The goal of this project is to delineate the mechanism underlying a novel regulation of HIV-1 release by CCL2 and to highlight CCR2 as a new HIV-1 drug target. Our recent work has shown that extracellular CCL2 modulates virus release efficiency by mobilizing ALIX, an ESCRT III adapter protein, from F-actin to the cytosol making it accessible to HIV-1 Gag, which in turn helps recruit ESCRT III complex to catalyze membrane scission at virus bud neck. CCL2 depletion, on the other hand, lead to a dramatic sequestration of ALIX to F-actin and an inhibition of virus production suggesting that CCL2 signaling must occur for virus production. CCL2-mediated enhancement in virus release is only observed in viruses with late motif LYPX, e.g., HIV-1 clade B (HIV-1B). Those lacking LYPX, e.g., HIV-1 clade C (HIV-1C), are unaffected. In this proposal, we will build upon these exciting observations through investigations along the following three specific aims.
Aim 1 : To understand the mechanism underlying CCL2-mediated ALIX mobilization, we will test the hypothesis that CCL2 signaling is obligatory for ALIX-mediated HIV-1 release. We will employ CCR2 and CCL2 single and double knockout HeLa cells to examine whether HIV-1 production and ALIX mobilization from F-actin to cytosol can occur in the absence of CCL2-signaling. We will delineate downstream events of CCL2 signaling, in which we will test the hypothesis that CCL2 signaling leads to ALIX phosphorylation, triggering ALIX mobilization from F-actin. It is known that serine phosphorylation converts a closed form of ALIX to an open conformation that can now bind to retroviral Gag protein and to CHMP4b, an ESCRT III protein. Separately, tyrosine phosphorylation of ALIX by Src is reported to mobilize ALIX from cytoskeleton to cytosol. As Src is downstream of CCL2 signaling pathway and the Src phosphorylation site on ALIX participates in the formation of ALIX closed form, we will test whether Src is a key mediator in CCL2 signaling that leads to an open conformation. We will test the effect of phosphorylation at both the tyrosine and serine residues implicated in ALIX mobilization by CCL2. We will study other post- translational modifications on ALIX protein.
Aim 2 : We will examine the effect of CCR2bV64I polymorphism on HIV replication. CCR2bV64I polymorphism is known to delay AIDS disease progression. Using PBMCs and macrophages from HIV-negative individuals with the genotypes, CCR2bV64/V64, CCR2bV64/64I or CCR2b64I/64I, we will test the hypothesis that V64I polymorphism abrogates CCL2-CCR2 signaling, inhibiting CCL2-binding or internalization and ALIX mobilization from F-actin, decreasing the ability of HIV-1B to respond to CCL2 and reducing virus production. Our preliminary data in HeLa cells already show that V64I abrogates the ability of HIV- 1B to respond to CCL2 and reduces the HIV-1B production levels.
Aim 3 : We will examine the effect of CCL2-2518G polymorphism, which is known to increase plasma CCL2 and the risk of HIV acquisition, on CCL2 signaling and HIV fitness. Employing HIV-susceptible cells from human volunteers with CCL2-2518/AA, CCL2-2518G/A and CCL2-2518G/G genotypes, we will test whether -2518G polymorphism leads to increased CCL2 expression, ALIX mobilization rates and virus fitness.
Research proposed here builds on a new way by which the amount of HIV-1 produced by infected cells is controlled by certain proteins called chemokines released by human immune cells. Interestingly, the genes for this chemokine (CCL2) and its receptor (CCR2) have been previously shown to influence HIV-1 disease ? but it was not known why. This application proposes to understand precisely how such chemokines influence HIV disease and develops a foundation for new drugs that can block HIV by targeting the chemokine.
