HIV interactions with host cell proteins in particle release

HIV与宿主细胞蛋白在颗粒释放中的相互作用

• 批准号: 10592276
• 负责人: Xinhong Dong
• 金额: $ 36.38万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592276
• 关键词: Biochemical; Biological Assay; Biology; Biophysics; CD4 Positive T Lymphocytes; Calorimetry; Cell Communication; Cell membrane; Cell surface; Cells; Confocal Microscopy; Coupled; Data; Development; Down-Regulation; Endocytosis; Exhibits; Flow Cytometry; Fluorescence Microscopy; Fractionation; Genetic; Goals; HIV; HIV-1; HIV/AIDS; Human; Image Cytometry; Knowledge; Laboratories; Location; Macrophage; Mediating; Membrane; Modification; Natural Immunity; Pathogenesis; Pathway interactions; Patients; Play; Prevention strategy; Proteins; Recycling; Reporting; Research; Role; Site; Site-Directed Mutagenesis; Specificity; Surface; T-Lymphocyte; Testing; Titrations; United States National Institutes of Health; Viral Physiology; Virus; Virus Replication; antagonist; experimental study; filamin; insight; live cell imaging; novel therapeutic intervention; particle; time use; trafficking; transmission process; treatment strategy; viral transmission; virus host interaction; vpu Protein

Project Summary/Abstract BST-2, also known as tetherin, inhibits HIV-1 release from the plasma membrane of infected cells. HIV-1 Vpu neutralizes this restriction through not-mutually exclusive mechanisms, including degradation/downregulation, sequestration, and displacement. However, the relative importance of each of these anti-tetherin mechanisms in virus replication and transmission remains to be defined. Our long-term goal is to understand the roles of Vpu and tetherin in HIV-1 pathogenesis. Specifically, we aim to understand the mechanisms of how host cell proteins are involved in the Vpu-tetherin interaction. Recent studies from our laboratory demonstrate that a specific host cell protein, filamin A (FLNa) can modulate the antiviral activity of tetherin, and the particle release-promoting activity of Vpu. These discoveries suggest that, an FLNa-dependent trafficking pathway of tetherin is the essential step for Vpu to overcome the restriction imposed by tetherin. In this proposal, we will further understand the details of how FLNa regulates the association of Vpu to tetherin. In Aim 1, we will determine the mechanism by which FLNa regulates the antiviral activity of tetherin. Using live cell imaging and flow cytometry, we will examine the effects of FLNa on trafficking dynamics of cell-surface tetherin and intracellular tetherin. By examining the role of FLNa in the antiviral activity of tetherin against different HIV-1 subtypes, we will define the action spectrum of FLNa. By performing rescue experiments coupled with site- directed mutagenesis, we will test the specificity of different FLNa domains in the antiviral activity of tetherin. In Aim 2, we will determine the mechanism by which FLNa modulates the activity of Vpu to relieve tetherin restriction. We will examine the role of FLNa in the anti-tetherin activity of Vpu derived from different genetic subtypes. Using isothermal titration calorimetry (ITC) and microscale thermophoresis (MST), we will quantify the FLNa-Vpu interaction. Using quantitative IP assays and quantitative confocal microscopy, we will examine potential roles of FLNa in the Vpu-tetherin interaction and in the Vpu-tetherin colocalization. Finally, we will evaluate the importance of FLNa in each of three different mechanisms involved in Vpu-mediated tetherin antagonism. In Aim 3, we will focus on human primary macrophages to define the potential role of FLNa in the association of Vpu to tetherin. We will determine the effects of FLNa in HIV-1 infected macrophages on virus release and virus transmission from macrophages to CD4+ T cells. Taken together, these studies will provide important insights into the Vpu-tetherin interaction in HIV-1 replication, transmission, and pathogenesis.

项目摘要/摘要 BST-2，也被称为Tetherin，可以抑制感染细胞质膜上的HIV-1释放。HIV-1 VPU 通过不相互排斥的机制，包括降级/下调监管，中和这一限制， 自动减支和转移。然而，这些反拴系机制中每一种的相对重要性 在病毒复制和传播方面仍有待界定。我们的长期目标是了解 VPU和Tetherin在HIV-1致病机制中的作用具体来说，我们的目标是了解宿主细胞如何 蛋白质参与VPU-tetherin的相互作用。我们实验室最近的研究表明， 特定的宿主细胞蛋白细丝A(Flna)可以调节Tetherin的抗病毒活性，而颗粒 VPU的促释放活性。这些发现表明，依赖于FLNA的运输途径 系留是VPU克服系留限制的关键步骤。在这项提案中，我们将 进一步了解FLNA如何规范VPU与Tetherin的关联的详细信息。在目标1中，我们将 确定Flna调节Tetherin抗病毒活性的机制。使用活细胞成像和 流式细胞术，我们将检测FLNA对细胞表面系链蛋白运输动力学和 细胞内系留蛋白。通过检测Flna在Tetherin对不同HIV-1的抗病毒活性中的作用 子类型，我们将定义FLNA的动作谱。通过结合现场进行救援实验- 定向诱变，我们将测试不同的Flna结构域在Tetherin抗病毒活性中的特异性。在……里面 目的2，我们将确定Flna调节VPU活性以缓解拴系的机制 限制。我们将研究Flna在不同基因来源的VPU的抗栓酶活性中的作用 子类型。利用等温滴定量热法(ITC)和微量热电泳法(MST)，我们将对 FLNA-VPU相互作用。使用定量IP分析和定量共聚焦显微镜，我们将检查 Flna在VPU-tetherin相互作用和VPU-tetherin共定位中的潜在作用。最后，我们会 评估Flna在VPU介导的系链中的三种不同机制中的重要性 对抗性。在目标3中，我们将集中在人的原代巨噬细胞上，以确定Flna在 VPU与Tetherin的关联。我们将确定Flna在HIV-1感染的巨噬细胞中对病毒的影响 巨噬细胞释放病毒并将其传递给CD4+T细胞。总而言之，这些研究将提供 VPU-tetherin在HIV-1复制、传播和发病机制中相互作用的重要见解。