The Role of the N-terminal Segment of CCR5 in HIV-1 Env-mediated Membrane Fusion and the Mechanism of Virus Adaptation to CCR5 Lacking This Segment

Background: HIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4). The critical determinants of CCR5 coreceptor function are the N-terminal domain (Nt) and the second extracellular loop. However, mutations in gp120 adapt HIV-1 to grow on cells expressing the N-terminally truncated CCR5(Delta 18) (Platt et al., J. Virol. 2005, 79: 4357-68).

Results: We have functionally characterized the adapted Env (designated Env(NYP)) using a quantitative cell-cell fusion assay. The rate of fusion with target cells expressing wild-type CCR5 and the resistance to fusion inhibitors was virtually identical for wild-type Env and Env(NYP), implying that the coreceptor affinity had not increased as a result of adaptation. In contrast, Env(NYP)-induced fusion with cells expressing CCR5(Delta 18) occurred at a slower rate and was extremely sensitive to the CCR5 binding inhibitor, Sch-C. Resistance to Sch-C drastically increased after pre-incubation of Env(NYP)- and CCR5(Delta 18)-expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env(NYP)-CD4-CCR5(Delta 18) complexes accumulate at sub-threshold temperature and that low-affinity interactions with the truncated coreceptor are sufficient for triggering conformational changes in the gp41 of Env(NYP) but not in wild-type Env. We also demonstrated that the ability of CCR5(Delta 18) to support fusion and infection mediated by wild-type Env can be partially reconstituted in the presence of a synthetic sulfated peptide corresponding to the CCR5 Nt. Pre-incubation of wild-type Env- and CCR5(Delta 18)-expressing cells with the sulfated peptide at sub-threshold temperature markedly increased the efficiency of fusion.

Conclusion: We propose that, upon binding the Nt region of CCR5, wild-type Env acquires the ability to productively engage the extracellular loop(s) of CCR5 - an event that triggers gp41 refolding and membrane merger. The adaptive mutations in Env(NYP) enable it to more readily release its hold on gp41, even when it interacts weakly with a severely damaged coreceptor in the absence of the sulfopeptide.