Efficient RNA silencing suppression activity of Potato Mop-Top Virus 8K protein is driven by variability and positive selection

Previously, we investigated the evolution of Potato mop-top virus (PMTV) ORFs. Results indicate that positive selection acts exclusively on an ORF encoding the 8K protein, a weak viral suppressor of RNA silencing (VSR). However, how the extraordinary variability contributes to 8K-mediated RNA silencing suppression remains unknown. Here, we characterized the RNA silencing suppression activity of the 8K protein from seven diverse isolates. We show that 8K encoded by isolate P1 exhibits stronger RNA silencing suppression activity than the 8K protein from six other isolates. Mutational analyses revealed that Ser-50 is critical for these differences. By comparing small RNA profiles we found a lower abundance of siRNAs with U residue at the 5'-terminus after expression of the P1 8K compared to expression of 8K from isolate P125, an isolate with weak VSR activity. These results provide new clues as to the role of positive selection in shaping activities of VSRs.