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

Gregory B Melikyan,David Kabat,Emily J Platt

doi:10.1186/1742-4690-4-55

Gregory B Melikyan, David Kabat + Show 1 more

Open Access

https://doi.org/10.1186/1742-4690-4-55

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Abstract

BackgroundHIV-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(Δ18) (Platt et al., J. Virol. 2005, 79: 4357–68).ResultsWe 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(Δ18) 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(Δ18)-expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env(NYP)-CD4-CCR5(Δ18) 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(Δ18) 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(Δ18)-expressing cells with the sulfated peptide at sub-threshold temperature markedly increased the efficiency of fusion.ConclusionWe 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.

Highlights

HIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4)
The adaptation to CCR5(∆18) does not alter the kinetics of Env-induced fusion or the apparent affinity for wildtype CCR5 Because the N-terminal domain (Nt) of CCR5 is necessary for high affinity binding of HIV-1 gp120(wt) [11,37,38,30,34,39,40], it is likely that the adaptive gp120 mutations that enable use of CCR5(∆18) would compensate by increasing viral interactions with extracellular loop 2 of a chemokine receptor (ECL2) or other undamaged regions of the
Our results indicate that adaptation of HIV-1 Env to use CCR5(∆18) for entry does not involve a tighter interaction with the wild-type CCR5

Summary

Introduction

HIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4). Sequential binding of the gp120 subunit of Env to CD4 and a coreceptor (CCR5 or CXCR4) triggers conformational changes in the transmembrane subunit, gp, which mediates membrane fusion [1,2]. Genetic analyses revealed that R5-tropic HIV-1 Env interact primarily with the N-terminal segment (Nt) and the second extracellular loop (ECL2) of CCR5 [11,18,19,20,21,22]. The tip (often referred to as crown) of the V3-loop appears to interact with the ECL2, whereas the bridging sheet and the conserved residues of the V3 stem are likely to engage the Nt domain of CCR5 [8,16,23]

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