Insights Into Hiv-1 Coreceptor Engagement And Its Contribution To Membrane Fusion

During HIV-1 entry, CD4 and a coreceptor (either CCR5 or CXCR4) must interact with the envelope glycoprotein (Env). The coreceptor N-terminus and extracellular loops are critical domains that interact cooperatively with Env to drive the release of gp41 for fusion with the host membrane. Conformational changes in Env that follow coreceptor engagement to trigger fusion are poorly understood. We explored the individual contribution of interactions between an HIV-1 Env and the coreceptor N-terminus with a variant, termed CEMAX, that mediated fusion and infection using a coreceptor construct that expressed only the first 27 residues of the CXCR4 N-terminus, fused to a CD4 anchoring molecule (X4-NT/CD4). CEMAX acquired mutations in gp41, but also contained a severely truncated V3 loop in gp120. Here we describe the derivation and characterization of HIV-1s with full V3 loops and combinations of these gp41 changes that enabled efficient utilization of an R5-NT/CD4 construct containing the first 19 residues of CCR5 fused to a CD4 anchoring molecule. The determinants for this gain of function were mapped to gp41 mutations in: the fusion peptide, an N-terminal helical domain, and heptad repeat-2. We demonstrated that these changes in heterologous HIV-1s conferred the same phenotype. We also evaluated the mechanism of this novel “N-terminus only” usage of CCR5. Although global changes in trimer architecture were not detected by several antibodies, the acquisition of N-terminus only usage was associated with reduced sensitivity to a gp41 fusion inhibitor, consistent with more rapid fusion kinetics. Our findings also indicated that infection of cells expressing CD4 and R5-NT/CD4 was markedly enhanced in the absence of Vpu and in the presence of tetherin, likely as a result of tetherin-mediated formation of cell-associated viral aggregates and facilitation of cell-to-cell spread. Based on new structural insights of the CD4-activated trimer in its final conformational state before coreceptor engagement, this novel phenotype is consistent with a model in which gp41 mutations destabilize interactions between the gp41 N-terminus and a CD4-induced pocket formed near the trimer core, lowering the threshold for triggering and enabling the coreceptor N-terminus to become sufficient for gp41 release and cell fusion