Viral Satellite RNAs for the Prevention of Cucumber Mosaic Virus (CMV) Disease in Field-Grown Pepper and Melon Plants.

A benign viral satellite RNA, in combination with a mild strain of cucumber mosaic virus (CMV-S), was used as a "vaccine" or "preinoculum" to demonstrate the feasibility of protecting pepper (Capsicum annuum cv. California Wonder) and melon (Cucurbita melo cv. Janus des Canaries) against two severe CMV strains, CMV-D and CMV-16, in the final 2 years of a 4-year pilot field and greenhouse experiment. In the field, healthy pepper and melon seedlings challenged with CMV-D and CMV-16 reduced yields by 33 to 60%; CMV-S caused only limited yield reduction in pepper and had no effect on the yield of melon. Different time intervals between preinoculation of pepper and melon seedlings with CMV-S and challenge inoculation with the severe CMV strains were tested. All plants challenged 3 weeks after vaccination showed nearly complete protection from subsequent infection by severe strains. The yield from preinoculated and challenged pepper plants was 80% that of untreated plants, while the yield from preinoculated and challenged melon plants was increased slightly over the untreated control plants. The use of this technology for biological control of plant viruses is discussed.

Cucumber mosaic virus from Ixora: infectious RNA transcripts confirm deficiency in satellite support and unusual symptomatology.

A cucumber mosaic virus isolate from an Ixora plant originating in the Philippines and first described 20 years ago (CMV-Ix) by Waterworth and Povish differs from other characterized CMV strains in its anomalous satellite support and in several biological and molecular properties. We describe the preparation of infectious transcripts from cloned complementary DNA and the characterization of progeny virus. The results confirm that CMV-Ix has a more limited host range than most CMV isolates. Virions of CMV-Ix are smaller than those of the control CMV-S, independent of the type of negative stain used. Furthermore, CMV-Ix from transcripts supports the replication of the satellite T-CARNA 5 but not of D-CARNA 5.

The complete sequence of a cucumber mosaic virus from Ixora that is deficient in the replication of satellite RNAs.

A cucumber mosaic virus (CMV-Ix) from Ixora is unusual in that it does not support the accumulation of some well-characterized CMV satellite RNAs in plants. CMV-Ix can support a particular satellite RNA variant which causes lethal tomato necrosis when inoculated with other CMV strains but not when inoculated with CMV-Ix. This difference in ability to support accumulation of specific satellite variants is apparent even when their sequences differ by only 10 nucleotides. Electroporation of tomato protoplasts with combinations of CMV-Ix or CMV-1 RNA plus the same satellite variants showed similar differences in accumulation, indicating a defect in satellite RNA replication and not movement or encapsidation. Pseudorecombinant virus infections between CMV-1 and CMV-Ix indicated that the genomic determinants responsible for this phenotype reside on RNA 1 since only combinations with CMV-Ix RNA 1 failed to replicate satellite RNA. The complete genome of CMV-Ix was cloned, sequenced and compared with the genomes of other cucumoviruses. CMV-Ix is most similar in RNA and protein sequence to subgroup 1 CMV-Fny and CMV-Y but slightly less similar than they are to each other. CMV-Ix and all cucumovirus strains sequenced thus far share a domain in the 3' untranslated portion of their genomic RNAs in which 39 of 40 bases are completely conserved.

Small satellite of arabis mosaic virus: autolytic processing of in vitro transcripts of (+) and (-) polarity and infectivity of (+) strand transcripts.

In vitro RNA transcripts of both (+) and (-) polarities were obtained from a circularly permuted dimer clone of the small satellite of arabis mosaic virus (sArMV). The transcripts show efficient self-cleavage at the two specific sites in the sequence where the monomers were joined. Autolytic processing of the full-length transcript in both orientations releases promoter-proximate fragments (+) or (-) P, promoter-distal fragments (+) or (-) D, and the monomer fragments (+) or (-) M. The presence of an OH group at their 5' ends and a 2',3' cyclophosphate at their 3' ends suggests that (+) and (-) M originated via two self-cleavage reactions within the full-length transcript of corresponding polarity. Infectivity assays showed that the (+) M fragment but not the (-) M fragment initiates replication as efficiently as the natural linear sArMV in Chenopodium quinoa. Two (-) fragments were identified which are the result of religation activity: a P-D fragment formed by religation of P and D, and c-M, which is the result of efficient self-ligation of (-) M. In contrast, linear (+) M self-ligates in vitro to a very limited extent but could be circularized enzymically in a wheat germ extract.

