Ternary complex formation of human immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured as an intermediate of membrane fusion.

Human immunodeficiency virus (HIV) Env-induced fusion is highly temperature dependent. When effector and target cells were coincubated at 37 degrees C, there was a kinetic delay before fusion commenced. When effector and target cells were coincubated for varied times at 23 degrees C, a temperature that does not permit fusion, a temperature-arrested stage was created. Raising temperature to 37 degrees C from the 23 degrees C intermediate eliminated the kinetic delay. Inhibitors (T22, AMD3100, and Sch-C) that block fusion by binding chemokine receptors were added after creating the intermediate so as to assess the extent of engagement between gp120 and chemokine receptors at that stage. For both CXCR4 and CCR5 as coreceptors, increasingly long times of coincubation at 23 degrees C reduced the efficacy of the coreceptor-binding inhibitors in blocking fusion. This implies that an increasing number of ternary Env/CD4/coreceptor complexes form over time at 23 degrees C. It also shows that ternary complex formation has a lower temperature threshold than the downstream steps that include Env folding into a six-helix bundle; this provides an experimental means to separate coreceptor binding by gp120 from the subsequent refolding of gp41 into a six-helix bundle structure. As the time of cell coincubation at 23 degrees C was prolonged, more cells quickly fused upon the raising of the temperature to 37 degrees C, and the increase quantitatively correlated with the greater percentage of fusion that was resistant to drugs. Therefore the pronounced kinetic delay in HIV Env-induced fusion is caused predominantly by the time needed for ternary complexes to form.

The cytoplasmic tail slows the folding of human immunodeficiency virus type 1 Env from a late prebundle configuration into the six-helix bundle.

Effects of the cytoplasmic tail (CT) of human immunodeficiency virus type 1 Env on the process of membrane fusion were investigated. Full-length Env (wild type [WT]) and Env with its CT truncated (DeltaCT) were expressed on cell surfaces, these cells were fused to target cells, and the inhibition of fusion by peptides that prevent Env from folding into a six-helix bundle conformation was measured. For both X4-tropic and R5-tropic Env proteins, DeltaCT induced faster fusion kinetics than did the WT, and peptides were less effective at inhibiting DeltaCT-induced fusion. We tested the hypothesis that the inhibitory peptides were less effective at inhibiting DeltaCT-induced fusion because DeltaCT folds more quickly into a six-helix bundle. Early and late intermediates of WT- and DeltaCT-induced fusion were captured, and the ability of peptides to block fusion when added at the intermediate stages was quantified. When added at the early intermediate, the peptides were still less effective at inhibiting DeltaCT-induced fusion but they were equally effective at preventing WT- and DeltaCT-induced fusion when added at the late intermediate. We conclude that for both X4-tropic and R5-tropic Env proteins, the CT facilitates conformational changes that allow the trimeric coiled coil of prebundles to become optimally exposed. But once Env does favorably expose its coiled coil to inhibitory peptides, the CT hinders subsequent folding into a six-helix bundle. Because of this facilitation of maximal exposure and hindrance of bundle formation, the coiled coil is optimally exposed for a longer time for WT than for DeltaCT. This accounts for the greater peptide inhibition of WT-induced fusion.