Cucumber mosaic virus-associated RNA 5. IX. The overtaking of viral RNA synthesis by CARNA 5 and dsCARNA 5 in tobacco.

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.