Imaging viral RNA genome replication at the single molecule level

描述（由申请人提供）：正链RNA病毒在大型膜锚定的“复制中心”复合体中复制其基因组并组装病毒粒子，这些复合体的分子组织本质上非常难以研究。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，在感染细胞中发生了大量的膜重排，病毒和宿主蛋白在这些膜上定位，但它们的分辨率不够高，无法确定特定的分子间接触。在光谱的另一端是核磁共振和晶体学方法，提供单个蛋白质和蛋白质- rna复合物的原子水平结构，但这些方法很难用于大型多组分复合物。为了弥补这两组结构方法之间的差距，我们提出了这个跨学科的R21发展项目，重点是使用单分子显微镜方法研究蛋白质与病毒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系在膜上的情况下的相互作用。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蛋白质- rna复合物的形成，这对病毒生长至关重要。如果成功，这项工作将打开广阔的大门

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.