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.