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Cymbidium ringspot virus (CymRSV) satellite RNA (satRNA) is a parasitic subviral RNA replicon that replicates and accumulates at the cost of its helper virus. This 621-nucleotide (nt) satRNA species has no sequence similarity to the helper virus, except for a 51-nt-long region termed the helper-satellite homology (HSH) region, which is essential for satRNA replication. We show that the accumulation of satRNA strongly depends on temperature and on the presence of the helper virus p19 silencing suppressor protein, suggesting that RNA silencing plays a crucial role in satRNA accumulation. We also demonstrate that another member of the Tombusvirus genus, Carnation Italian ringspot virus (CIRV), supports satRNA accumulation at a higher level than CymRSV. Our results suggest that short interfering RNA (siRNA) derived from CymRSV targets satRNA more efficiently than siRNA from CIRV, possibly because of the higher sequence similarity between the HSH regions of the helper and CIRV satRNAs. RNA silencing sensor RNA carrying the putative satRNA target site in the HSH region was efficiently cleaved when transiently expressed in CymRSV-infected plants but not in CIRV-infected plants. Strikingly, replacing the CymRSV HSH box2 sequence with that of CIRV restores satRNA accumulation both at 24 degrees C and in the absence of the p19 suppressor protein. These findings demonstrate the extraordinary adaptation of this virus to its host in terms of harnessing the antiviral silencing response of the plant to control the virus parasite satRNA.

RNA silencing is conserved in a broad range of eukaryotes and operates in the development and maintenance of genome integrity in many organisms. Plants have adapted this system for antiviral defense, and plant viruses have in turn developed mechanisms to suppress RNA silencing. RNA silencing-related RNA inactivation is likely based on target RNA cleavage or translational arrest. Although it is widely assumed that virus-induced gene silencing (VIGS) promotes the endonucleolytic cleavage of the viral RNA genome, this popular assumption has never been tested experimentally. Here we analyzed the viral RNA targeting by VIGS in tombusvirus-infected plants, and we show evidence that antiviral response of VIGS is based on viral RNA cleavage by RNA-induced silencing effector complex (RISC) programmed by virus-specific small interfering RNAs (siRNAs). In addition, we found that the RISC-mediated cleavages do not occur randomly on the viral genome. Indeed, sequence analysis of cloned cleavage products identified hot spots for target RNA cleavage, and the regions of specific RISC-mediated cleavages are asymmetrically distributed along the positive- and negative-sense viral RNA strands. In addition, we identified viral siRNAs containing high-molecular-mass protein complexes purified from the recovery leaves of the silencing suppressor mutant virus-infected plants. Strikingly, these large nucleoproteins cofractionated with microRNA-containing complexes, suggesting that these nucleoproteins are silencing related effector complexes.

Two plasmids from which the sequences coding for the 36- and 95-kDa proteins of Carnation Italian ringspot virus (CIRV) could be transcribed in vivo in the yeast Saccharomyces cerevisiae under the control of the ADH1 promoter and terminator were constructed. The two proteins, which constitute the viral replicase, were correctly translated and integrated into membranes of the yeast cells. An additional plasmid was introduced in yeasts expressing the CIRV replicase, from which a defective interfering (DI) RNA (DI-7 RNA) could be transcribed under the control of the GAL1 promoter and terminated by the Tobacco ringspot virus satellite ribozyme, which cleaved 19 nucleotides downstream of the 3' end of DI RNA. The DI-7 RNA transcripts were amplified by the viral replicase as demonstrated by the restoration of the authentic 3' end, the requirement of a specific cis-acting signal at this terminus, the preferential accumulation of molecules with the authentic 5' terminus (AGAAA), the synthesis of head-to-tail dimers, the presence of negative strands, and the incorporation of 5-bromo-UTP. Additionally, transformation with a dimeric construct of DI-7 RNA led to the synthesis of monomers, mimicking the activity of the viral replicase in plant cells.