Mapping sequences active in homologous RNA recombination in brome mosaic virus: Prediction of recombination hot spots

The mechanism of homologous recombination has been studied previously in brome mosaic virus (BMV), a tricomponent, positive-stranded RNA virus of plants, by using artificial sequences (reviewed by J. J. Bujarski and P. D. Nagy (1996). Semin. Virol. 7, 363-372). Here we extend these studies over BMV-derived sequences to obtain clues on prediction of homologous recombination hot spots. First, mismatch mutations, which reduced the AU content, were introduced into the common 60-nt recombination hot-spot sequence, either in the RNA2 or in both RNA2 and RNA3 components. This decreased the frequency of targeted homologous RNA2/RNA3 recombination and changed the distribution of junction sites. Second, several short BMV RNA1- or RNA2-derived sequences were introduced into the RNA3 component, and homologous recombination activity of these sequences was compared with that observed for previously characterized artificial sequences. Third, sequences at homologous recombinant junctions were compared among a large number of targeted and nontargeted recombinants. All these studies revealed several factors important for homologous recombination including the length of sequence identity, the extent of sequence identity, the AU content of the common sequences, the relative position of the AU-rich segment vs a GC-rich segment, and the presence of GC-rich sequences. Based on this novel model, we suggest that recombination hot spots can be predicted by means of RNA sequence analysis. In addition, we show that recombination can occur between positive and negative strands of BMV RNAs. This provides further clues toward the mechanism of recombination processes in BMV.