The replication of a necrogenic cucumber mosaic virus satellite is temperature-sensitive in tomato.

Lethal necrosis development in tomato plants infected with cucumber mosaic virus (CMV) strain D containing the necrogenic satellite D-CARNA 5 and held at 32 degrees C is shown to be impaired. CARNA 5 accumulation in tomato at 32 degrees C is reduced about 100-fold compared to accumulation in plants held at 24 degrees C, while viral RNA accumulation is reduced about 5-fold. CMV-infected tomato held for 3 days at 24 degrees C prior to shift to 32 degrees C do not develop lethal necrosis. Longer incubations at 24 degrees C prior to shift to 32 degrees C allow necrosis to develop. CMV-infected plants held for up to 4 weeks at 32 degrees C required an additional 8-10 days at 24 degrees C to develop necrosis. Necrogenic CMV-infected plants held at 24 degrees C and analyzed 3 days p.i. contained detectable amounts of ss- and ds-CARNA 5; upon shift to 32 degrees C, such CARNA 5 declined to undetectable levels and lethal necrosis did not occur. There appear to be temperature-sensitive factors that are required for efficient satellite replication which are not required for efficient viral RNA replication. Whether these factor(s) are of host or satellite origin is uncertain.

Effect of temperature on cucumber mosaic virus satellite-induced lethal tomato necrosis is helper virus strain dependent.

The effect of temperature on the response of tomato (Lycopersicon esculentum Mill. cv. Rutgers) to infections with the necrogenic cucumber mosaic virus (CMV) satellite D-CARNA 5 was investigated with each of four CMV strains D, 1, Y and S functioning as helper virus. At 24 degrees C lethal necrosis was observed in all infections. However, at 32 degrees C the response varied from total absence or reduction of necrosis with some strains to accelerated lethal necrosis with others. The total lack of necrotic response with CMV-S and the aggravated necrosis with CMV-Y at the higher temperature both turned out to be independent of the coinfecting satellite, and rather to correlate with the changing rate of viral RNA accumulation in tomato, which probably was responsible for the changes in pathogenic response. However, when CMV-D was helper virus, satellite accumulation decreased, while with CMV-1 it increased, respectively, while viral RNA accumulations were not seriously affected. Although these profound effects of temperature seem to link the necrotic response of tomato to the competitive replication dynamics of the infecting virus/satellite combination in the case of CMV-D/D-CARNA 5, temperature effects at other levels of disease induction probably play an important role as well.

Competition of viral and satellite RNAs of cucumber mosaic virus for replication in vitro by viral RNA-dependent RNA polymerase.

Cucumber mosaic virus (CMV) satellite RNA-induced viral symptom modulation is usually accompanied by a significant reduction of virus accumulation in plant tissue, which has led to the hypothesis that satellite RNA competes with the viral RNAs for replication by the viral replicase and thereby reduces viral RNA synthesis and viral symptoms. In this report, the RNA synthesis of the viral and satellite RNAs of CMV was studied in vitro using an RNA-dependent RNA polymerase (RdRp) purified from CMV-infected plants. Comparison of the kinetics of the CMV RdRp-catalysed RNA synthesis using as templates viral RNAs and satellite RNA, alone or in an appropriate mixture, showed that these RNAs competed with each other for RNA synthesis by the CMV RdRp. Determination of the rates of 32P incorporation into the viral and satellite double-stranded RNA products revealed an apparent replication advantage of the satellite RNA over viral RNAs. The results provide strong support for a previously proposed biochemical mechanism that attributes CMV satellite-induced viral symptom modulation to the replication competition between the satellite and viral RNAs.

Requirement of 3'-terminal guanosine in (-)-stranded RNA for in vitro replication of cucumber mosaic virus satellite RNA by viral RNA-dependent RNA polymerase.

The 3' terminus of the (-) RNA strand in the replicative forms of several (+)-stranded RNA viruses possesses an unpaired guanosine with unknown function. This unpaired guanosine is also found at the 3' terminus of the (-) strand in the double-stranded form of two cucumoviral satellite RNAs. Using a cucumber mosaic virus (CMV) RNA-dependent RNA polymerase capable of replicating the satellite RNA in vitro, the 3'-terminal guanosine of the satellite (-) strand was shown to be an absolute requirement for satellite (+) strand synthesis. If genomic RNA synthesis of CMV and other members of the alphavirus-like superfamily that produce (-) strands terminating in an unpaired 3' guanosine follows a similar strategy, the work reported here would represent the first experimental support for the notion of 3'-terminal guanosine functioning as an essential recognition signal for viral replicases, enabling (+) strand RNA synthesis to be initiated internally from a (-) strand template.

