| Abstract
| - The onset of cell fusion mediated by HIV-1 IIIB Env is preceded by a lag phase of 15−20min. Fusion mediated by the CD4-independent HIV-1 Env 8x, which is capable of interacting directlywith CXCR4, proceeds with a greatly reduced lag phase. We probed the intermediate steps during the lagphase in HIV-1 IIIB Env-mediated fusion with Leu3-a, an inhibitor of attachment of gp120 to CD4,AMD3100, an inhibitor of attachment of gp120 to CXCR4, and C34, a synthetic peptide that interfereswith the transition of gp41 to the fusion active state. Inhibitions of fusion as a function of time of additionof C34 and of AMD3100 were equivalent, indicating that engagement of gp120 by CXCR4 and formationof the gp41 six-helix bundle follow similar kinetics. The initial steps in fusion mediated by the CD4-independent Env 8x are too rapid for these inhibitors to interfere with. However, when 8x Env-expressingcells were incubated with target cells at 25 °C in the presence of AMD3100 or C34, prior to incubationat 37 °C, these inhibitors were capable of inhibiting 8x Env-mediated fusion. To further examine engagementof gp120 by CXCR4 and exposure of binding sites for C34, we have reversibly arrested the fusion reactionat 37 °C by adding cytochalasin B to the medium. We show that CXCR4 engagement and six-helixbundle formation only occur after the release of the cytochalasin arrest, indicating that a high degree ofcooperativity is required to trigger the initial steps in HIV-1 Env-mediated fusion.
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