Biochemistry of HIV-1 Budding

HIV-1 出芽的生物化学

• 批准号: 10295402
• 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/10295402
• 关键词: AMOT gene; ATP phosphohydrolase; Address; Affinity; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biogenesis; Biological Assay; Caliber; Cells; Collaborations; Complex; Cryoelectron Microscopy; Deposition; Excision; F-Actin; Factor-42; Family; Filament; Funding; Genetic; Goals; HIV; HIV Budding; HIV-1; Homo; 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; Site; Sorting - Cell Movement; 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; 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的组装是由病毒Gag多蛋白驱动的，宿主因子参与了重要的活动。我们和其他人已经证明， 转运所需的内体分选复合物（ESCRT）途径介导膜分裂反应， HECT泛素E3连接酶NEDD 4L刺激不能募集ESCRT的HIV-1构建体的释放 NEDD 4L结合蛋白的血管动蛋白（AMOT）家族成员促进Gag蛋白的组装进展， 从半球到膜包封的球体的晶格（称为“包封”）。最近，我们发现， 哺乳动物已经独立地进化出了CHMP 3（ESCRT-III）蛋白的截短的、逆转录转座的拷贝，其可以有效地抑制 HIV-1和其他ESCRT依赖性包膜病毒，无过度细胞毒性。我们现建议以这些意见为基础， 寻求互补的结构、生物化学、成像和功能方法来解决HIV-1生物发生中的三个核心问题：1） 组装的病毒粒子是如何被膜包裹的？2)ESCRT机制如何催化病毒膜收缩， 萌芽？3)哺乳动物如何保护自己免受依赖ESCRT的病毒的侵害？除了与艾滋病毒有关外， 这些过程在其他病毒和/或细胞系统中有类似之处，这应该会扩大我们研究的影响。 具体而言，我们建议表征AMOT-NEDD 4L复合物如何有助于HIV病毒体的抑制（目的1）， ESCRT-III细丝和VPS 4 ATP酶合作收缩膜并促进裂变（Aim 2），以及如何截短，逆转录 CHMP 3蛋白（retroCHMP 3）可以抑制逆转录病毒出芽而不诱导细胞毒性（Aim 3）。这些目标是由我们的 结构研究显示AMOT PPXY 1和NEDD 4L WW 3结构域如何形成高亲和力复合物（Aim 1），ESCRT-III蛋白如何 形成可溶性单体蛋白和膜结合丝（目标2和3），以及VPS 4 ATP酶如何结合这些丝并去除 ESCRT-III亚基（Aim 2）。每个目标还将得到旨在阐明AMOT-NEDD 4L复合物如何与细胞内的细胞因子结合的生物化学测定的支持。 重塑膜，稳定F-肌动蛋白，并激活NEDD 4L泛素E3连接酶活性（目的1），ESCRT-III亚基如何重要的共同作用， 组装和收缩膜（目的2），以及retroCHMP 3如何干扰ESCRT-III在病毒出芽位点的丝状体形成， 这些蛋白质是否在其自然环境中起限制因子的作用（目标3）。所有这些目标都将通过遗传和成像来实现 这将有助于揭示AMOT，NEDD 4L，ESCRT-III，VPS 4和retroCHMP 3变体的活性，因为它们在细胞位点起作用， HIV-1组装和出芽。我们的目标是使用这些互补的方法来生成和测试三个基本的机制模型 HIV-1生物合成的过程。 相关性（参见说明）： 像HIV-1这样的包膜病毒通过侵占宿主途径来促进它们从细胞中释放来传播感染。我们的目标 研究的目的是精确地了解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途径。 而不诱导细胞毒性。这些研究应该有助于揭示病毒和细胞如何重塑膜， 病毒特异性相互作用是治疗干预的有吸引力的靶点。