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The complete nucleotide sequence of a cucumber mosaic virus-associated RNA 5 (CARNA 5) that does not induce the tomato necrosis disease (J. M. Kaper, M. E. Tousignant, and S. M. Thompson, Virology 114, 526-533, 1981) has been determined and compared with the known sequence of a necrosis-inducing CARNA 5 (K. E. Richards, G. Jonard, M. Jacquemond, and H. Lot, Virology 89, 395-408, 1978). The nonnecrotic satellite RNA, (1)CARNA 5, is one nucleotide smaller yet 93% homologous with (n)CARNA 5, the necrosis-inducing satellite RNA. (1)CARNA 5 differs from (n)CARNA 5 by 21 substitutions, 3 deletions, and 2 insertions, with more changes in the 3' half than in the 5' half of the molecule. (1)CARNA 5, like (n)CARNA 5, is capped at its 5' terminus; the nucleotide sequences predict that putative translational products of the two RNAs could be significantly different.

Cucumber mosaic virus (CMV) and CMV-associated RNA 5 (CARNA 5)-related RNA synthesis was monitored under conditions of semisynchronous infection using a differential temperature inoculation technique (Dawson and Schlegel, 1973). Leaf strips sampled at specific intervals between 0 and 100 hr after temperature shift were vacuum infiltrated with [32P]phosphoric acid and actinomycin D and incubated during 4-hr periods. Total nucleic acid extracts were analyzed on polyacrylamide gels to compare the relative rates of 32P incorporation into CMV-RNA 3, CARNA 5, and dsCARNA 5. During the first 24 hr there was a rapid increase in the rate of 32P incorporation into all three RNAs. During the next 10-20 hr the rate of 32P incorporation into RNA 3 declined to minimal levels while that of CARNA 5 stayed at a plateau or declined slowly. In contrast, the rate of 32P incorporation into dsCARNA 5 increased steadily well beyond the first 48 hr after temperature shift. The distribution of radioactivity among its (+) and (-) strands was determined by isolating the dsCARNA 5 in each nucleic acid extract obtained from the 4hr-labeled tissues and determining its radioactivity after hybridization in the presence and in the absence of a large excess of unlabeled CARNA 5. It appeared that throughout the 100-hr experiment about 60% of the radioactivity of dsCARNA 5 was in its (+) strands. The rates of 32P incorporation into virus and CARNA 5-related RNAs were also compared in leaf strips from plants inoculated with the genomic CMV-RNAs alone and with a mixture of genomic RNAs and CARNA 5. Although in the infection with the genomic RNAs alone the synthesis of CARNA 5 and dsCARNA 5 was not prevented, the increase in their relative rates of synthesis seemed significantly slower and the relative rate of RNA 3 synthesis much greater than when the inoculum contained detectable CARNA 5.

A new satellite-like replicating RNA found in association with cucumber mosaic virus type 1 (CMV-1), one of the earliest CMV isolates described (Doolittle, 1920), was identified and partially characterized. CMV-1-associated RNA 5, or (1)CARNA 5, was isolated from CMV-1 propagated in tomato, where it failed to induce lethal necrosis. In this way it was distinguished from CARNA 5 with the tomato necrosis-causing ability, which was redesignated (n)CARNA 5. Although (1)CARNA 5 and (n)CARNA 5 could be separated by 7.5% polyacrylamide gel electrophoresis under certain conditions, the molecules are probably identical in size, or nearly so. (1)CARNA 5 and (n)CARNA 5 have only limited nucleotide sequence homology. This is also evident from the different multitransitional absorbance-temperature profiles that can be obtained during the thermal denaturation of their respective double-stranded forms. In mixed infections in tomato, the replication of (1)CARNA 5 was also supported by CMV strains WT or S, while (n)CARNA 5 was supported by CMV-1. The biologically different (1)CARNA 5 and (n)CARNA 5 can be considered sequence variants among a group of small satellite-like RNA molecules dependent upon CMV for their replication.

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.