The role of the N-terminal segment of CCR5 in HIV-I 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(. 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(. 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(. 18)- expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env( NYP)- CD4- CCR5(. 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(. 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(. 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.