Complex interplay of kinetic factors governs the synergistic properties of HIV-1 entry inhibitors.

The homotrimeric HIV-1 envelope glycoprotein (Env) undergoes receptor-triggered structural changes that mediate viral entry through membrane fusion. This process is inhibited by chemokine receptor antagonists (CoRAs) that block Env-receptor interactions and by fusion inhibitors (FIs) that disrupt Env conformational transitions. Synergy between CoRAs and FIs has been attributed to a CoRA-dependent decrease in the rate of viral membrane fusion that extends the lifetime of the intermediate state targeted by FIs. Here, we demonstrated that the magnitude of CoRA/FI synergy unexpectedly depends on FI-binding affinity and the stoichiometry of chemokine receptor binding to trimeric Env. For C-peptide FIs (clinically represented by enfuvirtide), synergy waned as binding strength decreased until inhibitor combinations behaved additively. Curiously, this affinity dependence on synergy was absent for 5-Helix-type FIs. We linked this complex behavior to the CoRA dependence of Env deactivation following FI binding. For both FI classes, reducing chemokine receptor levels on target cells or eliminating competent chemokine receptor-binding sites on Env trimers resulted in a loss of synergistic activity. These data imply that the stoichiometry required for CoRA/FI synergy exceeds that required for HIV-1 entry. Our analysis suggests two distinct roles for chemokine receptor binding, one to trigger formation of the FI-sensitive intermediate state and another to facilitate subsequent conformational transitions. Together, our results could explain the wide variety of previously reported activities for CoRA/FI combinations. These findings also have implications for the combined use of CoRAs and FIs in antiviral therapies and point to a multifaceted role for chemokine receptor binding in promoting HIV-1 entry.