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进入非分裂细胞的细胞核，在那里逆转录的病毒DNA被 整合到宿主基因组中。而核孔复合体（NPC）阻止被动 由于HIV-1是大分子的运输屏障，因此已经发展出有效的策略来穿透这一屏障。 HIV-1核输入是一个知之甚少的过程，涉及与HIV-1的复杂相互作用。 核输入机制，包括几种核孔蛋白、转运蛋白3和CPSF 6， 其结合病毒衣壳核心，所述病毒衣壳核心包含数百个拷贝的衣壳蛋白（CA）。 核孔蛋白敲低引起的多效性效应和HIV-1使用替代药物的能力 进口途径阻碍了对核进口的机制研究， 确定这一过程中涉及的所有东道国因素。我们的单粒子追踪 实验表明，HIV-1感染是通过CA依赖性对接在细胞膜上进行的。 核膜，然后是脱壳（CA的损失）和CA依赖性核运输到 融合的场所。然而，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 在核进口的过程中。因此，存在未满足的结构和功能需求。 研究HIV-1核输入的分子机制， 感染我们建议将我们高度合作开发的尖端方法与联合收割机相结合， 研究小组描绘了病毒和NPC的分子相互作用和结构变化， 核进口。具体而言，我们将：（1）确定宿主因素参与HIV-1输入使用 新的蛋白质组学和化学交联方法;（2）获得冷冻电子断层扫描 通过捕获的HIV-1核心/NPC复合物的（冷冻-ET）和交联质谱结构 通过我们的新的按需孔堵塞试验来检测传入的病毒;以及（3）开发相关的 荧光/冷冻ET成像管道，用于结构表征HIV-1中间体 核进口。在HIV-1核输入过程中分子相互作用的知识应该有所帮助 确定新的治疗靶点以阻断感染，为核免疫研究提供框架， 其他病毒的导入和对核孔功能的进一步基本理解。