Cucumber mosaic virus-associated RNA 5. XIII.--Opposite necrogenicities in tomato of variants with large 5' half insertion/deletion regions.

Two satellite RNA of cucumber mosaic virus (CMV) designated J876-CARNA-5 (for cucumber mosaic virus-associated RNA-5) and D27-CARNA-5 have been molecularly and biologically characterized. J876-CARNA-5 (387 nucleotides (nt)) and D27-CARNA-5 (391 nt) have nearly identical 5' half insertion/deletion regions where 120 nt replace approximately 70 nt of D-CARNA-5 (335 nt), the first variant described and sequenced. J876-CARNA-5 possesses the 15-nt conserved sequence element in its 3' half which is present in all tomato necrogenic variants and induces the same level of necrosis in tomatoes as the prototype necrogenic D-CARNA-5. D27-CARNA-5 lacks the 3' half necrosis-determining element and attenuates the CMV symptoms in tomato. Transcripts of cloned cDNA of J876-CARNA-5 were stably propagated in tomato in the presence of CMV-1. Purified J876-CARNA-5 progeny, inoculated with CMV-1 in a quantitative bioassay, induced tomato necrosis at the same dilution level as the natural satellite. Several computer-generated secondary structures of CMV satellites were examined and the possible correlation of a defined secondary structural element with necrosis induction is discussed.

Tomato necrosis and the 369 nucleotide Y satellite of cucumber mosaic virus: factors affecting satellite biological expression.

To determine which factors can affect biological expression of the Y satellite RNA of cucumber mosaic virus (CMV) in tomato, three laboratories collaboratively exchanged their natural satellite variants, the corresponding recombinant DNA clones and helper virus strains, as well as tomato varieties, on which different observations previously reported were based. The effects of these materials and the influence of temperature on symptom expression were systematically studied. The results show that in a standardized tomato bioassay at 24 degrees C, the Y satellite, when supported by either CMV-1 or CMV-Y, did not induce tomato necrosis in the Rutgers variety but elicited a slower necrotic response in the Best of All variety that was variably lethal, as compared to the faster inevitably lethal response induced by a prototype necrogenic D satellite variant in both tomato varieties. At higher temperatures (26.5 to 32 degrees C) an extremely fast-killing necrosis caused by CMV-Y itself was observed. The study demonstrates that in experiments on virus symptom modulation induced by CMV satellites, the nature of the helper virus, host plant varieties, as well as the environmental conditions should be precisely defined, and the effects of each parameter change determined separately.

Replication of satellite RNA in vitro by homologous and heterologous cucumoviral RNA-dependent RNA polymerases.

The RNA-dependent RNA polymerases (RdRp) of cucumber mosaic virus (CMV) and peanut stunt virus (PSV), members of the cucumovirus group, have been purified from virus infected plants and were used to study RNA synthesis in vitro using different viral RNAs, two cucumoviral satellites, and chimeric satellite cDNA clone transcripts as templates. The results show that solubilized RdRp preparations of CMV and PSV have a high degree of template dependency and catalyze (-) strand synthesis of the homologous cucumoviral RNAs with greater efficiency than the RNAs of heterologous cucumoviruses, although the PSV RdRp exhibits a lesser specificity than the CMV RdRp. On the other hand, both (-) and (+) strands of the satellite RNAs of CMV and PSV are synthesized by their homologous but not by the heterologous viral RdRps, indicating that recognition of satellites by the viral RdRp determines their replicative dependence upon specific helper viruses. Cucumoviral RdRp reactions using chimeric satellite transcripts suggest that the promoter structure for the satellite (-) strand synthesis resides in regions harboring the 3' termini of the two satellites.

Widely separated sequence elements within cucumber mosaic virus satellites contribute to their ability to induce lethal tomato necrosis.

