title: Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events.
abstract: Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated the K(d) of interaction 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 affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite 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 upon 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 coreceptor binding site in gp120 is exposed can be used for vaccine development.
authors.affiliation: Department of Pathology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. doranz@mail.med.upenn.edu
authors.forename: B J
authors.initials: BJ
authors.lastname: Doranz
authors.forename: S S
authors.initials: SS
authors.lastname: Baik
authors.forename: R W
authors.initials: RW
authors.lastname: Doms
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003470:Culture Media; D015686:Gene Products, env / Q000378:metabolism; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology*; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation; D019713:Receptors, CCR5 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D054299:env Gene Products, Human Immunodeficiency Virus
chemical_list: D015704:CD4 Antigens; D003470:Culture Media; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D009842:Oligopeptides; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1