Complete replication of a satellite RNA in vitro by a purified RNA-dependent RNA polymerase

The 334 nucleotide R satellite RNA was used as a template for purified RNA-dependent RNA polymerase (RdRp) from cucumber mosaic virus-infected tobacco plants. The products of the reaction were dsRNA and positive-strand RNA of the same size as the R satellite RNA. Similar products were obtained when T7 RNA polymerase positive-strand transcripts of a cDNA clone of the satellite RNA, designed to have the same 5' and 3' ends as the satellite RNA, were used as templates. The formation of the positive strands demonstrates complete replication of the satellite RNA. A positive-strand transcript with 65 and 255 additional nucleotides at the 5' and 3' ends of the satellite RNA respectively was also utilized as a template by the RdRp, but only dsRNA was formed. However, no products could be detected when the RdRp was programmed with transcripts corresponding to the negative-strand satellite RNA, either with no additional terminal nucleotides or with 24 and 310 additional nucleotides at the 5' and 3' ends respectively.