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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.

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.

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.

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.

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.

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.

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.

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.

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.

Cucumber mosaic virus (CMV), a widespread and economically important virus of vegetable crops, often contains a satellite RNA, here designated CARNA-5 (for CMV-associated RNA 5). Viral satellites are small nucleic acids that are sequence-unrelated to, but replicatively dependent upon, the viral genome. They essentially are molecular parasites of their helper viruses, and thereby frequently modulate viral symptom expression. Some isolates of CARNA-5 change normally moderate CMV symptoms in tomato into a lethal disease named tomato necrosis; others ameliorate CMV symptoms in tomato and other important crop plants. Here we report on the identification and molecular characterization of a 334-nucleotide necrogenic CARNA-5 isolated from tomato fields in southern Italy, where a massive outbreak of lethal necrosis occurred in the summer of 1988. This is the first time that direct evidence is given for the involvement of a viral satellite in a crop disease of epidemic scale. The possible molecular interrelationships between plant, virus, satellite and other factors that influence the satellite-induced symptom modulation underlying such a catastrophe are discussed.

The nucleotide sequence of the satellite of arabis mosaic virus was determined using the satellite RNA encapsidated in virions. The 300-nucleotide long sequence showed extensive homology (50%) with that of the 359-nucleotide satellite RNA of tobacco ringspot virus, which occurs both in a linear and a circular form. This homology also revealed the presence of conceived sequences believed to mediate self-cleavage of the latter as well as other viral satellite RNAs. A circular form of the arabis mosaic virus satellite can be isolated from infected tissues and partially converts to the linear form upon elution from denaturing gels.

The double-stranded forms of CARNA 5 and PARNA 5, viral satellites of the cucumovirus group, after denaturation and polyacrylamide gel electrophoresis under appropriate conditions allow their complementary strands to be separated and fractionated. These fractionated (+) and (-) strands are free of contaminating viral RNA fragments. They serve as templates for the preparation of cDNAs of opposite polarity, which are used to probe the viral RNA-dependent replication of CARNA 5 and PARNA 5.

Seven CARNA 5 (Cucumber mosaic virus-Associated RNA 5) variants were obtained from an Indonesian cucumber mosaic virus isolate (designated CMV-In) by serial passage of this virus in squash, Chenopodium quinoa, tobacco, and tomato. Bioassays of the isolated CARNA 5 variants for their capability to induce lethal necrosis in tomato allowed categorization of the CARNA 5 variants as either necrogenic or nonnecrogenic. Comparison of the CARNA 5 sequences with sequences previously published shows that in general CMV satellites display a high degree of nucleotide sequence conservation. Except for two sharply defined domains of variability, the sequences of the necrogenic CARNA 5s are essentially conserved, whereas those of the nonnecrogenic CARNA 5s possess at least nine additional hypervariable domains. In eight positions of the CARNA 5 sequences all necrogenic variants differ uniquely from those that are nonnecrogenic. All CMV satellites compared in this work possess open reading frames (ORFs), with equivalent initiation codons, capable of encoding polypeptides of significant lengths. Two equivalent or identical ORFs were found only in the necrogenic CARNA 5s, whereas a variable-length ORF was found in the sequences of all the CARNA 5 variants compared. These findings constitute a basis for experiments aimed at determining the biological significance, if any, of these ORFs.