Imaging viral RNA genome replication at the single molecule level

在单分子水平上对病毒 RNA 基因组复制进行成像

• 批准号: 8693304
• 负责人: Olve Breien Peersen
• 金额: $ 21.88万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8693304
• 关键词: Affect; Architecture; Binding; Binding Sites; Biochemistry; Biophysics; Cells; Complex; Crystallography; DNA Sequence Rearrangement; Data; Dengue; Development; Diffusion; Disease; Docking; Electron Microscopy; Environment; Enzymes; Food Poisoning; Genome; Growth; Hepatitis; Human; Human poliovirus; Image; In Vitro; Individual; Infection; Integration Host Factors; Internet; Laboratories; Lateral; Lead; Length; Measures; Membrane; Membrane Proteins; Methods; Microscopy; Molecular; Molecular Virology; Monitor; Natural Immunity; Norovirus; Poliomyelitis; Polioviruses; Polymerase; Polyproteins; Positioning Attribute; Protein Binding; Proteins; Proteolytic Processing; RNA; RNA Binding; RNA Viruses; RNA replication; RNA-Directed RNA Polymerase; RNA-Protein Interaction; Research Project Grants; Resolution; Role; Solutions; Structure; Structure-Activity Relationship; Surface; System; Techniques; Vesicle; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; West Nile virus; Work; base; biophysical techniques; electron tomography; experience; innovation; insight; light microscopy; method development; novel; optical imaging; particle; protein protein interaction; protein structure; public health relevance; research study; single molecule; two-dimensional; viral RNA

DESCRIPTION (provided by applicant): Positive strand RNA viruses replicate their genomes and assemble virions in the context of large membrane-anchored "replication center" complexes whose molecular organization is inherently very difficult to study. Electron microscopy and tomography as well as high-resolution light microscopy studies clearly show massive membrane rearrangements taking place in infected cells and the localization of viral and host proteins to these membranes, but their resolution is not high enough to define specific inter-molecular contacts. At the other end of the spectrum are NMR and crystallography methods that provide atomic level structures of individual proteins and protein-RNA complexes, but these approaches are difficult for large multi-component complexes. To bridge the gap between these two groups of structural methods, we propose this interdisciplinary R21 developmental project that is focused on using single molecule microscopy methods to study interactions between proteins and viral RNA. We will directly visualize a viral RNA-dependent RNA polymerase replicating a viral genome, determine where viral and host factors bind to the viral RNA, and investigate interactions between viral proteins in the context of being tethered to a membrane. This project is innovative in that it merges established single molecule methods with known protein-RNA interactions to directly visualize viral genome replication and the formation of protein-RNA complexes that are essential for virus growth. If successful, the work will open the door to a vast new set of methods that can be used to study viral RNA replication and replication center structures.

描述(申请人提供)：正链RNA病毒复制它们的基因组，并在大的膜锚定的“复制中心”复合体的背景下组装病毒粒子，其分子结构本身就很难研究。电子显微镜和断层扫描以及高分辨率光学显微镜的研究清楚地表明，感染细胞中发生了大规模的膜重排，病毒和宿主蛋白定位于这些膜上，但它们的分辨率不足以确定特定的分子间接触。光谱的另一端是核磁共振和结晶学方法，它们提供单个蛋白质和蛋白质-RNA复合体的原子级结构，但这些方法对大型多组分复合体来说是困难的。为了弥合这两组结构方法之间的差距，我们提出了这个跨学科的R21开发项目，重点是使用单分子显微镜方法来研究蛋白质和病毒RNA之间的相互作用。我们将直接可视化复制病毒基因组的依赖于病毒RNA的RNA聚合酶，确定病毒和宿主因子与病毒RNA结合的位置，并在被拴在膜上的背景下研究病毒蛋白质之间的相互作用。该项目的创新之处在于，它将已建立的单分子方法与已知的蛋白质-RNA相互作用结合起来，直接可视化病毒基因组复制和对病毒生长至关重要的蛋白质-RNA复合体的形成。如果成功，这项工作将打开一扇巨大的大门 一套可用于研究病毒RNA复制和复制中心结构的新方法。