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多聚蛋白驱动的，宿主因素贡献了基本的活动。我们和其他人已经证明了东道主 转运所需的内体分选复合体(ESCRT)途径的机械介导释放的膜裂变反应 Hect泛素E3连接酶NEDD4L刺激不能招募ESCRT的HIV-1结构物的释放 直接因素，以及NEDD4L结合蛋白的血管生成素(AMOT)家族成员促进组装GAG的进展 从半球到被膜包裹的球体的晶格(称为“包络”)。最近，我们发现了多个不同的 哺乳动物已经独立进化出CHMP3(ESCRT-III)蛋白的截短、反转录转座拷贝，这种蛋白可以有效地抑制CHMP3的释放 HIV-1和其他ESCRT依赖的包膜病毒没有过多的细胞毒性。我们现在建议在这些观察的基础上，通过 寻求互补的结构、生化、成像和功能方法，以解决艾滋病毒-1生物发生中的三个核心问题：1) 组装的病毒粒子如何包裹在膜上？2)ESCRT机制如何催化病毒膜的收缩和 萌芽？3)哺乳动物如何广泛地保护自己免受ESCRT依赖的病毒的侵袭？除了它们与艾滋病毒的相关性外，其中每一个 在其他病毒和/或细胞系统中也有类似的过程，这应该会扩大我们研究的影响。 具体地说，我们建议表征amot-NEDD4L复合体如何有助于HIV病毒粒子的包膜(目标1)，以及如何晚起作用 ESCRT-III细丝和Vps4 ATPase共同作用来收缩膜并促进分裂(目标2)，以及如何截断、反转座 CHMP3蛋白(逆转录CHMP3)可以抑制逆转录病毒的萌发，而不会引起细胞毒性(目标3)。这些目标得到了我们的 结构研究显示AMOT PPXY1和NEDD4L WW3结构域如何形成高亲和力复合体(目标1)，ESCRT-III蛋白如何 形成可溶的单体蛋白和膜结合的细丝(目标2和3)，以及Vps4 ATPase如何结合这些细丝和去除 ESCRT-III亚单位(目标2)。每个目标也将得到生化分析的支持，这些分析旨在阐明AMOT-NEDD4L复合体是如何 重塑细胞膜，稳定F-肌动蛋白，激活NEDD4L泛素E3连接酶活性(目标1)，ESCRT-III亚基如何参与 组装和压缩膜(目标2)，以及反转录CHMP3如何干扰ESCRT-III病毒发芽和感染部位的细丝形成 这些蛋白质在其自然环境中是否起到限制因素的作用(目标3)。所有的目标都将通过基因和成像来促进 有助于揭示AMOT、NEDD4L、ESCRT-III、Vps4和反转录CHMP3变异体在细胞内功能位点的活性 HIV-1组装和萌芽。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械模型 HIV-1生物发生的过程。 相关性(请参阅说明)： 像HIV-1这样的包膜病毒通过篡夺宿主途径来传播感染，以促进它们从细胞中释放。我们的目标是 研究是为了准确地了解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 加工过程中不会引起细胞毒性。这些研究应该有助于揭示病毒和细胞如何重塑细胞膜并识别 病毒特异性的相互作用是治疗干预的有吸引力的目标。