A novel strategy for creating recombinant infectious RNA virus genomes

Reverse transcriptases with RNase H activity are particularly apt to switch templates and generate recombinant molecules in vitro. This property has been exploited for the first time to create a library of recombinant RNAs 3 between two strains of Cucumber mosaic virus (CMV) or between CMV and Tomato aspermy virus (TAV), which share 75 and 63% sequence identity, respectively. The recombination events were almost entirely of the precise homologous type, and occurred at the same sites as those previously identified in co-infected plants, making it possible to use this strategy to create numerous cDNA fragments with crossovers similar to those occurring in vivo. Sub-cloning of recombinant fragments into an infectious full-length clone was accomplished by homologous recombination in yeast, alleviating the need for in vitro ligation at common restriction sites. Most of the recombinant genomes were infectious. Association of these two methods constitutes an efficient and practical means for generating numerous infectious viral genomes equivalent to ones that might arise by precise homologous recombination between two parental viral genomes in nature.