Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs.
RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.
Giner
Ana
A
Centre for Research in Agricultural Genomics, CRAG, CSIC-IRTA-UAB, Barcelona, Spain.
Lakatos
Lóránt
L
García-Chapa
Meritxell
M
López-Moya
Juan José
JJ
Burgyán
József
J
D020816:Amino Acid Motifs; D017124:Conserved Sequence; D020868:Gene Silencing; D019176:Potyviridae / Q000235:genetics*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000502:physiology*; D034743:RNA-Induced Silencing Complex / Q000037:antagonists & inhibitors*; D012097:Repressor Proteins; D014764:Viral Proteins / Q000502:physiology*
D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D012097:Repressor Proteins; D014764:Viral Proteins