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: 10490880
• 负责人: Chenxiang Lin
• 金额: $ 65.1万
• 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/10490880
• 关键词: Affinity; Architecture; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; Caliber; Capsid; Capsid Proteins; Cell Nucleus; Cells; Communities; Complement; Complex; Cryoelectron Microscopy; Cytoplasmic Protein; DNA; 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; 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; programs; 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.

项目摘要 人类免疫缺陷病毒1型（HIV-1）仍然对全球健康构成主要威胁。因此，这是必不可少的 我们充分了解病毒感染的机制，为治疗发育提供新的途径。 入侵非分散宿主细胞后，HIV-1必须进入遗传库核。为此， 包装在衣壳组件中的病毒基因需要通过核孔复合物（NPC）。 NPC是 充当细胞核的守门人的大量蛋白质通道。但是，HIV-1 CAPSID如何 违反NPC形成的障碍仍然知之甚少。以前的研究受到了 NPC结构的复杂性以及缺乏衣壳核苷相互作用的分子水平细节；那里 也是常规体外平台的一般无法捕获病毒的结构复杂性 Capsid，呈现出宿主因素认可的模式。因此，解锁更多的机械 HIV-1核进入的详细信息要求使用创新的体外方法，能够概括高阶 衣壳组件和核孔的本地环境。我们建议通过 利用我们最近建立的基于DNA-Origami的NPC模拟物，称为Nupods（核孔蛋白 由DNA组织），其中包含精确控制的孔隙尺寸和核孔的核苷 可编程的密度和方向，以及可编程的衣壳蛋白（CA）组件 忠实地重新创建选择性碎片或整个HIV-1帽质表面。我们将进一步验证我们的体外 通过感染实验和活细胞成像的发现。我们的多启发式团队将从我们的 各自的专业知识，包括HIV生物化学，结构生物学，DNA纳米技术，核运输， 和活细胞成像，以构建和应用此促成平台进行HIV-1 Capsid核运输的研究。 具体而言，我们将首先全面研究HIV-1衣壳之间的相互作用和各种各样的相互作用 HIV-1核进口涉及的细胞因素（AIM 1）。使用可溶性高阶CA组件和 重组核孔，我们将定义衣壳的生化和结构基础，辛核核心蛋白的结合， 为其余的研究奠定基础。然后，我们将建立一个随着结构性增加的nupods库 和组成复杂性，以确定HIV-1核进口的关键决定因素和相关的 病毒式衣壳和NPC的重塑（AIM 2）。这些Nupods将使用多种类型的 核苷位于具有可调尺寸的DNA-Origami通道上的指定位置 刚性。系统地改变Nupod设计并分析所得的Nupod-Capsid对接和 插入将有助于了解HIV-1核进口，并通过分子层的细节来进口。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（AIM 3）。总体而言，我们希望这个项目能够创建 强大的工具不仅有助于定义艾滋病毒核进入机制，还可以使我们能够探索 许多其他病毒的核运输。