The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication

Two of the five viral-coded proteins of tombusviruses, which are small, nonsegmented, plus-stranded RNA viruses of plants, are required for replication in infected cells. These replicase proteins, namely, p33 and p92, of cucumber necrosis virus are expressed directly from the genomic RNA via a readthrough mechanism. Their overlapping domains contain an arginine/proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). Site-directed mutagenesis of p33 expressed in Escherichia coli, followed by a gel shift assay, defined two of the four arginines as required for efficient RNA binding in vitro. In vivo testing of 19 RPR motif mutants revealed that the RPR motif, and therefore the ability to bind RNA, is important for the replication of tombusviruses and their associated defective interfering (DI) RNAs. Mutation within the RPR motif also affected the ratio of subgenomic versus genomic RNAs in infected cells. To test whether the RPR motif is essential for the function of either p33 or p92 in replication, we used a two-component system developed by Oster et al. (1998, J. Virol. 5845-5851), in which p92 was expressed from the genomic RNA of a tombusvirus, while p33 was expressed from a DI RNA. The protoplast experiments with the two-component system revealed that the RPR motif is essential for the replication function of both proteins. Interestingly, mutations within the RPR motif of p33 and p92 had different effects on RNA replication, suggesting different roles for the RNA-binding motifs of these proteins in tombusvirus replication.