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.

项目概要： 艾滋病毒/艾滋病仍然是一种全球流行病，全球每年有150万人感染，比最初的艾滋病毒/艾滋病流行40年多了。 病毒的发现。为了找到抗病毒治疗的新途径，重要的是要了解分子生物学。 以及病毒进入细胞的整个过程的结构细节。这项探索性 该项目的重点是HIV-1衣壳用于穿越核孔复合体的机制 (NPC)这是其进入细胞核进行脱壳、逆转录和整合的关键一步。一系列 最近的出版物表明，衣壳可以通过NPC易位， 放在原子核里面。这种教条式的转变是因为数据显示，全国人大实际上可以， 它可以容纳组装的HIV-1衣壳，而以前认为它太窄。虽然现在 虽然已经确定组装的衣壳可以通过NPC易位，但还不清楚是什么驱动了 这个过程的力量是。为了解决这一关键的知识差距，我们提出了一个新的概念框架， 我们专注于HIV-1衣壳和NPC转运屏障之间的相互作用，水凝胶（或 缩合物），其由含有苯丙氨酸-甘氨酸重复的NPC组分（FG-NUPs）组成。虽然FG- 已知几种FG-NUPs中的结构域与衣壳结合，但它们在衣壳转运中的作用仍然很差 理解大多数。当FG-NUPs组装成填充NPC的液体状水凝胶时， 具有>1000个潜在的FG-NUP相互作用位点，我们假设可以设想衣壳分配到 水凝胶，为易位提供热力学驱动力。为了揭示这背后的机制 过程中，我们提出了一个探索性的方法，结合生物化学，生物物理学和结构生物学。在 目标1，我们将对FG-衣壳相互作用进行全面的生化表征。中 定性分析，我们将分析控制HIV-1衣壳与 所有FG-Nup。在这里，我们假设，一个完整的衣壳上的数百个FG结合位点提供了 HIV-1衣壳进入NPC的主要驱动力。一个重要的因素是了解Nup 153， 在NPC的核侧，在指导HIV-1运输中起着独特的作用。在目标2中，我们将确定如何 组装的HIV-1胶囊样颗粒在体外与FG-水凝胶相互作用。初步数据显示， ~40 nm CA球可以快速进入具有NPC样性质的FG-水凝胶。这个系统使我们能够探测 决定因素是关键的核运输的形式，非常类似于在体内的情况， 考虑到由于许多动态相互作用同时发生而产生的亲合力效应。