THE ROLE OF TOMBUSVIRUS REPLICASE PROTEINS AND RNA IN REPLICASE ASSEMBLY, REPLICATION AND RECOMBINATION

Tombusviruses are single, positive strand RNA viruses of plants, often associated with parasitic defective interfering (DI) RNAs. Two viral- coded gene products, namely p33 and p92, are required for tombusvirus replication. The overlapping domains of p33 and p92 contain an arginine/proline-rich (RPR) RNA binding motif. In this study, the role of RPR motif and viral RNA in tombusvirus replication and recombination, as well as involvement of viral RNA in tombusvirus replicase assembly was examined. Using site-directed mutagenesis I generated a series of RPR mutants of Cucumber necrosis tombusvirus (CNV). Analysis of RPR mutants defined that wild type RPR motif, especially two of the four arginines, were required for efficient RNA binding in vitro, for replication of tombusviruses, their associated DI RNAs, subgenomic (sg)RNA synthesis and DI RNA recombination in vivo. Experiments using a two-component tombusvirus replication system showed that RPR motif is critical for functions of both p33 and p92 in replication, but its role in these proteins might not be identical. Recombination studies using a novel tombusvirus three-component system revealed that mutations in RPR motif of p33 replicase protein resulted in an altered viral RNA recombination rate. Identified DI RNA recombinants were mostly imprecise, with recombination sites clustered around a replication enchancer and an additional putative cis-acting element that might facilitate the template switching events by the tombusvirus replicase. To study the role of RNA during the assembly of functional tombusvirus replicase, recombinant CNV replicase that showed similar properties to plant-derived CNV replicase was purified from Saccharomyces cerevisiae. When in addition to p33 and p92 proteins DI RNA was co-expressed in yeast cells, the isolated replicase activity was increased ~40 fold. Further studies defined RNA motifs within two short DI RNA regions that enhanced active CNV replicase formation. In summary, this study showed that the conserved RNA binding motif of the tombusvirus replicase proteins and viral RNA are involved in replicase assembly, viral RNA replication, subgenomic RNA synthesis and RNA recombination. This data shed new light on the complex roles of the viral elements in replication, and will help future studies aimed at interfering with viral infections