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: 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
• 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

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.

本项目的目标是阐明CCL 2对HIV-1释放的新调节机制 并强调CCR 2作为新的HIV-1药物靶点。我们最近的研究表明，细胞外CCL 2调节 通过将阿利克斯（一种ESCRT III衔接蛋白）从F-肌动蛋白移动到细胞质，使其 HIV-1 Gag可接近，这反过来又有助于招募ESCRT III复合物催化病毒膜断裂， 芽颈。另一方面，CCL 2消耗导致阿利克斯对F-肌动蛋白的显著螯合和对细胞增殖的抑制。 表明病毒产生必须发生CCL 2信号传导。ccl 2介导 病毒释放的增强仅在具有晚期基序LYPX的病毒中观察到，例如，HIV-1进化枝B（HIV-1 B）。 那些缺乏LYPX的人，例如，HIV-1进化枝C（HIV-1C）不受影响。在本建议中，我们将在这些基础上 通过沿着以下三个具体目标进行的调查，获得了令人兴奋的观察结果。目标1：了解 我们将测试CCL 2信号转导是一种潜在的CCL 2介导的阿利克斯动员机制， 对于ALIX介导的HIV-1释放是必需的。我们将采用CCR 2和CCL 2单敲除和双敲除HeLa 细胞，以检查是否HIV-1的生产和阿利克斯从F-肌动蛋白动员到胞质溶胶可以发生在缺乏 CCL 2信号我们将描述CCL 2信号传导的下游事件，在其中我们将测试假设 CCL 2信号传导导致阿利克斯磷酸化，触发阿利克斯从F-肌动蛋白动员。已知的是 丝氨酸磷酸化将封闭形式的阿利克斯转化为开放构象， Gag蛋白和CHMP 4 b，一种ESCRT III蛋白。单独地，Src对阿利克斯的酪氨酸磷酸化是 据报道将阿利克斯从细胞骨架动员到胞质溶胶。由于Src是CCL 2信号通路的下游， 阿利克斯上的Src磷酸化位点参与阿利克斯闭合形式的形成，我们将测试Src是否 是CCL 2信号传导中的关键介体，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参与阿利克斯动员CCL 2。我们将研究其他后- 阿利克斯蛋白的翻译修饰。目的2：我们将检测CCR 2bV 64 I多态性对 艾滋病毒复制。CCR 2bV 64 I多态性被认为可以延缓艾滋病的进展。使用PBMC和 来自具有基因型CCR 2bV 64/V64、CCR 2bV 64/64 I或CCR 2b 64 I/64 I的HIV阴性个体的巨噬细胞， 将检验V64 I多态性消除CCL 2-CCR 2信号传导，抑制CCL 2结合或 内化和阿利克斯从F-肌动蛋白动员，降低HIV-1B对CCL 2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，V64 I消除了HIV-1的能力， 1B对CCL 2作出反应，并降低HIV-1B的产生水平。目标3：我们将研究 CCL 2 - 2518 G多态性，已知可增加血浆CCL 2和HIV感染风险， 信号和艾滋病毒适应性。使用来自具有CCL 2 -2518/AA的人类志愿者的HIV易感细胞， CCL 2 - 2518 G/A和CCL 2 - 2518 G/G基因型，我们将测试-2518G多态性是否导致CCL 2增加 表达、阿利克斯动员率和病毒适应性。