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

阐明 HIV-1 衣壳核进入机制的多尺度方法

• 批准号: 10794022
• 负责人: Chenxiang Lin
• 金额: $ 13.41万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-17 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10794022
• 关键词: Affinity; Architecture; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; Capsid; Capsid Proteins; Cell Nucleus; Cells; Communities; Complement; Complex; Cryoelectron Microscopy; Cytoplasm; DNA; Diameter; Dimensions; Disease; Docking; Environment; Face; Foundations; Gatekeeping; Genetic; Genome; Geometry; Goals; HIV; HIV Infections; HIV-1; In Vitro; Individual; Integration Host Factors; Invaded; Libraries; Molecular; Mutagenesis; NUP214 gene; Nanotechnology; Nuclear; Nuclear Import; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nuclear Proteins; Pattern; Pattern Recognition; Pore Proteins; Positioning Attribute; Process; Proteins; Recombinants; Research; Research Personnel; Research Project Grants; Rest; Series; Specificity; Structure; Surface; System; Techniques; Testing; Validation; Viral; Viral Genes; Virus; Virus Diseases; Virus Replication; Work; X-Ray Crystallography; antiretroviral therapy; base; density; design; experimental study; genetic approach; global health; inhibitor; innovation; insight; live cell imaging; nanopore; novel; nucleocytoplasmic transport; pathogen; protein structure; structural biology; success; therapeutic development; three dimensional structure; tool

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.

项目概要 1 型人类免疫缺陷病毒 (HIV-1) 仍然是全球健康的主要威胁。因此，有必要 我们充分了解病毒感染性的机制，为治疗开发提供新途径。 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，需要将 包装在衣壳组件中的病毒基因需要通过核孔复合体（NPC）。 NPC 是 大量的蛋白质通道，充当细胞核的看门人。然而，HIV-1衣壳如何 是否突破全国人大设立的屏障仍知之甚少。之前的研究受到以下因素的阻碍 NPC结构的复杂性以及衣壳-核孔蛋白相互作用的分子水平细节的缺乏；那里 传统体外平台普遍无法捕获病毒的结构复杂性 衣壳，它呈现出宿主因子识别的模式。因此，解锁更多机械 HIV-1进入核的细节需要能够重现高阶的创新体外方法 衣壳组件和核孔的原生环境。我们建议建立这样一个平台 利用我们最近建立的基于 DNA 折纸的 NPC 模拟物，称为 NuPOD（NucleoPorins） 由 DNA 组织），其中含有精确控制的孔尺寸和接枝的核孔蛋白 可编程密度和方向，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。我们将进一步验证我们的体外 感染性实验和活细胞成像的发现。我们的多位研究者团队将从我们的 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，构建并应用这个支持平台来研究 HIV-1 衣壳核运输。 具体来说，我们将首先全面研究 HIV-1 衣壳和各种物质之间的相互作用 参与 HIV-1 核输入的细胞因子（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-核孔蛋白结合的生化和结构基础， 为其余的学习打下基础。然后我们将建立一个结构不断增加的 NuPOD 库 和成分复杂性，以确定 HIV-1 核输入的关键决定因素以及相关的 病毒衣壳和 NPC 的重塑（目标 2）。这些 NuPOD 将由多种类型的 核孔蛋白位于 DNA 折纸通道上的指定位置，尺寸可调， 刚性。系统地改变 NuPOD 设计并分析由此产生的 NuPOD-衣壳对接和 插入将有助于了解 HIV-1 核输入的分子水平细节。此外，我们将验证 我们使用基于细胞的病毒学实验得出的主要发现（目标 3）。总的来说，我们希望这个项目能够创造 强大的工具不仅有助于定义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。