Polymerase-Membrane Interactions in Viral RNA Replication Complex Assembly.

Abstract

All characterized positive-strand RNA viruses associate with host intracellular membranes to facilitate viral genome RNA replication, yet the molecular mechanisms required for these essential interactions are poorly understood. To study positive-strand RNA virus replication complex assembly and function I used the established model alphanodavirus, Flock House virus (FHV). FHV establishes robust viral RNA replication in the budding yeast Saccharomyces cerevisiae where it assembles functional viral RNA replication complexes on the mitochondrial outer membrane. Using this powerful host-pathogen system I took complementary in vitro, genetic, and biochemical approaches to understand the polymerase-membrane interactions necessary for replication complex assembly and function. To investigate the initial steps in replication complex assembly, I established an in vitro FHV replicase membrane association assay with which we were able to recapitulate many of the in vivo characteristics of FHV biology in vitro. We found the FHV replicase to be a lipid-binding protein that associated with the mitochondrial outer membrane in a TOM complex independent manner via specificity for mitochondrial-enriched anionic phospholipids. In addition, we preformed a targeted genomic analysis to address the role of yeast mitochondrial outer membrane components in FHV RNA replication. We identified a deletion of the mitochondrial outer membrane protein Mim1 that led to a significant reduction in FHV RNA accumulation, and subsequent biochemical studies revealed a role for Mim1 in FHV replication complex assembly. Finally, we have begun to explore the composition and structure of FHV RNA replication complexes. We have developed a blue-native agarose gel electrophoresis system to identify the presence of two functionally distinct FHV RNA replication complexes in wildtype yeast. Furthermore, we have employed a lipidomic approach to investigate dynamic cellular and membrane-specific phospholipid changes associated with FHV infection and RNA replication. Thus far, we have identified significant FHV induced changes in global and membrane-specific phospholipid levels further implicating an important role for phospholipids in viral RNA replication. Taken together, this thesis provides significant and novel advances in the field of positive-strand RNA virus biology and identifies new avenues of focus for the development of anti-viral therapies targeted towards these viral pathogens.