Structural requirements for the nuclear export of HIV RNA

HIV RNA核输出的结构要求

• 批准号: 8231263
• 负责人: David Scott Booth
• 金额: $ 3.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231263
• 关键词: Affect; Architecture; Binding; Biochemical; Biological Assay; Biological Models; Carrier Proteins; Cell Nucleus; Cells; Cellular Assay; Charge; Complex; Computer Simulation; Cryoelectron Microscopy; Cytoplasm; Ensure; Eukaryotic Cell; Face; Foundations; Future; Gene Expression; Genome; Goals; HIV; In Vitro; Mammalian Cell; Maps; Mediating; Microinjections; Modeling; Nuclear Export; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Pathway interactions; Positioning Attribute; Production; Property; Proteins; Quality Control; RNA; RNA Processing; RNA Splicing; Recruitment Activity; Response Elements; Role; Running; Site; Staging; Structure; Testing; Viral Proteins; Virion; Work; Xenopus oocyte; base; cofactor; enzyme activity; grasp; intermolecular interaction; mutant; nucleocytoplasmic transport; pathogen; polyadenylated messenger RNA; prevent; protein function; public health relevance; receptor; reconstitution; research study; stoichiometry; success; therapeutic target; tool; viral RNA

DESCRIPTION (provided by applicant): Eukaryotic cells control the export of RNA from the nucleus to ensure that properly processed RNAs enter the cytoplasm for gene expression. Regulated RNA export restricts the lifecycle of the pathogen human immunodeficiency virus (HIV) because the host cell prevents export of the unspliced, viral RNA genome that encodes proteins for and packages into new virions. The viral protein Rev circumvents this inhibition by oligomerizing on an intronic RNA structure called the Rev Response Element (RRE) and by recruiting the host nuclear export adaptors Crm1 and Ran. To export the RRE, the ternary complex formed between these components must favorably interact with the nuclear pore complex (NPC) to compensate for the large size and polyanionic charge that are physical barriers for the translocation of any large ribonuceloprotein complex (RNPs) through the NPC. We do not yet clearly understand how export adaptors facilitate the transport of large cargoes, like the Rev-RRE complex, since structures of NPC substrates are limited to small cargoes with single adaptors while functional evidence suggests that multiple export receptors act in concert to transport large RNPs. Likewise, we hypothesize the Rev-RRE complex organizes multiple Crm1 adaptors to facilitate export. To investigate how the ternary HIV export complex translocates through the NPC, we propose reconstituting the quaternary complex, determining its structure by cryo-electron microscopy, and using the structure to guide biochemical and cellular assays to understand how the number and position of adaptors affects export. Together these experiments will provide the physical foundation for further interrogating how the NPC coordinates multiple stages of RNA export. PUBLIC HEALTH RELEVANCE: The progression of the human immunodeficiency virus (HIV) lifecycle depends on the viral protein Rev directing the nuclear export of the viral RNA through a Crm1-dependent pathway that circumvents the retention of unspliced RNA in the nucleus. Our goal is to understand how nuclear export adaptors assemble around the RNA cargo to facilitate its transport through the nuclear pore complex by solving the structure of the ternary export complex formed between Rev, a portion of the viral RNA, and export adaptors. These results will guide future work using the HIV export complex as a substrate for investigating the coordination of RNA export by the nuclear pore complex, potentially identifying specific nuclear transport proteins functioning as host cell cofactors that can be therapeutic targets.

描述(申请人提供)：真核细胞控制核糖核酸的输出，以确保经过适当处理的核糖核酸进入细胞质进行基因表达。受调控的RNA输出限制了病原体人类免疫缺陷病毒(HIV)的生命周期，因为宿主细胞阻止了未剪接的病毒RNA基因组的输出，该基因组编码蛋白质并包装成新的病毒粒子。病毒蛋白REV通过在称为REV反应元件(RRE)的内含子RNA结构上进行寡聚，并通过招募宿主核出口适配器CRM1和RAN来规避这种抑制。为了输出RRE，这些组分之间形成的三元复合体必须与核孔复合体(NPC)良好地相互作用，以补偿大尺寸和多阴离子电荷，这些电荷是任何大的核核蛋白复合体(RNPs)通过NPC转移的物理障碍。我们尚不清楚出口适配器如何促进大型货物的运输，如REV-RRE复合体，因为NPC底物的结构仅限于具有单个适配器的小货物，而功能证据表明多个出口受体协同作用运输大型RNP。同样，我们假设REV-RRE复合体组织了多个CRM1适配器以促进出口。为了研究HIV出口三元复合体是如何通过NPC转运的，我们建议重组四元复合体，用冷冻电子显微镜确定其结构，并利用该结构来指导生化和细胞分析，以了解接头的数量和位置如何影响出口。总之，这些实验将为进一步询问NPC如何协调RNA输出的多个阶段提供物理基础。 公共卫生相关性：人类免疫缺陷病毒(HIV)生命周期的进展取决于病毒蛋白REV，该蛋白通过依赖CRM1的途径绕过未剪接RNA在细胞核中的保留，指导病毒RNA的核输出。我们的目标是通过解决REV、病毒RNA的一部分和出口接头之间形成的三元出口复合体的结构，了解核出口适配子是如何围绕RNA货物组装的，以促进其通过核孔复合体的运输。这些结果将指导未来的工作，使用HIV输出复合体作为底物，研究核孔复合体对RNA输出的协调，有可能识别作为宿主细胞辅助因子的特定核运输蛋白，这些蛋白质可以成为治疗靶点。