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-dependentRNA polymerases

Tombusviruses are small, plus-sense, single-stranded RNA viruses of plants, RNA-dependent RNA polymerases (RdRp) of two tombusviruses, Tomato bushy st unt virus (TBSV) and Cucumber necrosis virus (CNV), have been partially pur ified from infected Nicotiana benthamiana plants. The obtained RdRp complex es are capable of de novo initiation of complementary RNA synthesis using e ither plus- or minus-strand templates derived from tombusvirus defective in terfering (DI) RNAs. In addition to template-sized products, shorter than f ull-length products were also generated efficiently apparently because of i nternal initiation of RNA synthesis by the tombusvirus RdRp. This property could be important for the formation of pi RNAs that are observed in tombus virus infections. The tombusvirus RdRp is also able to use heterologous RNA s 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 heterolo gous TCV-specific promoters. Reduced generation of a 3'-terminal extension product in the preceding assay suggests that the previously characterized r eplication enhancer present in sat-RNA C (Nagy et ai., 1999, EMBO J. 18, 56 53-5665) does not stimulate tombusvirus RdRp activity Taken together, these results suggest that template usage by the tombusvirus and carmovirus RdRp s are similar. but not identical. (C) 2000 Academic Press.