Molecular Interactions of HIV-1 with the Nuclear Pore Complex

HIV-1 与核孔复合物的分子相互作用

• 批准号: 10241258
• 负责人: Gregory B Melikian
• 金额: $ 136.92万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241258
• 关键词: Address; Adopted; Antiviral Therapy; Architecture; Binding; Biological; Biological Assay; Biology; Biotinylation; Caliber; Capsid; Capsid Proteins; Cell Nucleus; Cells; Chemicals; Complex; Computer Models; Cone; Cryo-electron tomography; Cryoelectron Microscopy; DNA; Data; Development; Dimerization; Docking; Electron Microscopy; Engineering; Event; Evolution; Factor V; Fluorescence; Fullerenes; Future; Gap Junctions; Gene Expression; Genome; HIV; HIV-1; Image; Infection; Integration Host Factors; Interphase Cell; Ions; Karyopherins; Knowledge; Label; Length; Light; Literature; Mediating; Methods; Molecular; Molecular Structure; Monitor; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Pathway interactions; Penetration; Peroxidases; Phytic Acid; Process; Proteins; Proteomics; Protocols documentation; Reporting; Research; Reverse Transcription; Structure; System; Techniques; Viral; Viral Genes; Virus; Virus Integration; Virus Replication; Visualization; analog; ascorbate; base; crosslink; design; experimental study; imaging approach; innovation; insight; integration site; knock-down; macromolecule; multidisciplinary; new therapeutic target; novel; novel strategies; novel virus; nucleocytoplasmic transport; particle; passive transport; pleiotropism; prevent; viral DNA

HIV-1 enters the nucleus of non-dividing cells where the reverse transcribed viral DNA is integrated into the host genome. Whereas the nuclear pore complex (NPC) prevents passive transport of large macromolecules, HIV-1 has evolved effective strategies to penetrate this barrier. The HIV-1 nuclear import is a poorly understood process that involves complex interactions with the nuclear import machinery, including several nucleoporins, transportin-3, and CPSF6, all of which bind the viral capsid core, which comprises hundreds of copies of the capsid protein (CA). Pleiotropic effects caused by nucleoporin knockdown and the ability of HIV-1 to use alternative import pathways have impeded the mechanistic studies of nuclear import and frustrated efforts to identify the full array of host factors involved in this process. Our single particle tracking experiments revealed that HIV-1 infection progresses through CA-dependent docking at the nuclear membrane, followed by uncoating (loss of CA) and CA-dependent nuclear transport to the sites of integration. However, very little is known regarding the molecular details and dynamics of virus-NPC interactions, including the structural changes in the architecture of both HIV-1 and the NPC in the course of nuclear import. There is thus an unmet need for structural and functional studies on the molecular mechanisms of HIV-1 nuclear import in the context of productive infection. We propose to combine cutting-edge approaches developed by our highly collaborative team to delineate the molecular interactions and structural changes in the virus and NPC during the nuclear import. Specifically, we will: (1) identify the host factors involved in HIV-1 import using novel proteomics and chemical cross-linking approaches; (2) obtain cryo-electron tomography (cryo-ET) and cross-linking mass-spec structures of the HIV-1 core/NPC complexes by capturing the incoming virus through our new on-demand pore clogging assay; and (3) develop correlative fluorescence/cryo-ET imaging pipeline to structurally characterize the intermediates of HIV-1 nuclear import. Knowledge of molecular interactions during the HIV-1 nuclear import should help identify novel therapeutic targets to block infection, provide a framework for studies of the nuclear import of other viruses and further fundamental understanding of the nuclear pore function.

HIV-1进入非分散细胞的核，其中反转录的病毒DNA为 集成到宿主基因组中。而核孔复合物（NPC）则防止被动 HIV-1的大型大分子的运输已发展出有效的策略以渗透到这一障碍物中。 HIV-1核进口是一个知之甚少的过程，涉及与之复杂的相互作用 核进口机械，包括几种核孔蛋白，Transportin-3和CPSF6 结合病毒衣壳核的结合，其中包含数百份衣壳蛋白（CA）的副本。 由核孔蛋白敲低和HIV-1替代的能力引起的多效效应 进口途径阻碍了核进口和沮丧的努力的机械研究 确定此过程中涉及的全部主机因素。我们的单个粒子跟踪 实验表明，HIV-1感染通过CA依赖的对接进行 核膜，然后取消涂层（CA的损失）和CA依赖性核转运 集成站点。但是，关于分子细节和动力学知之甚少 病毒-NPC相互作用，包括HIV-1和HIV-1结构的结构变化 NPC在核进口过程中。因此，对结构和功能有未满足的需求 研究HIV-1核进口的分子机制的研究 感染。我们建议将高度协作开发的尖端方法结合在一起 团队描述病毒和NPC的分子相互作用和结构变化 核进口。具体来说，我们将：（1）确定使用HIV-1导入的宿主因素 新颖的蛋白质组学和化学交联方法； （2）获得冷冻电子断层扫描 通过捕获HIV-1核/NPC复合物的（冷冻-ET）和交联的质谱结构 通过我们新的按需孔隙堵塞测定法的传入病毒； （3）发展相关性 荧光/冷冻成像管道以结构表征HIV-1的中间体 核进口。 HIV-1核进口期间分子相互作用的知识应有助于 确定新的治疗靶标，以阻止感染，为核研究提供了一个框架 进口其他病毒以及对核孔功能的进一步理解。