Molecular Interactions of HIV-1 with the Nuclear Pore Complex

HIV-1进入未分裂细胞的核，在那里反转录的病毒DNA 整合到宿主基因组中。而核孔复合体(NPC)防止被动 在大分子的运输方面，艾滋病毒-1已经演变出有效的策略来穿透这一障碍。 HIV-1核进口是一个知之甚少的过程，涉及与 核进口机制，包括几种核孔素、Transportin-3和CPSF6，都是 它结合病毒衣壳核心，该核心由数百个衣壳蛋白(CA)拷贝组成。 核孔蛋白基因敲除引起的多效性与HIV-1使用替代物的能力 进口途径阻碍了对核进口的机理研究，并挫败了 确定此过程中涉及的所有主机因素。我们的单粒子跟踪 实验表明，HIV-1感染是通过CA依赖的对接在 核膜，然后去涂层(CA丢失)和CA依赖的核运输到 整合的地点。然而，关于分子的细节和动力学，我们知之甚少。 病毒-鼻咽癌相互作用的研究，包括HIV-1和HIV-1 全国人大在核进口过程中。因此，对结构和功能的需求尚未得到满足 生产型背景下HIV-1核输入的分子机制研究 感染。我们建议将我们高度协作开发的尖端方法 研究小组研究病毒和鼻咽癌的分子相互作用和结构变化 核进口。具体地说，我们将：(1)确定艾滋病毒-1输入所涉及的宿主因素 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 捕获HIV-1核心/NPC复合体的(Cryo-ET)和交联质谱结构 通过我们新的按需毛孔堵塞试验来检测传入的病毒；以及(3)开发相关的 荧光/冷冻成像流水线用于HIV-1中间体的结构表征 核进口。了解HIV-1核进口过程中的分子相互作用应该会有所帮助 确定新的治疗靶点以阻断感染，为核研究提供框架 输入其他病毒，并进一步对核孔功能有基本的了解。

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.