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

项目摘要 人类免疫缺陷病毒1型(HIV-1)仍然是全球健康的主要威胁。因此，它是必不可少的 我们充分了解病毒感染性的机制，为治疗发展提供新的途径。 在入侵未分裂的宿主细胞后，HIV-1必须进入遗传库，即细胞核。要做到这一点， 病毒基因被包装在衣壳组装中，需要通过核孔复合体(NPC)。NPC是 作为细胞核守门人的大量蛋白质通道。然而，HIV-1衣壳是如何 突破全国人大形成的壁垒仍然知之甚少。之前的研究受到了 NPC结构的复杂性和衣壳-核孔蛋白相互作用的分子水平细节的缺乏； 也是传统的体外平台普遍无法捕捉到病毒的结构复杂性 衣壳，它呈现出被宿主因素识别的模式。因此，解锁更机械化 HIV-1核进入细节要求能够概括更高阶数的创新体外方法 衣壳组装和核孔的自然环境。我们建议通过以下方式建立这样一个平台 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)，它包含精确控制的孔尺寸和嫁接有 可编程密度和取向，以及可编程衣壳蛋白(CA)组件 忠实地重建选择性片段或整个HIV-1衣壳表面。我们将在体外进一步验证我们的 感染性实验和活细胞成像的发现。我们的多人调查组将从我们的 各自的专长，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，以建立和应用这一研究HIV-1衣壳核运输的使能平台。 具体地说，我们将首先全面研究HIV-1衣壳与一系列 参与艾滋病毒-1核进口的细胞因素(目标1)。使用可溶性高阶CA组件和 重组核孔蛋白，我们将定义衣壳与核孔蛋白结合的生化和结构基础， 为后面的研究奠定了基础。然后，我们将建立一个具有越来越多结构的NuPOD库 和成分复杂性，以确定艾滋病毒-1核进口的关键决定因素和相关的 病毒衣壳和鼻咽癌的重塑(目标2)。这些NuPOD将使用多种类型的 核孔素位于DNA-折纸通道上的指定位置，具有可调的尺寸和 僵硬。系统地改变了Nupod设计并分析了由此产生的Nupod-Capsid对接和 插入将有助于理解HIV-1核进口的分子水平细节。此外，我们还将验证 我们使用基于细胞的病毒学实验的主要发现(目标3)。总体而言，我们预计这个项目将创建 强大的工具，不仅将有助于确定艾滋病毒核进入的机制，而且还使我们能够探索 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.