Biochemistry of HIV-1 Budding

HIV-1 出芽的生物化学

基本信息

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
项目类别：
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

项目摘要

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 Gag 多蛋白驱动，宿主因素有助于宿主的基本活动。运输所需的内体分选复合物 (ESCRT) 途径的机制介导膜裂变反应，释放 HIV-1 病毒粒子（称为“出芽”），HECT 泛素 E3 连接酶 NEDD4L 刺激无法招募 ESCRT 的 HIV-1 构建体的释放 NEDD4L 结合蛋白的血管动蛋白 (AMOT) 家族成员促进 Gag 组装的进展最近，我们发现了从半球到膜包裹球体（称为“包裹”）的多种不同的晶格。哺乳动物独立进化出 CHMP3 (ESCRT-III) 蛋白的截短、逆转录转座副本，可有效抑制 HIV-1 和其他 ESCRT 依赖性包膜病毒没有过度的细胞毒性，我们现在建议在这些观察的基础上通过以下方法进行研究。寻求互补的结构、生化、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) 组装病毒粒子如何包裹在膜中？ 2）ESCRT 机制如何催化病毒膜收缩和收缩？ 3) 哺乳动物如何广泛保护自己免受 ESRT 依赖性病毒的侵害？这些过程在其他病毒和/或细胞系统中有类似物，这应该会扩大我们研究的影响。具体来说，我们建议描述 AMOT-NEDD4L 复合物如何促进 HIV 病毒颗粒包封（目标 1）、晚效作用如何 ESCRT-III 丝和 VPS4 ATP 酶协作收缩膜并促进裂变（目标 2），以及如何截短、逆转录 CHMP3 蛋白 (retroCHMP3) 可以抑制逆转录病毒出芽而不诱导细胞毒性（目标 3）。结构研究显示 AMOT PPXY1 和 NEDD4L WW3 结构域如何形成高亲和力复合物（目标 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 VPS4 ATP 酶如何结合这些丝并去除 ESCRT-III 亚基（目标 2）还将得到旨在阐明 AMOT-NEDD4L 复合物如何形成的生化测定的支持。重塑膜、稳定 F-肌动蛋白并激活 NEDD4L 泛素 E3 连接酶活性（目标 1），ESCRT-III 亚基的重要组成部分如何共同作用组装和收缩膜（目标 2），以及 RetroCHMP3 如何干扰病毒出芽和收缩部位的 ESCRT-III 丝形成这些蛋白质在自然环境中是否充当限制因素（目标 3）。遗传和成像将促进所有目标的实现。有助于揭示 AMOT、NEDD4L、ESCRT-III、VPS4 和 RetroCHMP3 变体在细胞位点发挥作用时的活性 HIV-1 组装和萌芽的目标是使用这些互补方法来生成和测试三个基本的机制模型。 HIV-1 生物合成过程。相关性（参见说明）： HIV-1 等包膜病毒通过侵占宿主途径来促进其从细胞中释放来传播感染。研究的目的是准确了解 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途径这些研究应该有助于揭示病毒和细胞如何重塑膜并识别。病毒特异性相互作用是治疗干预的有吸引力的目标。