Inducible Yeast System for Viral RNA Recombination Reveals Requirement for an RNA Replication Signal on Both Parental RNAs

Abstract

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 BMV RNA replication proteins 1a and 2a(pol) were engineered to transiently coexpress two independently inducible, overlapping, nonreplicating derivatives of BMV genomic RNA3. B3Delta3' lacked the coat protein gene and negative-strand RNA promoter. B3Delta5' lacked the positive-strand RNA promoter and had the coat gene replaced by the selectable URA3 gene. After 12 to 72 h of induction, B3Delta3' and B3Delta5' 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 B3Delta3' and B3Delta5'. Such recombinants were isolated only when 1a and 2a(pol) were expressed and after transient transcription of both B3Delta3' and B3Delta5', showing that recombination occurred at the RNA, not DNA, level. A minority of URA3-expressing replicons were derived from B3Delta5', independently of B3Delta3', by 5' truncation and modification, generating novel positive-strand promoters and demonstrating that BMV can give rise to subgenomic RNA replicons. Intermolecular B3Delta3'-B3Delta5' 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.