CCL2-CCR2b signaling in HIV-1 fitness and disease; Role of host genetic polymorphisms

HIV-1 健康和疾病中的 CCL2-CCR2b 信号传导；

基本信息

批准号：
10621769
负责人：
Vinayaka R. Prasad
金额：
$ 53.37万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10621769
关键词：
Acquired Immunodeficiency Syndrome Actins Adaptor Signaling Protein Affect Binding CCL2 gene CD4 Positive T Lymphocytes Cells Cohort Studies Cytoskeleton Cytosol Data Defect Disease Disease Progression Drug Targeting Electron Transport Complex III Enhancers Event F-Actin Foundations Genes Genetic Polymorphism Genotype Goals HIV HIV Infections HIV Seronegativity HIV-1 Hela Cells Human Human Volunteers Immune Individual Investigation Knock-out Lead Macrophage Mediating Mediator Membrane Molecular Conformation Mutation Neck Participant Peripheral Blood Mononuclear Cell Phosphorylation Phosphorylation Site Plasma Post-Translational Protein Processing Predisposition Production Proteins Publishing Regulation Reporting Research Risk Role Serine Signal Pathway Signal Transduction Surface Testing Tyrosine Tyrosine Phosphorylation Virion Virus Work antagonist chemokine drug candidate drug development experimental study extracellular fitness gag Gene Products in vitro testing in vivo mutant new therapeutic target novel novel therapeutics receptor recruit

项目摘要

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.

该项目的目标是描绘 CCL2 对 HIV-1 释放的新调控机制并强调 CCR2 作为新的 HIV-1 药物靶点。我们最近的工作表明细胞外 CCL2 调节通过将 ALIX（一种 ESCRT III 接头蛋白）从 F-肌动蛋白动员至胞质溶胶，提高病毒释放效率 HIV-1 Gag 可以接触到，这反过来又有助于招募 ESCRT III 复合物来催化病毒的膜断裂芽颈。另一方面，CCL2 耗尽会导致 ALIX 与 F-肌动蛋白的显着隔离并抑制病毒产生的过程表明 CCL2 信号传导必须发生才能产生病毒。 CCL2介导的仅在具有晚期基序 LYPX 的病毒中观察到病毒释放的增强，例如 HIV-1 clade B (HIV-1B)。那些缺乏 LYPX 的人，例如 HIV-1 C 型 (HIV-1C)，则不受影响。在本提案中，我们将在此基础上通过围绕以下三个具体目标的调查得出了令人兴奋的观察结果。目标 1：了解为了了解 CCL2 介导的 ALIX 动员的机制，我们将检验 CCL2 信号传导的假设 ALIX 介导的 HIV-1 释放所必需的。我们将采用CCR2和CCL2单敲除和双敲除HeLa 细胞来检查 HIV-1 的产生以及 ALIX 从 F-肌动蛋白到胞质溶胶的动员是否可以在不存在的情况下发生 CCL2 信号传导。我们将描述 CCL2 信号传导的下游事件，并在其中检验假设 CCL2 信号传导导致 ALIX 磷酸化，从而触发 F-肌动蛋白动员 ALIX。据了解丝氨酸磷酸化将 ALIX 的封闭形式转化为开放构象，现在可以与逆转录病毒结合 Gag 蛋白和 CHMP4b（一种 ESCRT III 蛋白）。另外，Src 对 ALIX 的酪氨酸磷酸化是据报道可将 ALIX 从细胞骨架动员到细胞质中。由于Src是CCL2信号通路的下游并且 ALIX上的Src磷酸化位点参与ALIX闭合形式的形成，我们将测试Src是否是 CCL2 信号传导中导致开放构象的关键介质。我们将测试磷酸化的效果涉及 CCL2 动员 ALIX 的酪氨酸和丝氨酸残基。我们将研究其他后期 ALIX 蛋白的翻译修饰。目标 2：我们将检查 CCR2bV64I 多态性对 HIV复制。已知 CCR2bV64I 多态性可延缓 AIDS 疾病的进展。使用 PBMC 和来自具有 CCR2bV64/V64、CCR2bV64/64I 或 CCR2b64I/64I 基因型的 HIV 阴性个体的巨噬细胞，我们将检验 V64I 多态性消除 CCL2-CCR2 信号传导、抑制 CCL2 结合或 F-肌动蛋白的内化和 ALIX 动员，降低了 HIV-1B 对 CCL2 的反应能力，减少病毒的产生。我们在 HeLa 细胞中的初步数据已经表明，V64I 消除了 HIV- 1B 对 CCL2 做出反应并降低 HIV-1B 的产生水平。目标3：我们将检查效果 CCL2-2518G 多态性已知会增加血浆 CCL2 和 CCL2 上 HIV 感染的风险信号传导和艾滋病毒适应性。使用来自人类志愿者的 HIV 易感细胞和 CCL2-2518/AA， CCL2-2518G/A和CCL2-2518G/G基因型，我们将测试-2518G多态性是否导致CCL2增加表达、ALIX 动员率和病毒适应性。