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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.
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Journal of General Virology vol. 91 , part 9, pp. 2393–2401
Supplementary Fig. S1. Systemic necrosis and death of an N. benthamiana plant inoculated with TBSV L plus satRNA L.
Supplementary Fig. S2. Northern blot analysis of satRNA L using a probe complementary to the 3′-terminal region or to the complete negative strand.
Supplementary Fig. S3. Positions of the 5′ or 3′ termini in the smaller-than-unit-length satRNA L molecules extracted from plants co-inoculated with TBSV L and grown at 24 or 15 °C.
Supplementary Fig. S4. Northern blot analysis of RNA extracts from protoplasts co-inoculated with satRNA L and selected CymRSV–CIRV hybrid clones with decreasing amounts of the 5′ portion of the CymRSV genome fused to the CIRV genome, CymRSV or CIRV.
Supplementary Fig. S5. Northern blot analysis of RNA extracts from plants co-inoculated with satRNA L and CIRV C80 or wild-type CIRV.
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