Biochemistry of HIV-1 Budding

HIV-1 出芽的生物化学

• 批准号: 10552530
• 负责人: WESLEY I. SUNDQUIST
• 金额: $ 55.71万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552530
• 关键词: AMOT gene; ATP phosphohydrolase; Address; Affinity; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biogenesis; Biological Assay; Cells; Collaborations; Complex; Cryoelectron Microscopy; Deposition; Diameter; Endosomes; Excision; F-Actin; Family; Family member; Filament; Funding; Genetic; Goals; HIV; HIV Budding; HIV-1; Homo; Homologous Gene; Human; Image; Impairment; Infection; Innate Immune System; Instruction; Integration Host Factors; Mammals; Mediating; Membrane; Modeling; Molecular; Neck; Pathway interactions; Process; Progress Reports; Proteins; Reaction; Reporting; Research; Resolution; Retrotransposition; Site; Sorting; Structure; System; Testing; Therapeutic Intervention; Toxic effect; Ubiquitin; Variant; Viral; Virion; Virus; Work; analog; constriction; design; gag Gene Products; genetic analysis; in vitro testing; inhibitor; member; membrane assembly; membrane model; monomer; pathogen; protein function; reconstitution; recruit; targeted treatment; ubiquitin-protein ligase; virus envelope

HIV-1 assembly is driven by the viral Gag polyprotein, with host factors contributing essential activities. We and others have shown that host machinery of the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway mediates the membrane fission reaction that releases HIV-1 virions (termed “budding”), that the HECT ubiquitin E3 ligase NEDD4L stimulates release of HIV-1 constructs that cannot recruit ESCRT factors directly, and that members of the Angiomotin (AMOT) family of NEDD4L-binding proteins promote progression of the assembling Gag lattices from hemispheres to membrane-enveloped spheres (termed “envelopment”). Most recently, we have discovered that multiple different mammals have independently evolved truncated, retrotransposed copies of a CHMP3 (ESCRT-III) protein that can potently inhibit release of HIV-1 and other ESCRT-dependent enveloped viruses without undue cellular toxicity. We now propose to build on these observations by pursuing complementary structural, biochemical, imaging, and functional approaches to address three central questions in HIV-1 biogenesis: 1) How do assembling virions become wrapped in membranes? 2) How does the ESCRT machinery catalyze viral membrane constriction and budding? 3) How can mammals protect themselves broadly against ESCRT-dependent viruses? In addition to their relevance for HIV, each of these processes has analogs in other viral and/or cellular systems, which should extend the impact of our studies. Specifically, we propose to characterize how AMOT-NEDD4L complexes contribute to HIV virion envelopment (Aim 1), how late-acting ESCRT-III filaments and VPS4 ATPases collaborate to constrict membranes and promote fission (Aim 2), and how truncated, retrotransposed CHMP3 proteins (retroCHMP3) can inhibit retroviral budding without inducing cellular toxicity (Aim 3). These Aims are buttressed by our structural studies showing how the AMOT PPXY1 and NEDD4L WW3 domains form a high affinity complex (Aim 1), how ESCRT-III proteins form soluble, monomeric proteins and membrane- bound filaments (Aims 2 and 3), and how VPS4 ATPases bind these filaments and remove ESCRT-III subunits (Aim 2). Each Aim will also be supported by biochemical assays designed to elucidate how the AMOT-NEDD4L complex remodels membranes, stabilizes F-actin, and activates NEDD4L ubiquitin E3 ligase activity (Aim 1), how essential ESCRT-III subunits co- assemble and constrict membranes (Aim 2), and how retroCHMP3 interferes with ESCRT-III filament formation at sites of virus budding and whether these proteins function as restriction factors in their natural settings (Aim 3). All of the Aims will be facilitated by genetic and imaging assays that will help reveal the activities of AMOT, NEDD4L, ESCRT-III, VPS4 and retroCHMP3 variants as they function at cellular sites of HIV-1 assembly and budding. Our goal is to use these complementary approaches to generate and test mechanistic models for three fundamental processes in HIV-1 biogenesis. RELEVANCE (See instructions): Enveloped viruses like HIV-1 spread infection by usurping host pathways to facilitate their release from cells. The goals of our research are to understand precisely how HIV-1 utilizes these host pathways, and how innate immune systems can inhibit these processes without inducing cell toxicity. These studies should help reveal how viruses and cells remodel membranes and identify virus-specific interactions that are attractive targets for therapeutic intervention.

HIV-1组装由病毒堵嘴多蛋白驱动，宿主因素促进了基本活动。我们和其他人已经表明了主机 运输（ESCRT）途径所需的内体分类复合物的机械介导了释放的膜裂变反应 HIV-1病毒（称为“芽”），泛素E3连接酶NEDD4L刺激无法募集ESCRT的HIV-1构建体的释放 直接的因素，以及NEDD4L结合蛋白的血管毒素（AMOT）家族的成员促进组装GAG的进展 从半球到膜开发球（称为“信封”）的晶格。最近，我们发现多个不同 哺乳动物已独立演化的截短，转译本的CHMP3（ESCRT-III）蛋白，可以潜在地抑制释放 HIV-1和其他依赖于ESCRT的包膜病毒，而没有细胞毒性。我们现在建议通过 追求完整的结构，生化，成像和功能性方法，以解决HIV-1生物发生中的三个核心问题：1） 组装病毒如何包裹在膜上？ 2）ESCRT机械如何催化病毒膜收缩和 萌芽？ 3）哺乳动物如何在依赖ESCRT的病毒中广泛保护自己？除了它们与艾滋病毒的相关性外 过程在其他病毒和/或细胞系统中具有类似物，这应该扩展我们的研究的影响。 具体而言，我们建议表征AMOT-NEDD4L复合物如何促进HIV病毒粒子环境（AIM 1） ESCRT-III细丝和VPS4 ATPases合作以收缩机制并促进裂变（AIM 2），以及如何截断，转化转导 CHMP3蛋白（retrochmp3）可以抑制逆转录病毒萌芽，而无需诱导细胞毒性（AIM 3）。这些目标由我们支撑 结构研究表明AMOT PPXY1和NEDD4L WW3结构域如何形成高亲和力络合物（AIM 1），如何ESCRT-III蛋白 形成固体，单体蛋白和膜结合丝（目标2和3），VPS4 ATPases如何结合这些丝并去除 ESCRT-III亚基（AIM 2）。每个目标也将由旨在阐明AMOT-NEDD4L复合物的生化测定法支持 重塑膜，稳定F-肌动蛋白，并激活NEDD4L泛素E3连接酶活性（AIM 1），必需的ESCRT-III亚基如何共同 组装和收缩的膜（AIM 2），以及逆行Chmp3如何在病毒萌芽和 这些蛋白质是否在其自然环境中起限制因素的作用（AIM 3）。所有目标都将通过遗传和成像制备 将有助于揭示AMOT，NEDD4L，ESCRT-III，VPS4和retrochmp3变体的活性的测定，因为它们在细胞位点起作用 HIV-1组装和萌芽。我们的目标是使用这些完整的方法来生成和测试三个基本的机械模型 HIV-1生物发生过程中的过程。 相关性（请参阅说明）： 诸如HIV-1（例如HIV-1）通过篡夺宿主途径以促进其从细胞释放而传播感染。我们的目标 研究旨在准确了解HIV-1如何利用这些宿主途径，以及先天免疫系统如何抑制这些宿主途径 没有诱导细胞毒性的过程。这些研究应有助于揭示病毒和细胞如何重塑膜并确定 病毒特异性相互作用是治疗干预的有吸引力的靶标。