RNA recombination between cucumoviruses: possible role of predicted stem-loop structures and an internal subgenomic promoter-like motif.

We previously analyzed hybrids of Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) that contained CMV RNA2 with the 3'-terminal sequence from TAV RNA2. In this article, we scrutinized the RNA3 molecules in these hybrid viruses by Northern hybridization and RT-PCR and found some recombinant CMV RNA3 molecules and various recombinant RNA4 molecules whose 3'-termini were derived from TAV RNA1 or 2. Sequence analyses revealed that most of the crossover sites for recombination were located near putative stem-loop structures and an internal subgenomic promoter-like motif. We inoculated in vitro transcripts synthesized from cDNA clones of the recombinant RNA3 onto N. benthamiana along with either CMV RNA1 and 2 or TAV RNA1 and 2. Although all of the hybrids were infectious, many sequence deletions and nucleotide substitutions were found when RNA1 and 2 from TAV were used, which suggests that fidelity of TAV replicase was lower than that of CMV replicase. The possible role of secondary structures and an internal subgenomic promoter-like motif in RNA recombination is discussed.