Conformational Differences between Functional Human Immunodeficiency Virus Envelope Glycoprotein Trimers and Stabilized Soluble Trimers

Abstract

Binding to the receptor CD4 triggers entry-related conformational changes in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, (gp120/gp41)3. Soluble versions of HIV-1 Env trimers (sgp140 SOSIP.664) stabilized by a gp120-gp41 disulfide bond and a change (I559P) in gp41 have been structurally characterized. Here, we use cross-linking/mass spectrometry to evaluate the conformations of functional membrane Env and sgp140 SOSIP.664. Differences were detected in the gp120 trimer association domain and C terminus and in the gp41 heptad repeat 1 (HR1) region. Whereas the membrane Env trimer exposes the gp41 HR1 coiled coil only after CD4 binding, the sgp140 SOSIP.664 HR1 coiled coil was accessible to the gp41 HR2 peptide even in the absence of CD4. Our results delineate differences in both gp120 and gp41 subunits between functional membrane Env and the sgp140 SOSIP.664 trimer and provide distance constraints that can assist validation of candidate structural models of the native HIV-1 Env trimer. IMPORTANCE HIV-1 envelope glycoprotein spikes mediate the entry of the virus into host cells and are a major target for vaccine-induced antibodies. Soluble forms of the envelope glycoproteins that are stable and easily produced have been characterized extensively and are being considered as vaccines. Here, we present evidence that these stabilized soluble envelope glycoproteins differ in multiple respects from the natural HIV-1 envelope glycoproteins. By pinpointing these differences, our results can guide the improvement of envelope glycoprotein preparations to achieve greater similarity to the viral envelope glycoprotein spike, potentially increasing their effectiveness as a vaccine.

Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function

“…Despite its relatively poor processing, we found that the EnvΔ712 DS mutant could mediate a moderate level of cell-cell fusion in a reducing environment, and this allowed us to investigate the conformation of the functional EnvΔ712 DS glycoprotein. Although these studies were performed with the cytoplasmic tail-deleted EnvΔ712, the ectodomain conformations of the EnvΔ712 and full-length Env from many HIV-1 strains, including HIV-1 JR-FL , are similar (42,101,132). Consistent with this similarity, the unmodified full-length Env and cytoplasmic tail-deleted EnvΔ712 exhibited nearly identical antigenic profiles ( Fig.…”

“…The cross-links observed in the sgp140 SOSIP.664 glycoprotein were completely compatible with multiple reported sgp140 SOSIP.664 structures (57)(58)(59)(60); thus, the available structural models accurately describe the conformation of sgp140 SOSIP.664 trimers in solution. In contrast, many cross-links observed in the membrane Env trimer could not be explained by the available high-resolution structural models of sgp140 SOSIP.664 trimers (101). Fourth, after comparing the results of double-electron-electron resonance (DEER) spectroscopic analyses of sgp140 SOSIP.664 trimers with the results of smFRET studies of native Env on HIV-1 virions, Stadtmueller et al speculated that existing high-resolution structures of HIV-1 Env trimers may represent state 2 conformations (127).…”

“…Second, the introduction of the I559P change, and, to a lesser extent, the SOS change, into membrane Envs has been shown to alter Env antigenicity (89,113,126). Third, cross-links introduced into an sgp140 SOSIP.664 glycoprotein and a mature membrane Env were analyzed by mass spectrometry (101). The cross-links observed in the sgp140 SOSIP.664 glycoprotein were completely compatible with multiple reported sgp140 SOSIP.664 structures (57)(58)(59)(60); thus, the available structural models accurately describe the conformation of sgp140 SOSIP.664 trimers in solution.…”

“…Immunoprecipitation of Envs in cell lysates and on cell surfaces. Virus-producing 293T cells were prepared as described above and used for immunoprecipitation of Env from the cell surface as described previously (101). In brief, at 48 h posttransfection, 293T cells were washed, scraped, resuspended in PBS, and incubated with 10 g/ml 2G12 antibody for 1 h at room temperature.…”

“…The antigenicity of HIV-1 JR-FL Env, EnvΔ712, and the corresponding SOS and DS mutants expressed on the surface of HOS cells was examined. The HIV-1 Env precursor is processed efficiently in HOS cells (78), allowing a better correlation between antibody binding to the cell surface Env and virus neutralization (42,89,101). We used a cell-based enzyme-linked immunosorbent assay (ELISA) to measure the recognition of cell surface Envs by a panel of broadly neutralizing antibodies as well as poorly neutralizing antibodies, in the absence and presence of 1 mM DTT.…”