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；

• 批准号: 10404019
• 负责人: Vinayaka R. Prasad
• 金额: $ 59.45万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 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/10404019
• 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; Mediating; Mediator of activation protein; 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 evaluation; macrophage; mutant; new therapeutic target; novel; novel therapeutics; receptor; recruit

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.

该项目的目的是描述CCL2对HIV-1释放的新型调节的基础机制 并强调CCR2是一种新的HIV-1药物靶标。我们最近的工作表明细胞外CCL2调节 病毒释放效率通过动员ALIX（一种ESCRT III衔接蛋白），从F-肌动蛋白到胞质醇 HIV-1 GAG可以使用，这又有助于招募ESCRT III复合物来催化病毒的膜分裂 芽脖子。另一方面，CCL2的耗竭导致ALIX对F-肌动蛋白的剧烈隔离和抑制作用 病毒生产表明CCL2信号必须用于病毒的产生。 CCL2介导的 病毒释放的增强只有在晚期基序LYPX的病毒中才能观察到，例如HIV-1进化枝B（HIV-1B）。 那些缺乏LYPX的人，例如HIV-1进化枝C（HIV-1C）不受影响。在此提案中，我们将建立在这些建议的基础上 通过以下三个特定目的进行调查来进行令人兴奋的观察。目标1：了解 CCL2介导的ALIX动员基础的机制，我们将测试CCL2信号的假设 ALIX介导的HIV-1释放的强制性。我们将采用CCR2和CCL2单敲门机HELA 细胞检查HIV-1的产生和ALIX的动员是否可以在不存在的情况下发生 CCL2信号。我们将描述CCL2信号的下游事件，其中我们将检验假设 CCL2信号传导导致ALIX磷酸化，从而触发F-肌动蛋白的ALIX动员。众所周知 丝氨酸磷酸化将ALIX的封闭形式转换为开放构象，现在可以结合到逆转录病毒 GAG蛋白和CHMP4B，一种ESCRT III蛋白。另外，SRC对ALIX的酪氨酸磷酸化为 报告将ALIX从细胞骨架到细胞质。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封闭形式的形成，我们将测试是否SRC 是CCL2信号传导中的关键介体，可导致开放构象。我们将测试磷酸化的影响 在酪氨酸和丝氨酸残基上，CCL2与ALIX动员有关。我们将研究其他后 ALIX蛋白的翻译修饰。 AIM 2：我们将研究CCR2BV64I多态性的影响 艾滋病毒复制。众所周知，CCR2BV64I多态性会延迟AIDS疾病的进展。使用PBMC和 来自HIV阴性个体的巨噬细胞，具有基因型CCR2BV64/V64，CCR2BV64/64I或CCR2B64I/64I，我们 将检验以下假设：V64i多态性废除CCL2-CCR2信号传导，抑制CCL2结合或 F-肌动蛋白的内在化和ALIX动员，降低了HIV-1B对CCL2和CCL2的能力 减少病毒产生。我们在HeLa细胞中的初步数据已经表明，V64i消除了HIV-的能力 1B对CCL2做出反应并降低HIV-1B的生产水平。目标3：我们将研究 CCL2-2518G多态性（已知会增加血浆CCL2和艾滋病毒获取风险）。 信号和艾滋病毒健身。使用CCL2-2518/AA的人类志愿者使用艾滋病毒敏感的细胞， 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。