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: 10380145
• 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/10380145
• 关键词: AIDS/HIV problem; 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; Human; Image Cytometry; Knowledge; Laboratories; Location; 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; Virus; Virus Replication; antagonist; experimental study; filamin; insight; live cell imaging; macrophage; 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.

项目概要/摘要 BST-2，也称为系链蛋白，可抑制 HIV-1 从受感染细胞质膜中释放。 HIV-1 Vpu 通过不相互排斥的机制（包括降解/下调）中和这种限制， 隔离和流离失所。然而，这些抗系绳蛋白机制的相对重要性 病毒复制和传播的作用仍有待确定。我们的长期目标是了解 HIV-1 发病机制中的 Vpu 和 Tetherin。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 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 filamin A (FLNa) 可以调节 Tetherin 的抗病毒活性，并且该颗粒 Vpu 的释放促进活性。这些发现表明，FLNa 依赖性转运途径 tetherin 是 Vpu 克服 tetherin 限制的重要步骤。在本提案中，我们将 进一步了解 FLNa 如何调节 Vpu 与 tetherin 关联的细节。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，我们将检查 FLNa 对细胞表面系链蛋白运输动力学的影响 细胞内系链素。通过检查 FLNa 在 tetherin 针对不同 HIV-1 的抗病毒活性中的作用 亚型，我们将定义 FLNa 的作用谱。通过进行救援实验并结合现场 定向诱变，我们将测试不同 FLNa 结构域在 tetherin 抗病毒活性中的特异性。在 目标2，我们将确定FLNa调节Vpu活性以缓解tetherin的机制 限制。我们将研究 FLNa 在源自不同遗传的 Vpu 的抗系链蛋白活性中的作用 亚型。使用等温滴定量热法 (ITC) 和微量热泳 (MST)，我们将量化 FLNa-Vpu 相互作用。使用定量 IP 测定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，我们将 评估 FLNa 在 Vpu 介导的系链蛋白涉及的三种不同机制中的重要性 对抗。在目标 3 中，我们将重点关注人类原代巨噬细胞，以确定 FLNa 在 Vpu 与系链蛋白的关联。我们将确定HIV-1感染的巨噬细胞中的FLNa对病毒的影响 释放和病毒从巨噬细胞传播到 CD4+ T 细胞。总的来说，这些研究将提供 对 Vpu-tetherin 相互作用在 HIV-1 复制、传播和发病机制中的重要见解。