Recognition of the core RNA promoter for minus-strand RNA synthesis by thereplicases of Brome mosaic virus and Cucumber mosaic virus

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the Bromovirus and Cucumovir us genera have a tRNA-like structure at the 3' end of their genomic RNAs th at interacts with the replicase and is required for minus-strand synthesis. In Brome mosaic vints (BMV), a stem-loop structure named C (SLC) is presen t within the tRNA-like region and is required for replicase binding and ini tiation of RNA synthesis in vitro. We have prepared an enriched replicase f raction from tobacco plants infected with the Fny isolate of Cucumber mosai c virus (Fny-CMV) that will direct synthesis from exogenously added templat es. Using this replicase, we demonstrate that the SLC-like structure in Fny -CMV plays a role similar to that of BMV SLC in interacting with the CMV re plicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5'CA3' dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are cri tical for interaction with the replicase. In the context of the entire tRNA -like element, both CMV SLC-like motifs are recognized by the BMV replicase . However, neither motif can direct synthesis by the BMV replicase in the a bsence of other tRNA-like elements, indicating that other features of the C MV tRNA can induce promoter recognition by a heterologous replicase.