Summary auto-generated
This study demonstrates the de novo generation of defective interfering (DI) RNA molecules from cymbidium ringspot virus (CyRSV) without requiring pre-existing DI RNA templates. Researchers inoculated Nicotiana clevelandii plants with infectious CyRSV RNA synthesized in vitro and performed serial passages through sap inoculation. Small RNA species (400-700 nucleotides) appeared by the third passage, which were identified as deletion mutants of the genomic RNA. Sequence analysis of six distinct DI RNA types revealed they originate from progressive deletions of three conserved genomic RNA blocks. The longest DI RNA (679 nt) contained three blocks derived entirely from genomic RNA with two major deletions. Smaller DI RNAs showed progressive reduction through additional deletions within these blocks. Computer-generated secondary structure analysis revealed that recombination sites were located within single-stranded sequences in interior or hairpin loops surrounded by stable secondary structures. The authors propose that DI RNA formation results from discontinuous RNA polymerase synthesis triggered by stable secondary structures that impede transcription, followed by template switching to initiation sites with conserved nucleotide sequences. This mechanism explains how DI RNAs evolve from larger to smaller molecules through a series of sequential deletions during viral replication.
Key findings
- De novo generation of six distinct DI RNA molecules (404-679 nt) from CyRSV genomic RNA without pre-existing DI RNA templates
- Progressive reduction in DI RNA size occurs through sequential deletions within conserved genomic RNA blocks, with smaller molecules contained within larger ones
- Recombination sites are located in single-stranded loops surrounded by stable secondary structures with negative free energy
- Conserved initiation sequences (GA-rich motifs) at recombination sites facilitate polymerase switching and template jumping during discontinuous RNA synthesis
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Abstract
Dipartimento di Protezione delle Piante dalle Malattie, Università degli Studi and Centro di Studio del CNR sui Virus e le Virosi delle Colture Mediterranee, Via Amendola 165/A, 70126 Bari, Italy
Nicotiana clevelandii plants were inoculated with cymbidium ringspot tombusvirus RNA synthesized in vitro, after which further passages were made by sap inoculation. During the third passage, low Mr RNA species appeared which had the characteristics of deletion mutants of genomic RNA. Sequence analysis of several of these defective interfering RNAs suggested a possible evolution of smaller from larger molecules. Computer-generated secondary structures of sequences surrounding recombination sites were extensive and stable and these sites occurred in interior or hairpin loops, thus providing a possible explanation for discontinuous RNA transcription and the formation of deletions in genomic RNA.
† Permanent address: Agricultural Biotechnology Center, P.O. Box 170, H-2101 Gödöllö, Hungary.