Inducible yeast system for viral RNA recombination reveals requirement for an RNA replication signal on both parental RNAs

To facilitate RNA recombination studies, we tested whether Saccharomyces cerevisiae, which supports brome mosaic virus (BMV) replication, also supports BMV RNA recombination. Yeast strains expressing BW RNA replication proteins la and 2a(pol) were engineered to transiently coexpress two independently inducible, overlapping, nonreplicating derivatives of BW genomic RNA3. B3 Delta 3' lacked the coat protein gene and negative-strand RNA promoter. B3 Delta 5' lacked the positive-strand RNA promoter and had the coat gene replaced by the selectable UR Delta 3 gene. After 12 to 72 h of induction, B3 Delta 3' and B3 Delta 5' transcription was repressed and Ura(+) yeast cells were selected. All Ura(+) cells contained recombinant RNA3 replicons expressing URA3. Most replicons arose by intermolecular homologous recombination between B3 Delta 3' and B3 Delta 5'. Such recombinants were isolated only when la and 2a(pol) were expressed and after transient transcription of both B3 Delta 3' and B3 Delta 5', showing that recombination occurred at the RNA, not DNA, level. A minority of URA3-expressing replicons were derived from B3 Delta 5', independently of B3 Delta 3', by 5' truncation and modification, generating novel positive-strand promoters and demonstrating that BW can give rise to subgenomic RNA replicons. Intermolecular B3 Delta 3'-B3 Delta 5' recombination occurred only when both parental RNAs bore a functional, cis-acting template recognition and recruitment element targeting viral RNAs to replication complexes. The results imply that recombination occurred in RNA replication complexes to which parental RNAs were independently recruited. Moreover, the ability to obtain intermolecular recombinants at precisely measurable, reproducible frequencies, to control genetic background and induction conditions, and other features of this system will facilitate further studies of virus and host functions in RNA recombination.