Structural Virology of Tripartite Motif Proteins

三联基序蛋白的结构病毒学

• 批准号: 10559905
• 负责人: Owen Pornillos
• 金额: $ 65.89万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10559905
• 关键词: AIDS/HIV problem; Address; Affect; Amino Acid Motifs; Avidity; Binding; Binding Proteins; Biochemical; Biology; Capsid; Cell Nucleus; Cell membrane; Cell-Free System; Cells; Competence; Complex; Cryoelectron Microscopy; Dependence; Development; Dissociation; Electrons; Engineering; Event; Funding; Goals; HIV; HIV-1; Human; In Vitro; Infection; Intercept; Interferons; Knowledge; Learning; Link; Measurement; Mediating; Modeling; Molecular; Mutation; Natural Immunity; Pathway interactions; Pattern; Pattern Recognition; Pattern recognition receptor; Pharmaceutical Preparations; Pharmacology; Phenocopy; Phenotype; Proteins; Recombinants; Reporting; Resolution; Retroviridae; Reverse Transcription; Role; Signal Transduction; Site; Specificity; Structure; System; TRIM Motif; TRIM5 gene; Techniques; Testing; Tomogram; Travel; Ubiquitin; Ubiquitination; Viral; Virus; Work; X-Ray Crystallography; cross-species transmission; dimer; fighting; human pathogen; inhibitor; insight; integration site; negative affect; novel; paralogous gene; premature; reconstitution; ubiquitin-protein ligase; virology

PROJECT SUMMARY Tripartite motif (TRIM) proteins are ubiquitin E3 ligases that function in development and innate immunity. TRIM5a and TRIMCyp proteins (collectively termed TRIM5) recognize and deactivate the incoming cores of retroviruses such as HIV- 1. Indeed, human TRIM5a is an important component of interferon-mediated control of HIV-1 infection. Previous work from our group suggested an unprecedented lattice-to-lattice mode of pattern recognition, in which TRIM5a dimers use the viral capsid surrounding the viral core as a template for higher-order assembly, ultimately forming a cage around the core. In the current funding period of this R01 project, we solved the first structure of the TRIM5a/capsid superlattice, developed and optimized a recombinant assembly and ubiquitination system for TRIM5/capsid complexes, and collaborated in studies that reconstituted the early events of HIV-1 replication in a cell-free system. In this renewal application, we now propose to: Aim 1, extend the resolution of our TRIM5a/capsid superlattice structure, in order to visualize contacts between the arrayed TRIM5a protein dimers and their bound viral CA protein hexamer subunits. Aim 2, utilize our novel in vitro ubiquitination and HIV-1 replication systems as experimental platforms to elucidate how TRIM5 deactivates its caged viral core. Aim 3, understand a novel mechanism of TRIM5 recognition, in which the homologous TRIM34 protein directs human TRIM5a to bind and inactive HIV-1 capsids bearing the N74D mutation. Collectively, these studies will fill in important missing gaps in our understanding of how TRIM5 proteins inhibit HIV-1 replication. Our developing understanding of TRIM5 has been giving important insights on HIV-1 capsid vulnerabilities and efforts to develop drugs that exploit these vulnerabilities.

项目总结 三部分基序(Trim)蛋白是泛素E3连接酶，在发育和先天免疫中发挥作用。TRIM5a和 TRIMCyp蛋白(统称为TRIM5)识别和失活逆转录病毒的传入核心，如HIV- 1.的确，人类TRIM5a是干扰素介导的HIV-1感染控制的重要组成部分。以前的工作 提出了一种史无前例的点阵到点阵模式识别，其中TRIM5a二聚体使用 围绕病毒核心的病毒衣壳，作为更高顺序组装的模板，最终形成围绕病毒核心的笼子 核心。在这个R01项目的当前资助期，我们解决了TRIM5a/衣壳超晶格的第一个结构， 开发并优化了TRIM5/衣壳复合体的重组组装和泛素化系统，以及 合作研究重建了无细胞系统中HIV-1复制的早期事件。在这次更新中 我们现在建议：目标1，扩展我们的TRIM5a/Capsid超晶格结构的分辨率，以便 可视化排列的TRIM5a蛋白二聚体与其结合的病毒CA蛋白六聚体亚基之间的联系。目标 2，利用我们的新型体外泛素化和HIV-1复制系统作为实验平台，阐明如何 TRIM5使其笼子内的病毒核心失活。目的3，了解TRIM5识别的一种新机制 同源的TRIM34蛋白引导人的TRIM5a结合携带N74D突变的灭活HIV-1衣壳。 总而言之，这些研究将填补我们在理解TRIM5蛋白如何抑制HIV-1方面的重要空白 复制。我们对TRIM5不断加深的了解对HIV-1衣壳的脆弱性提供了重要的见解 以及开发利用这些弱点的药物的努力。