title: Analysis of intermolecular RNA-RNA recombination by rubella virus.
abstract: 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.
authors.affiliation: Department of Biology, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Sandra D
authors.initials: SD
authors.lastname: Adams
authors.forename: Wen-Pin
authors.initials: WP
authors.lastname: Tzeng
authors.forename: Min-Hsin
authors.initials: MH
authors.lastname: Chen
authors.forename: Teryl K
authors.initials: TK
authors.lastname: Frey
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D012412:Rubella virus / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014709:Vero Cells
chemical_list: D020413:3' Untranslated Regions; D012367:RNA, Viral