cells containing an amino terminally deleted coreceptor in conjunction with the adapted virus HIV-1 JRCSF -Ad that efficiently infects these cells only when the tyrosine sulfate-containing amino terminal peptide Nt is present (18,36). 2G12 increased the Nt concentration required for entry approximately threefold (Fig.…”

“…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.…”

“…This standard assay is done in the presence of DEAE-dextran, a polycation that increases virion adsorption and enhances HIV-1 titers ∼20-fold (20,22,24). We prefer to stain and count infected foci in JC.53 cultures using antibodies to HIV-1, which allows us to compare titers in cell clones having distinct amounts of wild-type or mutant CD4 and/or CCR5 (18,21,25). This provides information about entry inhibitor mechanisms and factors that limit entry of diverse HIV-1 isolates.…”

“…Many factors that reduce titers including limiting CD4 and/or coreceptor concentrations or using mutant coreceptors or coreceptor antagonists slow entry (25). Conversely, adaptive mutations centered in the V3 region of gp120 overcome these limitations and accelerate entry (18,25). Recently, we found that the enhancement of titers caused by pretreating cultures with polycations results from the stabilization of virion adsorption, thus preventing a previously unknown competing process of rapid desorption and providing a longer opportunity for infection (24).…”