A multiscale approach for elucidating nuclear entry mechanisms of HIV-1 capsid

项目摘要 1型人体免疫机能丧失病毒（艾滋病毒-1）仍然是对全球健康的一个主要威胁。因此必须 我们完全了解病毒感染的机制，为治疗开发提供新的途径。 在侵入非分裂宿主细胞后，HIV-1必须进入基因库，即细胞核。为此， 包装在衣壳装配体中的病毒基因需要通过核孔复合物（NPC）。npc是 大量的蛋白质通道，作为细胞核的看门人。然而，HIV-1衣壳 突破由NPC形成的障碍仍然知之甚少。以前的研究受到了 NPC结构的复杂性和缺乏分子水平的capture-nucleoporin相互作用的细节; 也是常规体外平台一般无法捕获病毒的结构复杂性， 衣壳，其呈现被宿主因子识别的模式。因此，解锁更加机械化， HIV-1进入细胞核的细节需要创新的体外方法， 衣壳组装和核孔的天然环境。我们建议设立这样一个平台， 利用我们最近建立的基于DNA折纸的NPC模拟物，称为NuPODs（NucleoPorins 由DNA组织），其含有精确控制的孔尺寸和接枝有 可编程的密度和方向，以及可编程的衣壳蛋白（CA）组件， 忠实地重建选择性片段或整个HIV-1衣壳表面。我们将进一步验证我们的体外 感染性实验和活细胞成像的结果。我们的多调查员团队将从我们的 各自的专业知识，包括艾滋病毒生物化学，结构生物学，DNA纳米技术，核运输， 和活细胞成像，建立和应用这个使能平台，研究HIV-1衣壳核转运。 具体来说，我们将首先全面研究HIV-1衣壳与各种 参与HIV-1核输入的细胞因子（Aim 1）。使用可解高阶CA集合， 重组核孔蛋白，我们将定义帽蛋白-核孔蛋白结合的生物化学和结构基础， 为接下来的研究打下基础。然后，我们将建立一个NuPOD库， 和组成的复杂性，以确定HIV-1核输入的关键决定因素和相关的 病毒衣壳和NPC的重塑（目的2）。这些NuPOD将使用多种类型的 位于DNA-折纸通道上指定位置的具有可调尺寸的核孔蛋白， 刚度系统地改变NuPOD设计并分析所得的NuPOD-衣壳对接， 插入将有助于理解HIV-1核输入分子水平细节。此外，我们将验证 我们使用基于细胞的病毒学实验的关键发现（目标3）。总的来说，我们希望这个项目能创造 强大的工具，不仅有助于确定艾滋病毒进入核的机制，而且使我们能够探索 许多其他病毒的核运输。

Project Summary Human immunodeficiency virus type 1 (HIV-1) remains a major threat to global health. Therefore, it is essential that we fully understand the mechanism of viral infectivity to provide new avenues for therapeutic development. After invading a non-dividing host cell, HIV-1 must gain access to the genetic vault, the nucleus. To do this, the viral genes, packaged in a capsid assembly, need to pass through nuclear pore complexes (NPCs). NPCs are massive protein channels that function as the gatekeepers of the cell nucleus. However, how the HIV-1 capsid breaches the barrier formed by the NPC remains poorly understood. Previous studies were hampered by the complexity of the NPC structure and the lack of molecular-level details of capsid-nucleoporin interactions; there was also a general inability of conventional in vitro platforms to capture the structural complexity of the viral capsid, which presents patterns that are recognized by host factors. Therefore, unlocking more mechanistic details of HIV-1 nuclear entry calls for innovative in vitro approaches capable of recapitulating higher-order capsid assemblies and the native environment of nuclear pores. We propose to establish such a platform by leveraging our recently established DNA-origami-based NPC mimics, termed NuPODs (NucleoPorins Organized by DNA), which contain precisely controlled pore dimensions and nucleoporins grafted with programmable density and orientation, as well as the programmable capsid protein (CA) assemblies that faithfully recreate selective fragments or the entire HIV-1 capsid surface. We will further validate our in vitro findings by infectivity experiments and live-cell imaging. Our multi-investigator team will draw from our respective expertise, including HIV biochemistry, structural biology, DNA nanotechnology, nuclear transport, and live-cell imaging, to build and apply this enabling platform for the study of HIV-1 capsid nuclear transport. Specifically, we will first comprehensively study the interactions between HIV-1 capsids and an assortment of cellular factors involved in HIV-1 nuclear import (Aim 1). Using soluble high-order CA assemblies and recombinant nucleoporins, we will define the biochemical and structural basis of capsid-nucleoporin binding, laying the foundation for the rest of the study. We will then build a library of NuPODs with increasing structural and compositional complexity, to identify the key determinants of HIV-1 nuclear import and the associated remodeling of the viral capsid and the NPC (Aim 2). These NuPODs will be built with multiple types of nucleoporins positioned at designated positions on a DNA-origami channel with tunable dimensions and stiffness. Systematically varying the NuPOD design and analyzing the resultant NuPOD-capsid docking and insertion will help understand HIV-1 nuclear import with molecular-level details. Additionally, we will validate our key findings using cell-based virologic experiments (Aim 3). Overall, we expect this project to create powerful tools that will not only help define the mechanism of HIV nuclear entry, but also enable us to explore the nuclear transport of many other viruses.