The fusion domain of human immunodeficiency virus (HIV-1) envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N terminus of the transmembrane glycoprotein (gp41). A V2E mutant has been shown to dominantly interfere with wild-type envelope-mediated syncytium formation and virus infectivity. To understand this phenomenon, a 33-residue peptide (wild type, WT) identical to the N-terminal segment of gp41 and its V2E mutant were synthesized, fluorescently labeled, and characterized. Both peptides inhibited HIV-1 envelope-mediated cell-cell fusion and had similar ␣-helical content in membrane mimetic environments. Studies with fluorescently labeled peptide analogues revealed that both peptides have high affinity for phospholipid membranes, are susceptible to digestion by proteinase-K in their membrane-bound state, and tend to self-and coassemble in the membranes. In SDS-polyacrylamide gel electrophoresis the WT peptide formed dimers as well as higher order oligomers, whereas the V2E mutant only formed dimers. The WT, but not the V2E mutant, induced liposome aggregation, destabilization, and fusion. Moreover, the V2E mutant inhibited vesicle fusion induced by the WT peptide, probably by forming inactive heteroaggregates. These data form the basis for an explanation of the mechanism by which the gp41 V2E mutant inhibits HIV-1 infectivity in cells when co-expressed with WT gp41.

We have continuously measured CD4-induced conformational changes of cell surface-expressed human immunodeficiency virus type-1 envelope glycoprotein gp120-gp41 in situ using 4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid, a fluorescent probe that binds to hydrophobic groups. CD4-expressing human T cell lines induced significant and rapid conformational changes (<1 min delay) in gp120-gp41 from T cell-tropic strains, and little conformational changes in gp120-gp41 from macrophage-tropic strains, with equivalent levels of envelope expression. Conversely, CD4-expressing human macrophages induced significant and rapid conformational changes in gp120-gp41 from macrophage-tropic strains, and little conformational changes in gp120-gp41 from T cell-tropic strains. Thus, the conformational changes undergone by gp120-gp41, which lead to membrane fusion, are highly cooperative and require both receptor and co-receptor. We used a dye transfer assay to show that neither membrane lipid fusion or fusion pore formation can occur with host cells having different tropism from the envelope. HIV-11 gains entry into susceptible cells by means of fusion of viral and cellular membranes, which is mediated by the HIV-1 envelope glycoprotein, gp120-gp41 (1). The fusion reaction is triggered by the interaction of gp120-gp41 with host cell surface CD4 (2), and requires co-receptors such as CXCR4 and CCR5 for T-tropic and M-tropic HIV-1 strains, respectively (3-10). We report here new findings, which are relevant to the mechanisms by which the HIV-1 envelope glycoprotein gp120-gp41 mediates fusion of biological membranes. The HIV-1 fusion reaction proceeds along a series of multiple steps before the final event occurs, which results in delivery of the nucleocapsid into the cell (11). In previous studies with influenza hemagglutinin (HA), we have used quantitative fluorescence video microscopy to dissect the process and analyze the molecular interactions taking place in these intermediate steps (12,13). These include conformational changes in the viral envelope glycoprotein, assembly of envelope glycoproteins into prefusion pore aggregates, insertion of the fusion peptide into target membranes, destabilization of the target membrane, and formation and expansion of the fusion pore.The first step in the HIV-1 fusion cascade is the triggering of conformational changes in gp120-gp41 (14). We have measured the kinetics of conformational changes of cell surface-expressed gp120-gp41 triggered by interaction with target cell CD4 and co-receptor. The changes in conformation were revealed using the fluorescent probe 4,4Ј-dianilino-1,1Ј-binaphthyl-5,5Ј-disulfonic acid (bis-ANS), which displays little fluorescence in aqueous solution but lights up when bound to hydrophobic groups (15, 16). We used a fluorescence video microscopy set-up equipped with an intensified CCD camera to detect and quantify the changes in fluorescence upon interaction of gp120-gp41-expressing cells and appropriate target cells. To our knowledge, this is the first time conformat...

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A 16 kDa protein has been isolated in a homogeneous form as the major component of a paracrystalline paired membrane structure closely resembling the gap junction. The primary structure of this protein from arthropod and vertebrate species has been determined by protein and cDNA sequencing. The amino acid sequences are highly conserved and virtually identical to the amino acid sequence of the proteolipid subunit of the vacuolar H(+)-ATPases. The disposition of the protein in the membrane has been studied using proteases and the N,N'-dicyclohexylcarbodiimide reactive site identified. These data, together with secondary structure predictions, suggest that the 16 kDa protein is for the most part buried in the membrane, arranged in a bundle of four hydrophobic alpha-helices. Using computer graphics, a model has been constructed based on this arrangement and on the electron microscopic images of the paracrystalline arrays.