The potato virus X (PVX) system is an excellent model for understanding general mechanisms of RNA replication and potentially for designing novel antiviral strategies. PVX has a relatively small genome from which only a few subgenomic RNAs are produced. Both a tobacco protoplast replication system and a soluble template-dependent extract will be used to answer four questions. Which cis-acting sequences and structures affect PVX RNA accumulation in vitro? Sequences and structures that are important for synthesis of plus-and minus-strand RNAs will be determined by analysis of RNA accumulation in tobacco protoplasts inoculated with wild-type and mutant PVX transcripts. What are the template requirements for PVX RNA synthesis in vitro? The PVX template-dependent in vitro transcription system will be optimized and utilized to define template requirements for minus-and plus-strand RNA synthesis. What are the protein constituents and protein-protein interactions required for PVX RNA synthesis in vivo? The protein constituents required for PVX RNA synthesis in vitro will be purified and sequenced. Corresponding cDNA clones obtained by sequence analysis or from a yeast two-hybrid library screen will be used to assess protein-protein interactions required for RNA synthesis. Do host and/or viral proteins interact with regulatory elements in PVX RNA? The extent of binding of proteins from uninfected and infected plant or protoplast extracts to wild-type and mutant PVX transcripts will be determined by gel shift and immunoprecipitation assays. Purified binding proteins will be isolated by affinity chromatography for sequencing. Further analyses of RNA-protein interactions will utilize the cDNAs and proteins corresponding to sequenced proteins. Data obtained from these specific aims will define the components and mechanisms involved in RNA synthesis.
