Background:  The HIV-1 envelope glycoprotein (Env) mediates virus attachment to and membrane fusion with the target cell. While it has been shown that the binding of gp120 to CD4 and chemokine receptors triggers conformational changes in Env that drive virus entry, many aspects of HIV fusion remain unclear. Specifically, it remains to be determined whether the gp41 pre-hairpin intermediate forms after CD4 binding, or whether co-receptor binding is also required. Here we address this question using a novel approach. We previously used a 6-helix bundle-specific antibody with isolate-restricted Env reactivity to demonstrate that sCD4 was sufficient to induce conformational changes in Env that lead to the formation of a gp41 pre-hairpin intermediate. In the current study, we show that the exposure of the N-helical coiled-coil domain of gp41 by sCD4 in this system is not the result of receptor induced gp120 shedding.

Methods:  A 6-helix bundle-specific monoclonal antibody with isolate-restricted Env reactivity, T26, was used to characterize gp41 structure. A gp120 specific monoclonal antibody, D19, was used to measure gp120 shedding. Immunofluorescent staining and immunoprecipitation assays were employed to characterize antibody binding to cell-surface-expressed HIV envelope.

Results:  Here we report that sCD4-induced conformational changes in HIV Env expose the N-helical coiled-coil domain of gp41 at low temperature, in the absence of gp120 dissociation. We demonstrate this by using a peptide modeling the gp41 C-helix to trap a stable form of the pre-hairpin intermediate. By varying both time and temperature we differentiated sCD4-induced Env conformational changes from receptor-mediated gp120 shedding.

Conclusions:  These results extend our previous work demonstrating that sCD4 is sufficient to induce exposure of a gp41 pre-hairpin fusion intermediate. Specifically, we establish that the Env conformational changes leading to the exposure of this gp41 fusion intermediate are due solely to sCD4-induced changes in Env conformation and are not the result of gp120 shedding. These results strengthen the support for a model in which CD4, alone, is sufficient to induce formation of the gp41 pre-hairpin intermediate

Keywords: Entry; gp41; sCD4