A conserved RNA structure is essential for a satellite RNA-mediated inhibition of helper virus accumulation.

As a class of parasitic, non-coding RNAs, satellite RNAs (satRNAs) have to compete with their helper virus for limited amounts of viral and/or host resources for efficient replication, by which they usually reduce viral accumulation and symptom expression. Here, we report a cucumber mosaic virus (CMV)-associated satRNA (sat-T1) that ameliorated CMV-induced symptoms, accompanied with a significant reduction in the accumulation of viral genomic RNAs 1 and 2, which encode components of the viral replicase. Intrans replication assays suggest that the reduced accumulation is the outcome of replication competition. The structural basis of sat-T1 responsible for the inhibition of viral RNA accumulation was determined to be a three-way branched secondary structure that contains two biologically important hairpins. One is indispensable for the helper virus inhibition, and the other engages in formation of a tertiary pseudoknot structure that is essential for sat-T1 survival. The secondary structure containing the pseudoknot is the first RNA element with a biological phenotype experimentally identified in CMV satRNAs, and it is structurally conserved in most CMV satRNAs. Thus, this may be a generic method for CMV satRNAs to inhibit the accumulation of the helper virus via the newly-identified RNA structure.

Functional significance of a hepta nucleotide motif present at the junction of Cucumber mosaic virus satellite RNA multimers in helper-virus dependent replication.

Satellite RNAs (satRNA) associated with Cucumber mosaic virus (CMV) have been shown to generate multimers during replication. We have discovered that multimers of a CMV satRNA generated in the absence of its helper virus (HV) are characterized by the addition of a hepta nucleotide motif (HNM) at the monomer junctions. Here, we evaluated the functional significance of HNM in HV-dependent replication by ectopically expressing wild type and mutant forms of satRNA multimers in planta either in (+) or (-)-strand polarity. Comparative replication profiles revealed that (-)-strand multimers with complementary HNM (cHNM) are the preferred initial templates for HV-dependent replication than (-)-strand monomers and multimers lacking the cHNM. Further mutational analyses of the HNM accentuate that preservation of the sequence and native length of HNM is obligatory for efficient replication of satRNA. A model implicating the significance of HNM in HV-dependent production of monomeric and multimeric forms of satRNA is presented.

Regulation of a virus-induced lethal disease in tomato revealed by LongSAGE analysis.

Infection of Cucumber mosaic virus (CMV) and D satellite RNA (satRNA) in tomato plants induces rapid plant death, which has caused catastrophic crop losses. We conducted long serial analysis of gene expression (LongSAGE) in control and virus-infected plants to identify the genes that may be involved in the development of this lethal tomato disease. The transcriptomes were compared between mock-inoculated plants and plants infected with CMV, CMV/D satRNA, or CMV/Dm satRNA (a nonnecrogenic mutant of D satRNA with three mutated nucleotides). The analysis revealed both general and specific changes in the tomato transcriptome after infection with these viruses. A massive transcriptional difference of approximately 400 genes was found between the transcriptomes of CMV/D and CMV/Dm satRNA-infected plants. Particularly, the Long-SAGE data indicated the activation of ethylene synthesis and signaling by CMV/D satRNA infection. Results from inoculation tests with an ethylene-insensitive mutant and treatments with an ethylene action inhibitor further confirmed the role of ethylene in mediating the epinastic leaf symptoms and the secondary cell death in the stem. Results from Northern blot analysis demonstrated the partial contribution of ethylene in the induced defense responses in CMV/D satRNA-infected plants.

DCL4 targets Cucumber mosaic virus satellite RNA at novel secondary structures.

It has been reported that plant virus-derived small interfering RNAs (vsiRNAs) originated predominantly from structured single-stranded viral RNA of a positive single-stranded RNA virus replicating in the cytoplasm and from the nuclear stem-loop 35S leader RNA of a double-stranded DNA (dsDNA) virus. Increasing lines of evidence have also shown that hierarchical actions of plant Dicer-like (DCL) proteins are required in the biogenesis process of small RNAs, and DCL4 is the primary producer of vsiRNAs. However, the structures of such single-stranded viral RNA that can be recognized by DCLs remain unknown. In an attempt to determine these structures, we have cloned siRNAs derived from the satellite RNA (satRNA) of Cucumber mosaic virus (CMV-satRNA) and studied the relationship between satRNA-derived siRNAs (satsiRNAs) and satRNA secondary structure. satsiRNAs were confirmed to be derived from single-stranded satRNA and are primarily 21 (64.7%) or 22 (22%) nucleotides (nt) in length. The most frequently cloned positive-strand satsiRNAs were found to derive from novel hairpins that differ from the structure of known DCL substrates, miRNA and siRNA precursors, which are prevalent stem-loop-shaped or dsRNAs. DCL4 was shown to be the primary producer of satsiRNAs. In the absence of DCL4, only 22-nt satsiRNAs were detected. Our results suggest that DCL4 is capable of accessing flexibly structured single-stranded RNA substrates (preferably T-shaped hairpins) to produce satsiRNAs. This result reveals that viral RNA of diverse structures may stimulate antiviral DCL activities in plant cells.

