Retention of a small replicase gene segment in Tomato bushy stunt virus defective RNAs inhibits their helper-mediated trans-accumulation

Tomato bushy stunt virus (TBSV) and other tombusviruses are notorious for t heir propensity to accumulate defective interfering RNAs (Dls) upon serial passage through experimental Nicotiana species. Hallmarks of this occurrenc e include reduced levels of helper RNA and protein accumulation and amelior ation of the lethal necrosis induced upon infection of the host with the he lper viruses alone. The objective of this study was to determine whether th e prolific trans-accumulation of defective RNAs typically occurs for all re plicase-deficient TBSV mutants, or if this process is influenced by interna l cis-acting elements that have been excised from Dls. For this purpose, va rious replicase-deficient TBSV cDNA constructs were generated and their tra nscripts were tested for trans-accumulation competence in the presence of h elper virus. The results revealed that a region of ca. 150 nucleotides near the center of the replicase gene, with a predicted high degree of secondar y structure, was a potent inhibitor of trans-rescue (ITR) by TBSV. Relocati on of the ITR into efficiently trans-replicating Dls inhibited their accumu lation drastically, but only when inserted in the reverse orientation and w ith an intact 5' ITR-specific predicted hairpin structure. Insertion of the ITR element in the positive orientation yielded DI transcripts that were a ble to replicate, but failed to interfere noticeably with either accumulati on of the helper RNA or the onset of the lethal necrosis phenotype in N. be nthamiana. In conclusion, the ITR has an intrinsic capacity to inhibit tran s-accumulation of defective RNAs, but its stringency and biological effects are strongly influenced by the overall sequence context. (C) 2001 Academic Press.