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

Abstract

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.Support or Funding InformationR01 GM066682 to MJRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HIV-1 neutralization tiers are not relevant for inhibitors targeting the pre-hairpin intermediate

“…However, the L565Q virus showed increased sensitivity to 5-Helix (Fig. 2C), in line with previous reports 37,49 , indicating that epitope sequence is not the only determinant of sensitivity to 5-Helix. To further probe how sequence diversity impacts 5-Helix efficacy, we next assayed 5-Helix binding to nine peptides representing the CHR sequences of some of the strains tested across viral clades in our panel (Fig.…”

“…Therefore, we first sought to determine 5-Helix potencies using TZM-bl cells and corroborate our findings using HOS-CD4-CCR5 target cells for which 5-Helix potencies have been published [34][35][36][37] . We validated our 5-Helix preparations biochemically (SI Fig.…”

“…Although 5-Helix neutralization activity has been reported using a number of cell-cell fusion 30,34 and virus-cell fusion 34–37 assays, no reports to date have employed TZM-bl cells. Therefore, we first sought to determine 5-Helix potencies using TZM-bl cells and corroborate our findings using HOS-CD4-CCR5 target cells for which 5-Helix potencies have been published 34–37 . We validated our 5-Helix preparations biochemically (SI Fig.…”

“…Highly conserved residues are highlighted in red. Although 5-Helix neutralization activity has been reported using a number of cell-cell fusion 30,34 and virus-cell fusion [34][35][36][37] assays, no reports to date have employed TZM-bl cells.…”

“…The CHR-targeting designed protein called 5-Helix binds highly conserved CHR residues that form an α-helical conformation 31 (Fig. 1C); accordingly, 5-Helix has potent neutralizing activity at low nanomolar concentrations across distinct viral clades, though only a handful of viral strains have been examined to date 30,[34][35][36][37] .…”