Properties of a novel satellite RNA associated with tomato bushy stunt virus infections

The biological and molecular properties of a novel satellite RNA (satRNA L) associated with tomato bushy stunt virus (TBSV) are described. satRNA L consisted of a linear single-stranded RNA of 615 nt, lacked significant open reading frames (ORFs) and had no sequence identity with the helper genome other than in the 5'-proximal 7 nt and in a central region that is also conserved in all tombusvirus genomic, defective interfering and satellite RNAs. Secondary-structure analysis showed the presence of high-order domains similar to those described for other tombusvirus RNAs. Shorter-than-unit-length molecules were shown not to be related to a silencing mechanism. satRNA L did not modify the symptoms induced by TBSV under any of the temperature conditions tested. A full-length cDNA clone was constructed and used in co-inoculations with transcripts of carnation Italian ringspot virus (CIRV) and cymbidium ringspot virus (CymRSV). CIRV, but not CymRSV, supported the replication of satRNA L. Using CIRV-CymRSV hybrid infectious clones, two regions were identified as possible determinants of the different ability to support satRNA L replication. The first region was in the 5'-untranslated region, which folds differently in CymRSV in comparison with CIRV and TBSV; the second region was in the ORF1-encoded protein where a more efficient satRNA L-binding domain is suggested to be present in CIRV.