Topology of poliovirus RNA replication machinery
Rossignol, Evan Daniel
MetadataShow full item record
Viruses are obligate intracellular parasites that replicate utilizing the resources of host cells. The replication of positive sense RNA viruses is coupled with alterations to host cell membranes. These viruses are believed to replicate efficiently by using co-opted membrane structures assembled from viral and host cell proteins and lipids. Poliovirus is a prototypical positive-sense RNA virus, however the topological details of viral RNA replication are not well understood. In this work we use electron cryotomography, among other methods, to examine the ultrastructure of fractionated poliovirus RNA replication factories that were formed within infected cells, and to investigate oligomeric interactions within a three dimensional crystal formed by a poliovirus polymerase point mutant. Investigation of the ultrastructure of isolated viral RNA replication factories shows that the low resolution features of cryopreserved membrane structures are essentially identical to previously observed structures within plastic sections of infected cells. Furthermore, greater detail visible using electron cryotomography reveals pore-like structures and other high energy membrane conformations within the replication factories. We see a mix of single, double, and multi-membrane structures that are arranged with openings that connect their interior lumenal space to the exterior environment. The lumen of some of these membranous structures contains a linear polymeric density thought to be RNA. We conclude that the RNA replication of poliovirus may occur on the lumenal surface of vesicular membranes with an opening to the cytoplasm for metabolite and product exchange. Within the poliovirus replication machinery, the principal component is the RNA polymerase 3Dpol. This prototypical RNA-dependent RNA-polymerase forms homo-oligomeric interactions that are key to its functions. To investigate these interactions, previous studies focused on hollow helical structures formed by wild-type polymerase. Here, we investigate the structure of small three-dimensional crystals formed by 3Dpol with a mutation of a single residue, lysine 314, to alanine. Using electron cryotomography and volume averaging, we demonstrate that the crystal packing within this point mutant does not include physiological polymerase-polymerase interactions. Elucidation of the topology of poliovirus replication machinery provides a basis for future development of antiviral therapeutics.