VP3 Proteins Suppress RNA Silencing Figure 5. IBDV VP3 is able to functionally replace the HCPro silencing suppressor in a PPV infection. (A) Schematic representation of full-length cDNA clones derived from PPV and employed to infect N. benthamiana biolistically. The coding sequence of the GFP protein inserted between the NIb and CP cistrons is represented with a green rectangle. Black arrows indicate self-cleavages by the corresponding viral proteases, whereas the grey arrow indicates a cleavage in trans by the action of NIaPro. (B) GFP expression pattern of plants infected with the indicated viruses. Pictures of inoculated leaves collected at 7 days post inoculation (dpi), and the forth (+4) and fifth (+5) leaves above the inoculated one and the most apical leaves collected at 22 dpi were taken under an epifluorescence microscope. (C) Western blot analyses of plant tissue showing GFP foci collected at 9 dpi from inoculated leaves (upper panel) and at 22 dpi from upper non-inoculated leaves (+4 and +5 leaves) (lower panel) of two independent infected plants. (D) Western blot analysis of old (O) and young (Y) leaves collected at 38 dpi from two independent infected N. benthamiana plants. A polyclonal serum specific for PPV CP was used for assessment of virus accumulation, whereas immunoreaction with polyclonal sera specific for IBDV VP3 confirmed the identity of the infecting chimera. The membranes stained with Ponceau red showing the Rubisco are included as loading controls. doi:10.1371/journal.pone.0045957.g005 genomes exist in the nature, which might demand additional protective measures to ensure efficient infection. The VP3 protein produced by members of the family Birnaviridae, is known to be involved in forming filamentous ribonucleoproteins that shield genomic dsRNAs [55]. In this report, we demonstrate that the VP3 proteins of several birnaviruses are also able to counteract RNA silencing by preventing formation of siRNAs from dsRNA silencing inducers. Plants have been previously used to identify and study VSRs derived from vertebrate-infecting viruses given that the interpre- tation of results is not distorted by the presence of other dsRNA- related antiviral defences, like the interferon pathway [26,27,40,69]. Thus, we made use of two co-agroinfiltration assays in N. benthamiana plants. In the first assay, RNA silencing is triggered by the sense transcript of the GFP reporter gene, which is converted into dsRNA by a host RdRp, while in the second, RNA silencing is induced by an inverted-repeated construct that directly forms a GFP-specific dsRNA. IBDV VP3 protected GFP mRNA from RNA silencing-mediated degradation in both systems (Fig. 1). We therefore conclude that VP3 acts downstream the dsRNA formation, which is in agreement with the dsRNA nature of birnaviral genomes. Many VSRs bind dsRNA [33,70], but not all dsRNA binding proteins have RNA silencing suppression activity [26,29]. Whereas some VSRs bind long dsRNA molecules without size specificity and interfere with Dicer cleavage activity [70–74], others disturb silencing by specific sequestration of siRNAs [64,75–78]. To establish the mechanism of action by which VP3 suppresses RNA silencing, we found that, in addition to its capacity to interact with long-dsRNAs [66,67], IBDV VP3 can effectively bind ds-small RNA molecules of different sizes (Fig. 2B). This includes dsRNAs with a length of 21-nt, which is the typical siRNA size class produced from RNA viruses during the infection cycle. Moreover, whereas the plant VSR P1b, which suppresses silencing by specific interaction with 21-nt small RNAs [64], mainly affected the accumulation of secondary siRNAs, both primary and secondary GFP-derived siRNAs were barely detected in leaves expressing either IBDV VP3 or Influenza virus NS1 (Fig. 1B). These data support the idea that VP3 and NS1 could use a dual mode of action, which would be different to that used by P1b, based on perturbing Dicer-mediated cleavage of long dsRNAs to produce siRNAs whilst also blocking the incorporation of these siRNAs into RNA-induced silencing complexes. A similar mechanism of action has been suggested for the VSR B2 from the insect nodavirus Flock house virus [71,79]. By using a mutagenic approach, we have PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45957