Viral pathogenesis is intimately linked with dynamic and complex host-pathogen interactions. The mechanisms underlying these essential interactions, in particular those shared by viruses with similar genomic structures, represent attractive potential targets for antiviral drugs. Viruses that contain a positive-sense single-stranded RNA genome, such as hepatitis C virus, West Nile virus, and the SARS coronavirus, represent a diverse group of pathogens responsible for significant human diseases for which few effective therapies exist. Despite the varied clinical syndromes caused by these viruses, all characterized positive-strand RNA viruses use intracellular membranes for viral RNA replication complex formation and function. However, the mechanisms whereby viral RNA replication complexes assemble on specific intracellular membranes are not well understood. The long-term objectives of this project are to elucidate the host-pathogen interactions that facilitate positive-strand RNA virus replication complex assembly and function. A detailed understanding of these interactions will provide insight into the mechanisms of viral pathogenesis, and potentially identify novel targets for broadly effective antiviral drugs. The specific focus of this proposal is to define the early events in viral RNA replication complex assembly. The targeting, transport, and initial interactions of virus-encoded RNA replication complex proteins with intracellular membranes are essential steps in replication complex assembly, and therefore are important determinants of viral pathogenesis. The general strategy of the proposed research is to use Flock house virus, an established and versatile model used to study positive-strand RNA virus structure and replication, to investigate the mechanisms of viral RNA replication complex assembly. Biochemical, molecular, and genetic approaches will be used to investigate the targeting and transport of the Flock house virus RNA-dependent RNA polymerase to intracellular membranes.
The specific aims of this proposal are designed to accomplish two goals: 1) understand the role of cellular chaperone proteins in Flock house virus RNA replication complex assembly;and 2) define the membrane receptor responsible for Flock house virus RNA replication complex intracellular localization.
|Castorena, Kathryn M; Stapleford, Kenneth A; Miller, David J (2010) Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication. BMC Genomics 11:183|
|Weeks, Spencer A; Shield, William P; Sahi, Chandan et al. (2010) A targeted analysis of cellular chaperones reveals contrasting roles for heat shock protein 70 in flock house virus RNA replication. J Virol 84:330-9|
|Stapleford, Kenneth A; Rapaport, Doron; Miller, David J (2009) Mitochondrion-enriched anionic phospholipids facilitate flock house virus RNA polymerase membrane association. J Virol 83:4498-507|
|Weeks, Spencer A; Miller, David J (2008) The heat shock protein 70 cochaperone YDJ1 is required for efficient membrane-specific flock house virus RNA replication complex assembly and function in Saccharomyces cerevisiae. J Virol 82:2004-12|
|Castorena, Kathryn M; Weeks, Spencer A; Stapleford, Kenneth A et al. (2007) A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells. J Virol 81:8412-20|