New defective RNAs from citrus tristeza virus: evidence for a replicase-driven template switching mechanism in their generation.

Defective RNAs (D-RNAs) ranging in size from 1968 to 2759 nt were detected in four citrus tristeza virus (CTV) isolates by hybridization of electroblotted dsRNAs with two probes specific for the 5'- and 3'-terminal genomic regions. The RNAs that hybridized with both probes were eluted, cloned and sequenced. Comparison with the sequences of the corresponding genomic regions of the helper virus showed, in all cases, over 99% nucleotide identity and direct repeats of 4-5 nt flanking or in the vicinity of the junction sites. The presence of the repeats from two separate genome locations suggests a replicase-driven template switching mechanism for the generation of these CTV D-RNAs. Two of the CTV isolates that differed greatly in their pathogenicity contained an identical D-RNA, suggesting that it is unlikely that this D-RNA is involved in symptom modulation, which may be caused by another factor.