The Multiple Functions of Vpu at the Membrane

HIV病毒蛋白U(VPU)是一种重要的跨膜病毒蛋白，具有多种功能。 宿主细胞环境，以实现最佳病毒复制。首先，VPU在细胞重塑中起着关键作用 通过去除抑制病毒复制的膜结合宿主蛋白，包括CD4，BST-2/Tetherin， 和MHC分子。它通过将cul1-βTrCP-Skp1-Rbx1E3连接酶复合体招募到 膜，随后泛化并通过再运输将目标蛋白从细胞表面移除 和/或退化。其次，众所周知，VPU可以影响免疫信号，特别是通过放松对 NFkB。这通过NFkB TrCP的隔离发生，否则将通过降解激活β 通过下调BST-2，从而激活NFkB。 其胞质结构域与TRAF的相互作用。第三，也是最后一点，VPU被认为是一种同源低聚物 高尔基体中的病毒孢素离子通道改变膜电位并潜在地增强 病毒粒子释放。这三种功能都依赖于细胞膜上多个事件的协调。 与一系列特征性的、未知的宿主蛋白复合体结合。一个不完美的人 了解所涉及的宿主复合体和处理膜蛋白的固有困难 在体外，我们理解VPU如何执行这些不同的过程的能力被扼杀了。为了更好地 为了描述VPU的多功能特性，我们建议采用一种结合最新状态的总体策略 ART蛋白质组的结构/生物物理机制研究和原代细胞遗传学研究 验证。在目标1中，我们将使用包括翻译后修饰(Ptm)在内的全局蛋白质组学技术。 基于抗坏血酸过氧化物酶的邻近生物素标记质谱仪(APEX-MS)鉴定 VPU和选择性功能分离突变体(Core)参与的宿主复合体和信号通路 1和5)。在目标2中，我们将采用高通量突变和抗体衍生结合的组合。 获得单体和齐聚物低温电子显微镜和X射线衍射结构的伙伴稳定化方法 原子分辨率的VPU络合物(核心3、4、6和7)。确定的候选宿主结合因子 在AIM 1中，将通过荧光尺寸排除色谱(FSEC)和 同样用于结构审问。在目标3中，我们将使用主单元格CRISPR/Cas9编辑 剔除目标1中确定的每个候选宿主因子的方法，以测试它们对 复制一系列VPU突变病毒(核心2)。蛋白质-蛋白质相互作用及其在细胞内的定位 VPU的存在和不存在将通过共聚焦显微镜在体内得到验证。使用Core 5，我们的数据 将为结构-功能假说的生成进行整理，我们最终将在原代细胞中进行测试 遗传模型。该项目将整个HARC的专业知识联系起来，并将产生 以及对艾滋病毒VPU多功能性质的机械论洞察。

HIV Viral Protein U (Vpu) is an essential, transmembrane viral protein with several functions in modulating the host cellular environment for optimal viral replication. First, Vpu plays a critical role in remodeling the cell surface by removing membrane-bound host proteins that inhibit viral replication, including CD4, BST-2/Tetherin, and MHC molecules. It does this by recruitment of the Cul1-βTrCP-Skp1-Rbx1 E3 ligase complex to the membrane, which subsequently ubiquitinates and removes target proteins from the cell surface by retrafficking and/or degradation. Second, Vpu is known to influence immune signaling, particularly through deregulation of NFKb. This occurs through both sequestration of βTrCP, which would otherwise activate NFKb by degradation of its IKK inhibitor, and through down-regulation of BST-2, which is thought to activate NFKb through an interaction between its cytoplasmic domain and TRAF. Third and finally, Vpu is thought to act as a homooligomeric viroporin ion channel in the Golgi apparatus to alter membrane potential and potentially enhance virion release. All three functions rely on the coordination of multiple events at the cell membrane in conjunction with a series of characterized and yet unknown host protein complexes. An imperfect understanding of the host complexes involved and the inherent difficulties of working with membrane proteins in vitro has stifled our ability to understand how Vpu carries out each of these distinct processes. To better characterize the multifunctional nature of Vpu, we propose to employ an overall strategy that couples state-of-the art proteomic discovery with structural/biophysical mechanistic interrogation and primary cell genetic validation. In Aim 1, we will employ global proteomic techniques including post-translational modification (PTM) profiling and Ascorbate Peroxidase-based proximity biotin labeling mass spectrometry (APEX-MS) to identify the host complexes and signaling pathways engaged by Vpu and select separation-of-function mutants (Core 1 and 5). In Aim 2, we will employ a combination of high-throughput mutagenesis and antibody-derived binding partner stabilization approaches to obtain cryo-EM and X-ray diffraction structures of monomeric and homooligomeric Vpu complexes at atomic resolution (Cores 3, 4, 6 and 7). Candidate host binding factors identified in Aim 1 will be tested for Vpu binding in vitro by Fluorescence Size Exclusion Chromatography (FSEC) and similarly used for structural interrogation. In Aim 3, we will employ primary cell CRISPR/Cas9 editing approaches to knock-out each of the candidate host factors identified in Aim 1 to test their impact on the replication of a series of vpu mutant viruses (Core 2). Protein-protein interactions and localization in the presence and absence of Vpu will be validated in vivo by confocal microscopy. Working with Core 5, our data will be collated for structure-function hypothesis generation that we will ultimately test in our primary cell genetic model. This project bridges expertise across the entire HARC collaborative and will generate structural and mechanistic insight into the multifunctional nature of HIV Vpu.