5' sequences of rubella virus RNA stimulate translation of chimeric RNAs and specifically interact with two host-encoded proteins.
Sequences at the 5' and 3' ends of the rubella virus (RV) genomic RNA can potentially form stable stem-loop (SL) structures that are postulated to be involved in virus replication. We have analyzed the function of these putative SL structures in RNA translation by constructing chimeric chloramphenicol acetyltransferase (CAT) RNAs, flanked either by both 5'- and 3'-terminal sequence domains from the RV genome or several deletion derivatives of the same sequences. After in vitro transcription of chimeric RNAs, the translational efficiencies of these RNAs were compared by the rabbit reticulocyte lysate translation system. For in vivo translation studies, the level of CAT activity was measured for chimeric RV/CAT RNAs expressed in transfected cells by the adenovirus major late promoter. Both in vivo and in vitro translation activities of the chimeric RNAs revealed that the presence of 5' and 3' SL sequences of RV RNA, in correct (+) orientation and context [5'(+)SL and 3'(+)SL, respectively] was necessary for efficient translation of chimeric RV/CAT RNAs. The presence of the RV 5'(+)SL sequence had the primary enhancing effect on translation. To identify host proteins which interact with the 5'(+)SL which may be involved in RV RNA translation, RNA gel-shift and UV cross-linking assays were employed. Two host proteins 59 and 52 kDa in size, present in cytosolic extracts from both uninfected and RV-infected cells, specifically interacted with the RV 5'(+)SL RNA. Direct binding comparisons between wild-type and mutant 5'(+)SL RNAs demonstrated that sequences in and around the bulge region of the terminal stem domain of this structure constituted a protein binding determinant. Human serum, qualified for anti-Ro/SS-A antigen specificity, immunoprecipitated 59- and 52-kDa protein-RNA complexes containing the RV 5'(+)SL RNA. However, poly- and monoclonal antisera raised against the recombinant 60- and 52-kDa Ro proteins failed to precipitate complexes containing the 5'(+)SL RNA. The identity of the proteins binding this RV cis-acting element remains to be determined; however, their role in RV translation is discussed.
Pogue
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GP
Laboratory of Molecular Pharmacology, Food and Drug Administration, Bethesda, Maryland 20892.
Cao
X Q
XQ
Singh
N K
NK
Nakhasi
H L
HL
D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000187:drug effects*; D012313:RNA / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics / Q000494:pharmacology*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D012412:Rubella virus / Q000235:genetics*; D017384:Sequence Deletion; D014709:Vero Cells
D012367:RNA, Viral; C039919:RNA, recombinant; D016601:RNA-Binding Proteins; D012313:RNA