An RNA domain within the 5 ' untranslated region of the tomato bushy stuntvirus genome modulates viral RNA replication

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in vira l RNA replication. Computer-aided thermodynamic analysis of secondary struc ture, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a promi nent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5 ' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a m odel subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20- fold. Compensatory-type mutational analysis of S1 within this replicon reve aled a strong correlation between formation of the predicted S1 structure a nd efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disru pted Sis, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was si gnificantly more detrimental to DI RNA accumulation than (-)-strand destabi lization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mut ant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5' terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role fo r the TSD, and in particular its S1 sub-element, in tombusvirus RNA replica tion. (C) 2001 Academic Press.