Structural basis for HIV-1 Gag interactions with cellular and viral constituents

HIV-1 Gag 与细胞和病毒成分相互作用的结构基础

基本信息

批准号：
9979755
负责人：
Jamil Subhi Saad
金额：
$ 45.65万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-15 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9979755
关键词：
Achievement Antiviral Agents Attention Binding Biochemical Capsid Capsid Proteins Cell membrane Complex Cryoelectron Microscopy Cytoplasmic Tail Data Defect Deuterium Development Drug Design Engineering Funding Genetic HIV HIV-1 HIV-2 Human Hydrogen Infection Investigation Knowledge Life Lipids Mass Spectrum Analysis Mediating Membrane Modeling Molecular Mutation Outcome Peptides Phase Phosphatidylinositols Phosphatidylserines Play Process Production Proteins Proteome Public Health Recombinants Rest Roentgen Rays Role SP1 gene Safety Signal Transduction Site Solid Structure Technical Expertise Testing Therapeutic Agents Transmembrane Domain Viral Virion Virus Vision X-Ray Crystallography biochemical tools biophysical properties biophysical techniques biophysical tools crosslink env Gene Products experience gag Gene Products in vivo insight membrane assembly membrane model molecular imaging molecular rearrangement mutant nanodisc technology nanodisk novel strategies novel therapeutics particle pathogen reconstitution recruit structural biology three dimensional structure

项目摘要

During the late phase of HIV-1 infection, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on specific interactions between its matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a signaling lipid localized on the inner leaflet of the PM. Concurrent or subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles, a process that is dependent on Gag assembly. Several lines of evidence suggest that incorporation of Env is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of gp41 (gp41CT), a mechanism that remains to be elucidated. For over a decade, we have pioneered approaches to investigate at the molecular level how retroviral (HIV-1, HIV-2, MLV, and ASV) MA domains of Gag interact with lipids (e.g., PI(4,5)P2) and membranes, a key requirement for understanding Gag assembly and ultimately Env incorporation. A major barrier to characterizing gp41CT–MA interaction has been the unavailability of a recombinant gp41CT protein and the inability to reconstitute the proper conditions for the interaction to occur. During this funding period we have recorded a breakthrough by determining the three- dimensional structure of HIV-1 gp41CT and characterized its interaction with membrane. This achievement, an elusive task for over two decades, filled a major gap by providing the structure of the last segment of HIV-1 proteome. In this renewal, we set our sights on elucidating the molecular mechanism by which Gag mediates the recruitment of HIV-1 Env into assembly sites. We will test the hypothesis that trimerization of the MA domain plays an important role in gp41CT interaction and therefore Env recruitment into particles. We will employ a battery of structural biology, biophysical, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to membrane and how it interacts with gp41CT. Our specific aims are: (i) Engineer HIV-1 MA trimer and hexamer and characterize their binding to membranes, (ii) characterize the interactions between HIV-1 MA and gp41CT, and (iii) determine the structure of MA–gp41(TM-CT)–membrane complex by single-particle cryo-EM. This proposal rests on a solid premise of: (1) 25 years of functional data that is waiting for a molecular interpretation to provide sharper insights into Gag assembly on the PM and mechanisms of Env incorporation, (2) highly technical skill sets for working with proteins, membranes, structural and biophysical tools, (3) strong preliminary data, and (4) an outstanding collaborative team with 20-30 years of experience in cryo-EM, x-ray crystallography, and mass spectrometry. Therefore, the proposed structural studies will provide a detailed understanding of HIV-1 Gag assembly on the PM and Gag–mediated Env incorporation. We hope that the outcome of this proposal will help in the development of new antiviral therapeutic agents that inhibit assembly, Env incorporation and ultimately virus production.

在HIV-1感染的后期，将GAG多蛋白转运到质膜（PM）的集会。 PM上的og靶标和组装取决于其矩阵（MA）之间的特定相互作用结构域和磷脂酰肌醇4,5-双磷酸（PI（4,5）P2），一种信号脂质位于内部的脂质上下午。同时或在插科打插件之后，将信封（Env）蛋白招募到PM 掺入病毒颗粒中，这一过程取决于插科打剂的组件。几条证据表明ENV的融合结合是由GAG的MA结构域与细胞质之间的相互作用介导的 GP41（GP41CT）的尾部，该机制尚待阐明。十多年来，我们开创了开创性的在分子水平进行研究的方法逆转录病毒（HIV-1，HIV-2，MLV和ASV） GAG与脂质（例如Pi（4,5）P2）和机制相互作用，这是理解插科打gssbly的关键要求并最终设有Enving。表征GP41CT – MA相互作用的主要障碍是重组GP41CT蛋白的无法获得，并且无法重新建立适当的条件发生的相互作用。在这一资金期间，我们通过确定三个 - HIV-1 GP41CT的尺寸结构，并表征了其与膜的相互作用。这项成就，一个超过二十年的难以捉摸的任务，通过提供HIV-1的最后一部分结构来填补一个重大差距蛋白质组。在此续约中，我们将目光投向了阐明插科打媒体的分子机制将HIV-1环境招募到组装站点。我们将测试MA结构域的修剪化的假设在GP41CT相互作用中起重要作用，因此在颗粒中募集了ENV。我们将采用一个结构生物学，生物物理和生化工具的电池，以产生大分子的图面 GAG的MA结构域与膜结合及其与GP41CT的相互作用。我们的具体目的是：（i）工程师 HIV-1 Ma Trimer和Hexamer并表征它们与膜的结合，（ii）表征了相互作用在HIV-1 MA和GP41CT之间，以及（iii）确定MA – GP41（TM-CT） - 膜复合物的结构。单粒子冷冻EM。该建议基于以下前提：（1）等待25年的功能数据为了进行分子解释，可以在PM和Env机理上对插科打插入的洞察力进行更清晰的见解合并，（2）使用蛋白质，膜，结构和生物物理的高度技术技能工具，（3）强大的初步数据，以及（4）一个出色的合作团队，拥有20 - 30年的经验冷冻EM，X射线晶体学和质谱法。因此，拟议的结构研究将提供对PM和GAG介导的Env收入的HIV-1插科打插件的详细理解。我们希望那样该提案的结果将有助于开发新的抗病毒治疗剂，这些治疗剂抑制组装，设想并最终将病毒产生。