Subgenomic mRNA regulation by a distal RNA element in a (+)-strand RNA virus

Subgenomic (sg) mRNAs are synthesized by (+)-strand RNA viruses to allow fo r efficient translation of products encoded 3' in their genomes, This strat egy also provides a means for regulating the expression of such products vi a modulation of sg mRNA accumulation. We have studied the mechanism by whic h sg mRNAs levels are controlled in tomato bushy stunt virus, a small (+)-s trand RNA virus which synthesizes two sg mRNAs during infections. Neither t he viral capsid nor movement proteins were found to play any significant ro le in modulating the accumulation levels of either sg mRNA, Deletion analys is did, however, identify a 12-nt-long RNA sequence located approximately 1 ,000 nt upstream from the site of initiation of sg mRNA2 synthesis that was required specifically for accumulation of sg mRNA2. Further analysis revea led a potential base-pairing interaction between this sequence and a sequen ce located just 5' to the site of initiation for sg mRNA2 synthesis. Mutant genomes in which this interaction was either disrupted or maintained were analyzed and the results indicated a positive correlation between the predi cted stability of the base-pairing interaction and the efficiency of sg mRN A2 accumulation. The functional significance of the long-distance interacti on was further supported by phylogenetic sequence analysis which revealed c onservation of base-pairing interactions of similar stability and relative position in the genomes of different tombusviruses. It is proposed that the upstream sequence represents a cis-acting RNA element which facilitates sg mRNA accumulation by promoting efficient synthesis of sg mRNA2 via a long- distance RNA-RNA interaction.