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: 10077424
• 负责人: Vinayaka R. Prasad
• 金额: $ 69.92万
• 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/10077424
• 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; 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.

这个项目的目标是描述CCL2对HIV-1释放的一种新的调节机制 并强调CCR2是新的HIV-1药物靶点。我们最近的工作表明，细胞外CCL2调制 通过将ESCRT III适配蛋白Alix从F-肌动蛋白动员到胞浆使其具有释放病毒的效率 可与HIV-1 Gag结合，进而帮助招募ESCRT III复合体催化病毒的膜断裂 巴德·尼奇。另一方面，CCL2的耗尽导致Alix与F-肌动蛋白的戏剧性隔离和抑制 病毒的产生表明CCL2信号必须发生才能产生病毒。CCL2介导的 病毒释放的增强只在具有晚期基序LYPX的病毒中观察到，例如HIV-1分支B(HIV-1B)。 那些缺乏LYPX的人，例如HIV-1 C分支(HIV-1C)，不受影响。在这项建议中，我们将建立在这些基础上 通过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 CCL2介导Alix动员的机制，我们将检验CCL2信号是 对于Alix介导的HIV-1释放是必需的。我们将采用CCR2和CCL2单、双基因敲除HeLa 细胞检测HIV-1的产生和Alix从F-肌动蛋白到胞浆的动员是否可以在缺乏的情况下发生 CCL2信令。我们将描述CCL2信令的下游事件，在其中我们将检验假设 CCL2信号导致Alix磷酸化，触发F-肌动蛋白的Alix动员。众所周知， 丝氨酸磷酸化将封闭形式的Alix转化为现在可以与逆转录病毒结合的开放构象 GAG蛋白和ESCRT III蛋白CHMP4b。另外，Src对Alix的酪氨酸磷酸化是 据报道，将Alix从细胞骨架动员到细胞溶胶。AS Src位于CCL2信号通路下游， 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 是导致开放构象的CCL2信号的关键介体。我们将测试磷酸化的效果 与CCL2动员Alix有关的酪氨酸和丝氨酸残基。我们将研究其他岗位- Alix蛋白的翻译修饰。目的2：我们将研究CCR2bV64I基因多态性对 艾滋病毒复制。CCR2bV64I基因多态性被认为可以延缓艾滋病的进展。使用PBMC和 携带CCR2bV64/V64、CCR2bV64/64I或CCR2b64I/64I基因的HIV阴性个体的巨噬细胞 将检验这样一种假设，即V64I多态性废除CCL2-CCR2信号，抑制CCL2结合或 F-肌动蛋白的内化和Alix动员，降低HIV-1B对CCL2和 减少病毒的产生。我们在HeLa细胞中的初步数据已经表明，V64I可以消除HIV- 1B应对CCL2，减少HIV-1B的生产水平。目标3：我们将检查 CCL2-2518G多态，已知会增加血浆CCL2和HIV感染风险 信号和艾滋病毒健康。使用来自携带CCL2-2518/AA的人类志愿者的艾滋病毒敏感细胞， CCL2-2518G/A和CCL2-2518G/G，我们将测试-2518G多态是否会导致CCL2增加 表达、Alix动员率和病毒适合性。