Evidence that ORF 1 and 2 are the only virus-encoded replicase genes of cymbidium ringspot tombusvirus.

The 33-kDa and its 92-kDa readthrough protein genes (open reading frames (ORFs) 1 and 2) of cymbidium ringspot tombusvirus (CyRSV) were introduced into Nicotiana benthamiana plants. Protoplasts derived from transgenic plants expressing the first two ORFs were inoculated with in vitro transcripts of defective interfering (DI) RNA. The efficient replication of input DI RNA, which is incapable of autonomous replication, confirmed the biological activity of the expressed viral replicase. In addition, in vitro transcripts of a genomic RNA deletion mutant clone encoding only ORFs 1 and 2 were able to replicate on their own and could support the replication of CyRSV DI RNAs in N. benthamiana protoplasts.