NONHOMOLOGOUS RNA RECOMBINATION IN TOMBUSVIRUSES - GENERATION AND EVOLUTION OF DEFECTIVE INTERFERING RNAS BY STEPWISE DELETIONS

We used a protoplast system to study the mechanisms involved in the ge neration and evolution of defective interfering (DI) RNAs of tomato bu shy stunt tombusvirus (TBSV). Synthetic transcripts corresponding to d ifferent naturally occurring TBSV DI RNAs, or to various artificially constructed TBSV defective RNAs, were analyzed. The relative levels of competitiveness of different DI RNAs were determined by coinoculating their corresponding transcripts into protoplasts along with helper ge nomic RNA transcripts and monitoring the level of DI RNA accumulation. Further studies were performed to assess the contribution of naked DI RNA stability and DI RNA encapsidation efficiency to the observed lev els of competitiveness. In addition, the ability of various defective RNAs to evolve to alternative forms was tested by serially passaging p rotoplast infections initiated with transcripts corresponding to helpe r genomic RNA and a single type of defective RNA. These studies, and t he analysis of the sequences of observed recombinants, indicate that ( i) replication competence is a major factor dictating DI RNA competiti veness and is likely a primary determinant in DI RNA evolution, (ii) D I RNAs are capable of evolving to both smaller and larger forms, and t he rates at which various transitions occur differ, (iii) DI RNA-DI RN A recombination and/or rearrangement is responsible for the formation of the evolved RNA molecules which were examined, and (iv) sequence co mplementarities between positive- and negative-sense strands in the re gions of the junctions suggest that, in some cases, base pairing betwe en an incomplete replicase-associated nascent strand and acceptor temp late may mediate selection of recombination sites. On the basis of our data, we propose a stepwise deletion model to describe the temporal o rder of events leading to the formation of tombusvirus DI RNAs.