Analysis of intermolecular RNA–RNA recombination by rubella virus

To investigate whether rubella virus (RUB) undergoes intermolecular RNA–RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5′-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3′-proximal structural protein (SP) ORF, and maintained the 3′ end of the genome, including the putative 3′ cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3′ half of the genome including the SP-ORF and 3′ CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA–RNA recombination. Using transcripts tagged with a 3′-terminal deletion, it was found that recombinants contained the 3′ end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3′ CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3′ CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3′ end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating selection pressure against duplications in other regions of the genome.