Replication of peanut stunt virus and its associated RNA 5 in cowpea protoplasts

Peanut stunt virus (PSV) RNA containing PSV-associated RNA 5 (PARNA 5) was used as the inoculum in the successful infection of cowpea protoplasts. Total nucleic acid extracts of protoplast samples at different times after inoculation were analyzed for the presence of PSV genomic RNAs and PARNA 5 using glyoxal denaturation, agarose gel electrophoresis, blotting to nitrocellulose, and hybridization to specific probes. It appears that (+)-stranded PSV genomic RNAs are synthesized up to 36 hr after inoculation after which their synthesis levels off, whereas PARNA 5 synthesis continues much later during infection. Oligomers of PARNA 5 were found in the infected protoplasts, in double-stranded RNA preparations from PSV-infected tissues, and in single-stranded PSV-RNA preparations isolated from purified virus. However, we were unable to demonstrate the presence of circular PARNA 5 molecules in infected protoplasts or tissues. These results leave open the question whether PARNA 5 is replicated via a rolling circle type replication mechanism, as proposed for viroids and tobacco ringspot virus satellite, or via a virus-like replication mechanism, as certain structural features of PARNA 5 would indicate. It is not impossible that both types of mechanisms are operative at different phases of PARNA 5 replication.