The Multiple Functions of Vpu at the Membrane

Vpu 在膜上的多种功能

• 批准号: 10229570
• 负责人: Robert M Stroud
• 金额: $ 3.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10229570
• 关键词: Amino Acids; Antibodies; Antiviral Agents; Bacteriophages; Binding; Binding Proteins; Biochemical; Biological Assay; Biophysics; Biotin; Biotinylation; CD4 Antigens; CD4 Positive T Lymphocytes; CRISPR/Cas technology; CUL1 gene; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Complement; Complex; Couples; Cryoelectron Microscopy; Crystallization; Cullin Proteins; Cytoplasmic Tail; Data; Data Set; Defect; Down-Regulation; Environment; Event; Fab Immunoglobulins; Filtration; Fluorescence; Fluorescence Microscopy; Generations; Genes; Genetic; Genetic Models; Golgi Apparatus; HIV; HIV Infections; HIV Receptors; Homo; IL27RA gene; Immune signaling; In Vitro; Infection; Integration Host Factors; Ion Channel; Knock-out; Knowledge; Label; Left; Length; Libraries; Mass Spectrum Analysis; Membrane; Membrane Potentials; Membrane Proteins; Modeling; Molecular Sieve Chromatography; Monitor; Mutagenesis; Nature; Peroxidases; Phosphorylation; Play; Post-Translational Protein Processing; Process; Proteins; Proteomics; Resolution; Role; Series; Serine; Signal Pathway; Signal Transduction; Structure; System; T-Lymphocyte; Techniques; Testing; Therapeutic Intervention; Ubiquitination; Validation; Viral Physiology; Viral Proteins; Virion; Virus; Virus Replication; X ray diffraction analysis; ascorbate; base; behavior in vitro; beta-Transducin Repeat-Containing Proteins; cell determination; cell type; chemical genetics; genetic manipulation; improved; in vitro Assay; in vivo; inhibitor/antagonist; insight; mutant; next generation; protein complex; protein protein interaction; recruit; reflectance confocal microscopy; therapeutic candidate; trafficking; ubiquitin-protein ligase; virology; virus core

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.

HIV病毒蛋白U（Vpu）是一种必需的跨膜病毒蛋白，在调节HIV感染中具有多种功能。 宿主细胞环境以获得最佳病毒复制。首先，Vpu在重塑细胞中起着关键作用 通过去除抑制病毒复制的膜结合宿主蛋白，包括CD 4，BST-2/Tetherin， 和MHC分子。它通过将Cul 1-β TrCP-Skp 1-Rbx 1 E3连接酶复合物募集到 膜，随后泛素化并通过再运输从细胞表面去除靶蛋白 和/或降解。第二，已知Vpu影响免疫信号传导，特别是通过免疫调节的失调。 NFKb。这通过螯合βTrCP发生，否则βTrCP将通过降解激活NFKb 通过下调BST-2，它被认为是通过一种新的途径激活NFKb。 其胞质结构域和TRAF之间的相互作用。第三，也是最后一点，Vpu被认为是一种同源寡聚体， 高尔基体中的病毒孔蛋白离子通道改变膜电位并可能增强 病毒粒子释放。所有这三种功能都依赖于细胞膜上多个事件的协调， 与一系列表征的但未知的宿主蛋白质复合物结合。一个不完美 理解所涉及的宿主复合物和与膜蛋白一起工作的固有困难 体外实验已经扼杀了我们理解Vpu如何执行这些不同过程的能力。更好地 特征的多功能性质的Vpu，我们建议采用一个整体战略，夫妇的状态， 利用结构/生物物理机制询问和原代细胞遗传学的蛋白质组学发现 验证。在目标1中，我们将采用包括翻译后修饰（PTM）在内的整体蛋白质组学技术 分析和基于抗坏血酸过氧化物酶的邻近生物素标记质谱法（APEX-MS）， 由Vpu参与的宿主复合物和信号传导途径，并选择功能分离突变体（Core 1和5）。在目标2中，我们将采用高通量诱变和抗体衍生结合的组合 配偶体稳定化方法，以获得单体和均聚物的低温EM和X射线衍射结构 原子分辨率的Vpu复合物（核心3，4，6和7）。鉴定的候选宿主结合因子 将通过荧光尺寸排阻色谱法（FSEC）测试目标1中的Vpu体外结合， 类似地用于结构性询问。在目标3中，我们将采用原代细胞CRISPR/Cas9编辑， 方法敲除目标1中确定的每个候选宿主因子，以测试它们对 一系列vpu突变病毒的复制（核心2）。蛋白质-蛋白质相互作用和定位 Vpu的存在和不存在将通过共聚焦显微镜在体内验证。使用Core 5，我们的数据 将被整理用于产生结构-功能假设，我们最终将在我们的原代细胞中进行测试 遗传模型该项目跨越整个HARC合作的专业知识，并将产生结构性的 和对HIV Vpu多功能性质的机械洞察。