Evaluation of cis-acting elements in the rubella virus subgenomic RNA that play a role in its translation.
The subgenomic (SG) mRNA of rubella virus (RUB) contains the structural protein open reading frame (SP-ORF) that is translated to produce the three virion structural proteins: capsid (C) and glycoproteins E2 and E1. RUB expression vectors have been developed that express heterologous genes from the SG RNA, including replicons which replace the SP-ORF with a heterologous gene, and these expression vectors are candidate vaccine vectors. In the related alphaviruses, translational enhancing elements have been identified in both the 5' untranslated region (UTR) of the SG RNA and the N-terminal region of the C gene. To optimize expression from RUB vectors, both the 5'UTR of the SG RNA and the C gene were surveyed for translational enhancing elements using both plasmids and replicons expressing reporter genes from the SG RNA. In replicons, the entire 5'UTR was necessary for translation; interestingly, when plasmids were used the 5'UTR was dispensable for optimal translation. The RUB C gene contains a predicted long stem-loop starting 62 nts downstream from the initiation codon (SLL) that has a structure and stability similar to SL's found in the C genes of two alphaviruses, Sindbis virus (SIN) and Semliki Forest virus, that have been shown to enhance translation of the SG RNA in infected cells. However, a series of fusions of various lengths of the N-terminus of the RUB C protein with reporter genes showed that the SLL had an attenuating effect on translation that was overcome by mutagenesis that destabilized the SLL or by adding downstream sequences of the C gene to the fusion. Thus, for optimal expression efficiency from RUB expression vectors, only the 5'UTR of the SG RNA is required. Further investigation of the differing effects of the SLL on RUB and alphavirus SG RNA translation revealed that the SIN and RUB SLLs could enhance translation when expressed from a SIN cytopathic replicon, but not when expressed from a plasmid, a RUB replicon, or a SIN noncytopathic replicon. Thus, the SLL only functions in a "cytopathic environment" in which cell translation has been altered.
Pappas
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CL
Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA.
Tzeng
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Frey
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TK
D000818:Animals; D002522:Chlorocebus aethiops; D016679:Genome, Viral; D014176:Protein Biosynthesis / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D012412:Rubella virus / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014709:Vero Cells
D012367:RNA, Viral