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 their propensity to accumulate defective interfering RNAs (DIs) upon serial passage through experimental Nicotiana species. Hallmarks of this occurrence include reduced levels of helper RNA and protein accumulation and amelioration of the lethal necrosis induced upon infection of the host with the helper viruses alone. The objective of this study was to determine whether the prolific trans-accumulation of defective RNAs typically occurs for all replicase-deficient TBSV mutants, or if this process is influenced by internal cis-acting elements that have been excised from DIs. For this purpose, various replicase-deficient TBSV cDNA constructs were generated and their transcripts were tested for trans-accumulation competence in the presence of helper virus. The results revealed that a region of ca. 150 nucleotides near the center of the replicase gene, with a predicted high degree of secondary structure, was a potent inhibitor of trans-rescue (ITR) by TBSV. Relocation of the ITR into efficiently trans-replicating DIs inhibited their accumulation drastically, but only when inserted in the reverse orientation and with an intact 5' ITR-specific predicted hairpin structure. Insertion of the ITR element in the positive orientation yielded DI transcripts that were able to replicate, but failed to interfere noticeably with either accumulation of the helper RNA or the onset of the lethal necrosis phenotype in N. benthamiana. In conclusion, the ITR has an intrinsic capacity to inhibit trans-accumulation of defective RNAs, but its stringency and biological effects are strongly influenced by the overall sequence context.