RNA determinants of junction site selection in RNA virus recombinants and defective interfering RNAs

RNA recombination plays an important role in the diversification and evolution of RNA viruses. Most of these events are believed to be mediated by an actively copying viral replicase switching from a donor template to an acceptor template, where it resumes synthesis. In addition, intramolecular replicase-mediated events (i.e., rearrangements) can lead to the generation of replicable deleted forms of a viral genome, termed defective interfering (DI) RNAs. To gain further insight into the recombination process, the effect of various primary and secondary structures on recombination site selection in vivo was examined using plant RNA tombusviruses. The effect of sequence identity and complementarity on deletion events that generate DI RNAs was also investigated. Our results suggest that (1) 5' termini and strong hairpin structures in donor templates represent preferred sites for recombinations, (2) junction sites in acceptor templates do not occur in double-stranded regions, (3) nucleotide homology can shift donor and acceptor recombination sites closer to regions of identity and, (4) both sequence identity and complementarity can direct deletion sites in DI RNAs. These results further define RNA determinants of tombusvirus RNA recombination and rearrangement.