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Papers In Press, published online ahead of print May 3, 2004
Biology, York University, Toronto, Ontario M3J 1P3
Corresponding Author: kawhite{at}yorku.ca
Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the family Tombusviridae. The (+)-strand RNA genome of TBSV lacks both a 5’ cap and a 3’ poly(A) tail and instead contains a 3’-terminal RNA sequence that acts as a cap-independent translational enhancer (3’CITE). In this study, we have determined the RNA secondary structure of the translation-specific central segment of the 3’CITE, termed region 3.5 (R3.5). MFOLD structural modeling combined with solution structure mapping and comparative sequence analysis indicate that R3.5 adopts a branched structure that contains three major helices. Deletion and substitution studies revealed that two of these extended stem-loop (SL) structures are essential for 3’CITE activity in vivo. In particular, the terminal loop of one of these SLs, SL-B, was found to be critical for translation. Compensatory mutational analysis showed that SL-B functions by base-pairing with another SL, SL3, in the 5’ untranslated region (UTR) of the TBSV genome. Thus, efficient translation of TBSV mRNA in vivo requires a 5’-3’ RNA-RNA interaction that effectively circularizes the message. Similar types of interactions are also predicted to occur in TBSV subgenomic mRNAs between their 5’ UTRs and the 3’CITE, and both genomic and subgenomic 5’-3’ interactions are well conserved in all members of the genus Tombusvirus. In addition, a survey of other genera in Tombusviridae revealed the potential for similar 5’-3’ RNA-RNA-based interactions in their viral mRNAs, suggesting that this mechanism extends throughout this large virus family.
J. Biol. Chem, 10.1074/jbc.M401272200
Submitted on February 4, 2004
Revised on April 21, 2004
Accepted on May 3, 2004
5'-3' RNA-RNA interaction facilitates cap- and poly(A) tail-independent translation of tomato bushy stunt virus mRNA: A potential common mechanism for tombusviridae
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