Independent replication of cowpea mosaic virus bottom component RNA: in vivo instability of the viral RNAs.
We compared the accumulation of cowpea mosaic virus (CPMV) RNAs after inoculation of cowpea and Chenopodium quinoa protoplasts with bottom (B) or middle (M) component virions alone, or with a mixture of the M and B components. RNA extracted from infected protoplasts was subjected to quantitative spot hybridization after electrophoresis and blotting. Compared to the complete M plus B mixture, the B inoculum induced (1) a reduced rate and extent of accumulation of B-RNA of both the (+) and (-) polarities concomitant with a balance between synthesis and degradation and (2) B-RNA that decreased in amount after further RNA synthesis was prevented by administration of cordycepin. In contrast, the (-) B-RNA apparently was stable, possibly sequestered in double-stranded form. M-component-inoculated cowpea protoplasts were subsequently inoculated with B component at intervals, and, after further incubation, RNA was analyzed by electrophoresis and hybridization. Rescue of the ability to form M-RNAs was lost when the interval between inoculations was greater than 17 hr, although the protoplasts remained sensitive to the complete inoculum. The data suggest that both of the genomic RNAs are unstable in protoplasts when inoculated alone, i.e., under conditions that leave viral RNA unprotected by coat protein.