RNA directed RNA synthesis is essential for survival of many procaryotic and eucaryotic RNA viruses. a large percentage of eucaryotic viruses are plus-strand RNA viruses that replicate in the cytoplasm of host cells, and many of these cause diseases that are of medical and agricultural significance. Despite their importance, only a few animal and plant viruses have been studied in terms of replication and very little is known about the replication mechanisms. Potato virus X (PVX) is an excellent model system for understanding the virus/host interactions necessary for RNA replication and disease development. This virus contains one genomic RNA that is functionally monocistronic. The viral replicase, P1, is the only viral protein required for RNA synthesis, and potentially encodes several activities required for replication. Thus, PVX P1 is an excellent model for a replication module that evolved as a coordinated entity. PVX is useful for biochemical and genetic studies because the virus replicates well in tobacco plants and protoplasts, and infectious transcripts can be generated form a PVX cDNA clone for mutational analyses. This system will be used to determine the RNA/RNA and RNA/protein interactions associated with PVX RNA synthesis, the extent of host protein involvement in replication, and the multifunctional nature of P1 by addressing three questions. What are the cis-acting regulatory sequences and structures necessary for RNA replication? The sequences and structures that are important for synthesis of plus and minus strand RNAs will be determined by introducing deletions at the termini of an infectious, PVX cDNA clone. Transcripts derived from these clones will be inoculated onto tobacco protoplasts and evaluated for synthesis of RNA. The significance of predicted secondary structures in regulatory regions will be determined by analyzing RNA synthesis in protoplasts inoculated with transcripts containing site- directed mutations, and by analysis of structures in solution. Do P1 and/or host proteins interact specifically with PVX RNA? The P1 RNA binding domain and the precise region of PVX RNA that is bound by P1 or host proteins will be determined. The functional significance of the P1 RNA binding domain to replication will be analyzed by mutational analysis in protoplasts. Which biochemical reactions necessary for replication are catalyzed by P1 or P1-containing extracts? P1 could function as an RNA binding protein, RNA dependent RNA polymerase, helicase and capping enzyme. Infected tobacco extracts and P1 or portions of P1 isolated from E. coli or insect cells will be assayed for activities related to each of these functions. These studies will form a basis for future research on analysis of P1 functional domains, host protein functions, interactions required for replication complex formation, potential P1/host membrane interactions, and the development of an in vitro replication system to define mechanisms of RNA replication.
