Partial Purification and Characterization of Cucumber necrosis virus and Tomato bushy stunt virus RNA-Dependent RNA Polymerases: Similarities and Differences in Template Usage between Tombusvirus and Carmovirus RNA-Dependent RNA Polymerases

AbstractTombusviruses are small, plus-sense, single-stranded RNA viruses of plants. RNA-dependent RNA polymerases (RdRp) of two tombusviruses, Tomato bushy stunt virus (TBSV) and Cucumber necrosis virus (CNV), have been partially purified from infected Nicotiana benthamiana plants. The obtained RdRp complexes are capable of de novo initiation of complementary RNA synthesis using either plus- or minus-strand templates derived from tombusvirus defective interfering (DI) RNAs. In addition to template-sized products, shorter than full-length products were also generated efficiently apparently because of internal initiation of RNA synthesis by the tombusvirus RdRp. This property could be important for the formation of DI RNAs that are observed in tombusvirus infections. The tombusvirus RdRp is also able to use heterologous RNAs derived from satellite RNAs associated with Turnip crinkle virus (TCV) as templates. Generation of full-length, complementary RNA by the tombusvirus RdRp suggests that it can correctly and efficiently recognize the heterologous TCV-specific promoters. Reduced generation of a 3′-terminal extension product in the preceding assay suggests that the previously characterized replication enhancer present in sat-RNA C (Nagy et al., 1999, EMBO J. 18, 5653–5665) does not stimulate tombusvirus RdRp activity. Taken together, these results suggest that template usage by the tombusvirus and carmovirus RdRps are similar, but not identical