To determine the structural requirements for cucumber mosaic virus (CMV) satellites to elicit lethal tomato necrosis, three satellite variants D, S and Y were used in the construction and cloning of chimeric cDNAs. D and S are necrogenic and non-necrogenic 'prototype' variants, respectively, and Y possesses the 3' conserved necrosis-determining region but does not cause lethal tomato necrosis. Its 5' half harbours an insertion/deletion region that results in a molecule about 30 nucleotides longer than other variants. Tomato bio-assays were conducted with RNA transcripts of all six chimeric combinations of the 5' and 3' halves of the three satellite variants divided by a common restriction site, as well as with a mutated chimera. None of the chimeras containing the 5' half of Y induced lethal necrosis in tomato even when their 3' halves were that of the D variant with the conserved necrogenic element. Chimeras with the 3' half of Y elicited only partial or restricted necrosis which was much less severe than that induced by prototype variant D, and often was not lethal. Site-directed mutation of a single nucleotide in proximity to the necrogenic element of such a chimera containing the 3' half of Y restored much lethal necrogenicity. The results revealed the presence of structural elements in CMV satellite variant Y that modulate or even suppress the expression of the 3' conserved necrosis-determining element. They indicate that in CMV satellites widely separated sequence elements constituting a three-dimensional requirement are responsible for eliciting lethal necrosis in tomato.

Replication footprint analysis of cucumber mosaic virus electroporated into tomato protoplasts.

Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.

Replication of cucumber mosaic virus satellite RNA in vitro by an RNA-dependent RNA polymerase from virus-infected tobacco.

An RNA-dependent RNA polymerase purified from tobacco infected with cucumber mosaic virus catalyzes the synthesis of (-) and (+) strands of the viral satellite RNA, CARNA 5, but fails to replicate the satellite RNA of peanut stunt virus (PSV). The enzyme replicates the genomic RNAs of the three principal cucumoviruses CMV, PSV and tomato aspermy virus (TAV) with varying efficiencies. The specificity with which CMV RdRp replicates different sequence-unrelated RNA templates suggests that the site of their recognition requires secondary or higher level structural organization.

Cucumber-mosaic-virus-associated RNA-5. XII. Symptom-modulating effect is codetermined by the helper virus satellite replication support function.

In tomato, the disease-modulating effects of a cucumber mosaic virus (CMV) satellite isolate from Belgium, here designated T-CARNA-5 (CARNA-5 = CMV-associated RNA-5), were found to be different depending on the supporting helper virus strain. With two CMV strains, T-CARNA-5 induced lethal necrosis, but with a third strain from Ixora spp. (CMV-Ix), aggravated stunting was observed. However, the primary structure of the T-CARNA-5 contained within virus isolated from tobacco or tomato infected with each of these three CMV strains, conformed to the conserved sequence profile of CARNA-5 isolates which are necrogenic in tomato. Dilution endpoint bioassay of T-CARNA-5 established a direct cause-effect relationship between it and tomato necrosis or stunting, depending on the helper virus. Total nucleic acid extracts taken at different times from tomato plants infected with the above CMV strains and T- or S-CARNA-5 (used as non-necrogenic control) showed viral RNA, ssCARNA-5 and dsCARNA-5 to be present in significant amounts, but in sometimes dissimilar proportions depending on the combination; except in CMV-Ix/S-CARNA-5 infection where neither ss-nor dsCARNA-5 was found. The experiments established that CARNA-5 biological expression studies in CMV-infected tomato have to take into account the helper virus satellite replication support function, which may be a primary codeterminant of quantitative or qualitative differences in the symptom modulation observed.

Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus.

The two satellite RNAs associated with CYMV infections were sequenced. The larger (sCYMV-L1) has only linear molecules 1145 nucleotides long, a poly(A) tail, a long open reading frame (ORF) coding for a protein of Mr 39,636 resembling in composition those of other large nepovirus satellite RNAs, a 5' leader sequence of 16 nucleotides and a 3' non-coding region of 40 nucleotides. In vitro translation of sCYMV-L1 yielded a protein product with a size that corresponded to that predicted from the sequence. The smaller satellite (sCYMV-S1) is 457 nucleotides long, has no ORF of significant length and no in vitro messenger activity. Both linear and circular forms of this satellite RNA were detected in infected tissues. Comparison of the sCYMV-S1 primary structure with the sequences of other small nepoviral satellites reveals large regions of homology. Analysis of the secondary structures derived from the sequences of the plus and minus strands suggests possible consensus sequences for their self-cleavage.

Transformed tomato plants express a satellite RNA of cucumber mosaic virus and produce lethal necrosis upon infection with viral RNA.

Tomato plants transformed with a single copy of a tomato necrosis causing satellite RNA of cucumber mosaic virus (CMV) express the satellite sequence, but the plants show no disease symptoms and have a normal appearance. Upon challenge infection of the F1 progeny with a CMV strain free of any detectable encapsidated satellite the plants accumulated single and double-stranded forms of satellite RNA and developed lethal necrosis.