Enhancer-like properties of an RNA element that modulates tombusvirus RNA accumulation

Prototypical defective interfering (DI) RNAs of the plus-strand RNA virus t omato bushy stunt virus contain four noncontiguous segments (regions I-IV) derived from the viral genome. Region I corresponds to 5'-noncoding sequenc e, regions II and III are derived from internal positions, and region IV re presents a 3'-terminal segment We analyzed the internally located region II I in a prototypical DI RNA to understand better its role in DI RNA accumula tion. Our results indicate that (1) region III is not essential for DI RNA accumulation, but molecules that lack it accumulate at significantly reduce d levels (similar to 10-fold lower), (2) region III is able to function at different positions and in opposite orientations, (3) a single copy of regi on III is favored over multiple copies, (4) the stimulatory effect observed on DI RNA accumulation is not due to region Ill-mediated RNA stabilization , (5) DI RNAs lacking region III permit the efficient accumulation of head- to-tail dimers and are less effective at suppressing helper RNA accumulatio n, and (6) negative-strand accumulation is also significantly depressed for DI RNAs lacking region III. Collectively, these results support a role for region III as an enhancer-like element that facilitates DI RNA replication . A scanning-type mutagenesis strategy was used to define portions of regio n III important for its stimulatory effect on DI RNA accumulation. Interest ingly, the results revealed several differences in the requirements for act ivity when region III was in the forward versus the reverse orientation. In the context of the viral genome, region III was found to be essential for biological activity. This latter finding defines a critical role for this e lement in the reproductive cycle of the virus, (C) 1999 Academic Press.