[Studies on construction of artificial mutants of Cucumber mosaic virus satellite RNA and their biological activity].

Based on the full length cDNA clone of a Cucumber mosaic virus satellite RNA, which was 369nt in size, artificial mutants were developed by the method of error-prone PCR and DNA shuffling. The new satellite cDNAs were transcribed in vitro into ssRNA and pseudo-recombined with a helper Cucumber mosaic virus, which contains no satellite RNA. Sequence analysis showed that A to T/G or G to A replacement all the four mutants, named MS1, MS5, MS6 and MS11 respectively, and there is no C to G or G to C replacement, but amongst, only the mutants MS11 could replicated when recombined with the helper virus strain. No satellite RNA could be detected by RT-PCR amplification and double-stranded RNA analysis for those pseudo-recombination constitution of Cucumber mosaic virus strain with mutants MS1, MS5 and MS6.Sequence homological comparison showed that the single replacement of mutants MS1, MS5 and MS6 occurred in the highly conservative regions and the T to A replacement of mutant MS11 was located in the normal-variation region. This is the first artificial mutation of satellite RNA of plant RNA viruses. The results indicated that single base in the region of satellite RNA maybe important to maintaining the biological activity of satellite RNA for its replication and stability. The variation and evolution of satellite RNA could be hopefully studied through combination directed evolution by DNA shuffling with pseudo-recombination in vitro.

Circular dichroism studies of CMV-D and CMV-S: two strains of cucumber mosaic cucumovirus with a different biological behaviour.

Cucumber mosaic cucumovirus is a plant virus in which a typical satellite RNA system is present, displaying a dualistic biological behaviour. In fact, it has been shown that satRNA is able either to aggravate or attenuate the viral disease symptomatology with a modulating capability going from death of the host plant to a surprising absence of symptoms. D-satRNA and S-satRNA have been considered the prototype necrogenic and non necrogenic satRNAs respectively. On the basis of circular dichroism spectroscopy, it is suggested that the different biological behaviours can be explained by taking into account the different capabilities exerted by S- and D-satRNAs in inducing structuring effects onto CMV-S and CMV-D genomic RNAs.

Structural analysis of a necrogenic strain of cucumber mosaic cucumovirus satellite RNA in planta.

Structural studies of plant viral RNA molecules have been based on in vitro chemical and enzymatic modification. That approach, along with mutational analysis, has proven valuable in predicting structural models for some plant viruses such as tobacco mosaic tobamovirus and brome mosaic bromovirus. However, in planta conditions may be dramatically different from those found in vitro. In this study we analyzed the structure of cucumber mosaic cucumovirus satellite RNA (sat RNA) strain D4 in vivo and compared it to the structures found in vitro and in purified virions. Following a methodology developed to determine the structure of 18S rRNA within intact plant tissues, different patterns of adenosine and cytosine modification were found for D4-sat RNA molecules in vivo, in vitro, and in virions. This chemical probing procedure identifies adenosine and cytosine residues located in unpaired regions of the RNA molecules. Methylation data, a genetic algorithm in the STAR RNA folding program, and sequence alignment comparisons of 78 satellite CMV RNA sequences were used to identify several helical regions located at the 5' and 3' ends of the RNA molecule. Data from previous mutational and sequence comparison studies between satellite RNA strains inducing necrosis in tomato plants and those strains not inducing necrosis allowed us to identify one helix and two tetraloop regions correlating with the necrogenicity syndrome.

Contribution of mutation and RNA recombination to the evolution of a plant pathogenic RNA.

The nucleotide sequence of 17 variants of the satellite RNA of cucumber mosaic virus (CMV-satRNA) isolated from field-infected tomato plants in the springs of 1989, 1990, and 1991 was determined. The sequence of each of the 17 satRNAs was unique and was between 334 and 340 nucleotides in length; 57 positions were polymorphic. There was much genetic divergence, ranging from 0.006 to 0.141 nucleotide substitutions per site for pairwise comparisons, and averaging 0.074 for any pair. When the polymorphic positions were analyzed relative to a secondary structure model proposed for CMV-satRNAs, it was found that there were significantly different numbers of changes in base-paired and non-base-paired positions, and that mutations that did not disrupt base pairing were preferred at the putatively paired sites. This supports the concept that the need to maintain a functional structure may limit genetic divergence of CMV-satRNA. Phylogenetic analyses showed that the 17 CMV-satRNA variants clustered into two subgroups, I and II, and evolutionary lines proceeding by the sequential accumulation of mutations were apparent. Three satRNA variants were outliers for these two phylogenetic groups. They were shown to be recombinants of subgroup I and II satRNAs by calculating phylogenies for different molecular regions and by using Sawyer's test for gene conversion. At least two recombination events were required to produce these three recombinant satRNAs. Thus, recombinants were found to be frequent ( approximately 17%) in natural populations of CMV-satRNA, and recombination may make an important contribution to the generation of new variants. To our knowledge this is the first report of data allowing the frequency of recombinant isolates in natural populations of an RNA replicon to be estimated.