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

基本信息

批准号：
8828553
负责人：
Olve Breien Peersen
金额：
$ 18.16万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8828553
关键词：
Affect Architecture Binding Binding Sites Biochemistry Biophysics Cells Complex Crystallography Data Dengue Development Diffusion Disease Docking Electron Microscopy Environment Enzymes Food Poisoning Genome Growth Health 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 research study single molecule two-dimensional viral RNA

项目摘要

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（由申请人提供）：正链RNA病毒在大型膜锚定“复制中心”复合体的背景下复制其基因组并组装病毒粒子，其分子组织本质上很难研究。电子显微镜和断层扫描以及高分辨率光学显微镜研究清楚地显示了受感染细胞中发生的大规模膜重排以及病毒和宿主蛋白在这些膜上的定位，但它们的分辨率不足以定义特定的分子间接触。另一方面，核磁共振和晶体学方法可以提供单个蛋白质和蛋白质-RNA 复合物的原子级结构，但这些方法对于大型多组分复合物来说很困难。为了弥合这两类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，该项目的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病毒RNA依赖性RNA聚合酶，确定病毒和宿主因子与病毒RNA结合的位置，并研究病毒蛋白在被束缚在膜上的情况下之间的相互作用。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 相互作用相结合，以直接可视化病毒基因组复制和病毒生长所必需的蛋白质-RNA 复合物的形成。如果成功的话，这项工作将为广阔的领域打开大门。一组可用于研究病毒 RNA 复制和复制中心结构的新方法。