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.
A large number of diseases are caused by viruses that have RNA genomes, including poliomyelitis, West Nile and Dengue fevers, hepatitis, and food poisoning by noroviruses. These viruses replicate their genomes with a unique RNA-dependent RNA polymerase enzyme that is not found in human cells. The focus of this pilot research project is to use state of the art microscopy techniques to image single molecules of the viral polymerase as it makes new copies of the virus genome. If successful, these experiments will lay the groundwork for expanded studies to directly visualize where viral and human proteins interact with the virus genome during infection and how we may interfere with these interactions to limit virus infections.
|Krapf, Diego; Campagnola, Grace; Nepal, Kanti et al. (2016) Strange kinetics of bulk-mediated diffusion on lipid bilayers. Phys Chem Chem Phys 18:12633-41|
|Campagnola, Grace; Nepal, Kanti; Schroder, Bryce W et al. (2015) Superdiffusive motion of membrane-targeting C2 domains. Sci Rep 5:17721|