In vitro synthesis of double-stranded RNA by carnation cryptic virus-associated RNA-dependent RNA polymerase

Partially purified carnation cryptic virus (CarCV) preparations possessed RNA-dependent RNA polymerase activity which was absent in comparable preparations from virus-free carnations. Enzyme activity was dependent upon the presence of virus particles, Mg2+, and the four ribonucleoside triphosphates, and was insensitive to inhibitors of DNA-dependent RNA polymerases. The 32P-labeled enzyme reaction products were largely dsRNAs as indicated by resistance to S1 nuclease and RNase A at high but not low ionic strength. The in vitro synthesized dsRNAs hybridized specifically with CarCV genomic dsRNAs, and the radioactive products present in the polymerase reaction mixture sedimented with the virus particles in sucrose density gradients. The data suggest that the RNA-dependent RNA polymerase associated with CarCV particles is a replicase which catalyzes the synthesis of copies of the genomic dsRNAs.