RNA silencing suppression mechanisms of Triticum mosaic virus P1: dsRNA binding property and mapping functional motifs

摘要

Triticum mosaic virus (TriMV) is the exemplar strain of the type species of the genus Poacevirus in the family Potyviridae infecting wheat in the Great Plains region of the USA. Previously, we reported that the P1 protein of TriMV is a viral suppressor of RNA silencing. Mutational analyses of P1 showed that deletion of 55 N-terminal amino acids, and a single amino acid at the C-terminus retained its ability to suppress ssGFP-induced RNA silencing. These data suggest that the N-terminal region but not the C-terminal region of P1 is flexible for suppression of RNA silencing activity. Computational analyses revealed that TriMV P1 contains LXK/RA and zinc finger motifs at the N-terminal region and a domain containing the GW motif at the C-terminal region. Mutational analysis of TriMV P1 suggested functional roles for these motifs in suppression of RNA silencing. Electrophoretic mobility shift assays with bacterially expressed P1 protein revealed that P1 binds to 180-nt and 21- and 24-nt ds-siRNAs derived from green fluorescent protein sequence. Additionally, TriMV P1 protected the 655-nt long dsRNA derived from TriMV coat protein from dicing by the human Dicer enzyme into siRNAs. Disruption of the GW motif in TriMV P1 with a W332A mutation abolished silencing suppression, pathogenicity enhancement and viability of TriMV, suggesting a functional role for the GW motif in suppression of RNA silencing.