Mechanism of nuclear pore passage of the HIV-1 capsid

HIV-1衣壳核孔通过机制

• 批准号: 10762097
• 负责人: Thomas Schwartz
• 金额: $ 23.71万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-22 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10762097
• 关键词: Address; Antiviral Therapy; Applications Grants; Avidity; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biophysics; Buffers; Capsid; Capsid Proteins; Cell Nucleus; Cells; Characteristics; Chronic Disease; Collaborations; Complex; Cryo-electron tomography; Data; Diameter; Disease; Elements; Environment; Epidemic; Eukaryotic Cell; Foundations; Future; Glycine; Goals; HIV; HIV-1; HIV/AIDS; Hand; Health; Human; Hydrogels; In Vitro; Individual; Infection; Kinetics; Knowledge; Life Cycle Stages; Liquid substance; Molecular; Molecular Target; Nuclear; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Peptides; Phenylalanine; Physical condensation; Play; Positioning Attribute; Process; Property; Proteins; Publications; Research; Reverse Transcription; Role; Series; Shapes; Side; Site; System; Testing; Therapeutic; Thermodynamics; Translating; Viral; Virus; Work; design; driving force; in vivo; individualized medicine; interest; mutant; novel; nucleocytoplasmic transport; particle; programs; receptor; structural biology

PROJECT SUMMARY: HIV/AIDS remains a global epidemic, with 1.5 M infections per year globally, more than 40 years after the initial discovery of the virus. To find new avenues for antiviral therapies, it is important to understand the molecular and structural details of the entire process that the virus uses to enter and traffic the cell. This exploratory project is focused on the mechanisms that the HIV-1 capsid uses to traverse the nuclear pore complex (NPC), a critical step on its journey to the nucleus for uncoating, reverse transcription, and integration. A series of recent publications suggest that the capsid can translocate through the NPC and that uncoating likely takes place inside of the nucleus. This dogmatic shift came about as data showed that the NPC can, in fact, accommodate an assembled HIV-1 capsid, while it was previously perceived to be too narrow. While it is now established that the assembled capsid can translocate through an NPC, it is unclear what the driving force of that process is. To address this critical knowledge gap, we propose a new conceptual framework, in which we focus on interactions between the HIV-1 capsid and the transport barrier of the NPC, a hydrogel (or condensate) made up of phenylalanine-glycine-repeat containing NPC components (FG-NUPs). While FG- domains within several FG-NUPs are known to bind to the capsid, their role in capsid transport is still poorly understood for most. As FG-NUPs assemble into liquid-like hydrogels that fill the NPC, and as each HIV-1 capsid has >1000 potential FG-NUP interaction sites, we hypothesize that one can envision the capsid partitioning into the hydrogel, providing a thermodynamic driving force for translocation. To reveal the mechanisms behind this process, we propose an exploratory approach that combines biochemistry, biophysics, and structural biology. In Aim 1, we will perform a comprehensive biochemical characterization of the FG-capsid interaction. In a qualitative assay, we will analyze the dynamic parameters that govern the interaction of the HIV-1 capsid with all FG-Nups. Here we hypothesize that the many hundreds of FG-binding sites on an intact capsid provide the main driving force for HIV-1 capsids to enter NPCs. An important element will be to understand whether Nup153, at the nuclear side of the NPC, plays a distinct role in directing HIV-1 transport. In Aim 2, we will determine how assembled HIV-1 capsid-like particle interacts with the FG-hydrogel in vitro. Preliminary data indicates that intact ~40 nm CA spheres can rapidly enter an FG-hydrogel with NPC-like properties. This system enables us to probe the determinants that are critical for nuclear transport in a format that closely resembles the in vivo situation, taking into account avidity effects as a result of the many dynamic interactions occurring simultaneously.

项目摘要： 艾滋病毒/艾滋病仍然是一种全球流行病，全球每年1.5 m感染，最初的40多年 发现病毒。要找到用于抗病毒疗法的新途径，了解分子很重要 以及病毒用于输入和流动细胞的整个过程的结构细节。这个探索性 项目的重点是HIV-1 Capsid用来穿越核孔复合物的机制 （NPC），这是其前往核心脱落，逆转录和整合的关键步骤。系列 最近出版物表明，衣壳可以通过NPC转移，不涂层可能需要 放置在核内。随着数据显示，NPC实际上可以 容纳组装的HIV-1帽胶，而以前被认为太窄。虽然现在 确定组装的衣壳可以通过NPC转运，目前尚不清楚什么驾驶 该过程的力量是。为了解决这个关键的知识差距，我们提出了一个新的概念框架 我们专注于HIV-1 CAPSID与NPC的传输屏障之间的相互作用，水凝胶（或 冷凝物）由含有NPC成分（FG-NUPS）的苯丙氨酸 - 甘氨酸重复组成。而FG- 已知几个FG-NUP中的域与CAPSID结合，它们在衣壳运输中的作用仍然很差 大多数人都了解。当FG-Nups组装成填充NPC的液体样水凝胶中，每个HIV-1帽壳 具有> 1000个潜在的FG-NUP交互作用位点，我们假设可以设想将衣壳分配到 水凝胶为易位提供热力学驱动力。揭示其背后的机制 过程，我们提出了一种结合生物化学，生物物理学和结构生物学的探索方法。在 AIM 1，我们将对FG-CAPSID相互作用进行全面的生化表征。在 定性测定，我们将分析控制HIV-1 CAPSID相互作用的动态参数 所有FG-Nups。在这里，我们假设在完整的capsid上的数百个FG结合位点提供了 HIV-1 CAPSIDS进入NPC的主要驱动力。重要的要素是了解NUP153是否 在NPC的核方面，在指导HIV-1运输中起着独特的作用。在AIM 2中，我们将确定如何 组装的HIV-1衣壳状颗粒在体外与FG-Hydrogel相互作用。初步数据表明完整 〜40 nm ca球体可以迅速进入具有NPC样性质的FG-Hydrogel。该系统使我们能够探测 对核运输至关重要的决定因素，其格式与体内情况非常相似， 考虑到同时发生的许多动态相互作用而导致的亲身效应。