The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.The regulatory systems of gene expression mediated by sequence-specific RNA silencing are mechanisms conserved in a wide variety of eukaryotic organisms (50). These pathways are triggered by double-stranded RNA (dsRNA), which is recognized by Dicer-like RNases to produce short dsRNAs of 21 to 26 nucleotides (nt) in length (small interfering RNAs [siRNAs]) (3, 32). One strand of these small RNAs is then incorporated into different silencing effector complexes, guiding them, by sequence complementarity, to degrade mRNA, inhibit RNA translation, or interfere with transcription by chromatin rearrangements (47).Different RNA silencing pathways have been identified, and one of them, active in the cytoplasm, has been shown to play an antiviral role in eukaryotes, where viruses generate dsRNA tracts in replicative intermediates, highly structured mRNAs, or other RNA molecules as a consequence of the action of cellular RNA-dependent RNA polymerases (10, 56). In plants, fungi, and Caenorhabditis elegans at least, this is a non-cellautonomous process, which spreads to remote cells or tissues (57, 59). The systemic silencing signal has still not been identified, although the sequence specificity of this pathway suggests the involvement of a nucleic acid component (19,34).To cou...