HIV interactions with host cell proteins in particle release

项目总结/摘要 BST-2，也称为tetherin，抑制HIV-1从感染细胞的质膜释放。HIV-1 VPU 通过不相互排斥的机制，包括降解/下调， 隔离和转移然而，这些抗栓蛋白机制中的每一个的相对重要性 病毒的复制和传播仍有待确定。我们的长期目标是了解 Vpu和Tetherin在HIV-1发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-系链蛋白相互作用。我们实验室最近的研究表明， 特异性宿主细胞蛋白细丝蛋白A（FLNa）可以调节系链蛋白的抗病毒活性，并且该颗粒 促进Vpu释放的活性。这些发现表明，FLNA依赖的转运途径， tetherin是Vpu克服tetherin所施加的限制的必要步骤。在本提案中，我们将 进一步了解FLNa如何调节Vpu与tetherin的关联的细节。在目标1中，我们 确定FLNa调节tetherin抗病毒活性的机制。使用活细胞成像， 流式细胞术，我们将检查FLNa对细胞表面连接蛋白的运输动力学的影响， 细胞内拴蛋白通过检测FLNa在tetherin抗不同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，加上现场- 通过定向诱变，我们将测试不同FLNa结构域在系链蛋白的抗病毒活性中的特异性。在 目的2：探讨FLNa调节Vpu活性，减轻Tetherin的作用机制 限制.我们将研究FLNa在来源于不同基因的Vpu的抗栓蛋白活性中的作用。 亚型使用等温滴定量热法（ITC）和微量热泳（MST），我们将定量 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。最后我们将 评估FLNa在Vpu介导的栓系蛋白的三种不同机制中的重要性 对抗在目标3中，我们将重点关注人原代巨噬细胞，以确定FLNa在细胞凋亡中的潜在作用。 Vpu与系链蛋白的结合。我们将确定FLNa在HIV-1感染的巨噬细胞中对病毒的影响。 从巨噬细胞释放和病毒传播到CD 4 + T细胞。这些研究将提供 对HIV-1复制、传播和发病机制中Vpu-tetherin相互作用的重要见解。

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.