Role of RNA secondary structures in tombusvirus subgenomic mRNA transcriptio

Tomato bushy stunt virus (TBSV) is a messenger-sensed, single-stranded (ss) RNA plant virus. Its RNA genome is ∼4.8 kilobasepairs in length and encodes five viral proteins. Proteins encoded at the 5' end of the genome are translated directly from this message, whereas proteins encoded near the 3' end of the genome are translated from two viral subgenomic (sg) mRNAs that are transcribed during infection. Sg mRNA transcription is mediated by the formation of long distance RNA-RNA interaction. Base-pairing between a sequence just 5' to the initiation site for sg mRNA transcription (termed receptor sequence, RS) and an upstream sequence (termed activator sequence, AS) acts as a signal for the viral RNA-dependent RNA polymerase to stall during genome replication and generate the (-)-strand RNA template that is used to transcribe sg mRNAs. Different sets of AS/RS interactions are involved in the transcription of sg mRNA1 (i.e. the AS1/RS1 interaction) and sg mRNA2 (AS2/RS2 interaction) and both sets of interaction are essential for transcription of these two sg mRNAs. Although the importance of these interactions has been demonstrated, the possible role of the RNA sequences and structures that surround these RNA elements have not been analyzed. In this study, I have investigated whether the RNA contexts of the AS and RS elements are important for sg mRNA transcription in TBSV. Comparative sequence analysis of related viral tombusvirus species suggested that each of the AS elements is located in the terminal loop of a stem-loop (SL) RNA structure. I hypothesized that this position could aid formation of the AS/RS interactions by presenting the AS elements in the terminal loop in ss RNA form. To investigate the possible importance of the proposed AS-associated SL structure, nucleotide substitutions were introduced into wt and modified viral genomes. These mutants were then inoculated into plant protoplasts and the effect on sg mRNA transcription was assessed by Northern blot analysis of isolated viral RNAs. Using this approach, I was able to show that formation of the AS1 SL structure was important for sg mRNA1 transcription, however because no clear correlation between maintenance of the AS2 SL structure and sg mRNA2 transcription was observed. Therefore, my results indicate that only the AS1 SL is important for efficient sg mRNA transcription.;Comparative RNA sequence analysis of the sequence surrounding RS1 indicated that a SL, called SL1-sg1, is present just 5' to this element. Interestingly, the position of this RNA helix immediately next to RS1 could allow it to coaxially stack with the RNA helix formed by the long-distance AS1/RS1 interaction. I hypothesized that this type of helix-helix interaction could potentially stabilize the AS1/RS1 interaction and facilitate sg mRNA1 transcription. To test this hypothesis mutations were introduced into SL1-sg1 that would either maintain or decrease its stability. When mutant viral genomes containing these modifications were tested in plant protoplasts, a positive correlation between maintenance of SL1-sg1 stability and efficient sg mRNA1 transcription levels was observed. This result supports the concept that SL1-sg1 exists and is important for sg mRNA transcription.;Overall, my results have led to the identification of RNA structure associated with the AS and RS elements that are important for sg mRNA transcription. These findings provide important new details about the role of RNA context in the function of AS and RS elements and extend our understanding of the regulation of sg mRNA transcription in TBSV.