Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events

Binding of the extracellular subunit of human immunodeficiency type 1 (HIV- 1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exp osure of a highly conserved coreceptor binding site in gp120 that helps med iate membrane fusion, Characterizing the structural features involved in gp 120-coreceptor binding and the conditions under which binding occurs is imp ortant for understanding the fusion process, the evolution of pathogenic st rains in vivo, the identification of novel anti-MN compounds, and the devel opment of HIV vaccines that utilize triggered structures of Ens. Here we us e the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion, Using saturation bi nding and homologous competition analysis, we estimated the K-d of interact ion between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly af fected by the pH of binding, G-protein coupling of CCR5, or partial gp120 d eglycosylation, Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despit e its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost u pon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120 -soluble CD4 complex was remarkably stable over time and temperature ranges , offering the possibility that complexes in which the highly conserved cor eceptor binding site in gp120 is exposed can be used for vaccine developmen t.