identified a region of basic residues, called here as Patch 1, involved in the dsRNA binding activity of IBDV VP3 and shown that these residues are also relevant for RNA silencing suppression (Fig. 2C and Fig. 3). Whereas these results support the contribution of dsRNA binding to the RNA silencing suppression activity of VP3, they do not provide clues on the relative importance of long dsRNA protection and siRNA sequestration since all mutations had similar effects on the VP3 binding to the different tested RNA molecules (Fig. 2C and Fig. 3). Interestingly, whereas mutations in another region of basic residues, here called as Patch 2, produced a partial reduction in the capacity of IBDV VP3 to interact with both long and short dsRNAs, they did not affect the ability of this protein to suppress the degradation of GFP mRNA by silencing (Fig. 2C and Fig. 3). This result indicates that a diminished dsRNA binding is enough to counteract the silencing machinery, as previously observed for the VSR P1b [64], and supports the idea that the VP3 Patch 2 could also be involved in RNA interaction. The expression of VSRs is the general strategy used by plant viruses to counteract antiviral RNA silencing and some of them, like the potyvirus PPV, are unable to establish an infection in the absence of an active VSR [80,81]. Here, we found that IBDV VP3 is able to replace HCPro, the well-characterized VSR, in a PPV infection (Fig. 5). This result demonstrates the effectiveness of the RNA silencing suppression activity of IBDV VP3 to counteract host defences in a viral infection context. However, it is important to note that although the antisilencing activities of IBDV VP3 and PPV HCPro expressed from the PPV polyprotein are similar in an agroinfiltration assay (Fig. S2), the infection efficiency of PPV-VP3 chimera is somewhat lower than that of wild type PPV. N. benthamiana plants infected with this chimerical virus did eventually recover from the infection (Fig. 5D). These findings are in agreement with recent results obtained with PPV chimeras expressing different heterologous VSRs [82], supporting the idea that the capacity of a given VSR to replace HCPro in a potyviral infection does not rely only in the strength of suppressors, but also in the particular molecular mechanism that they use to suppress the RNA silencing. It is well established that RNA silencing plays a key role in the antiviral innate immunity of invertebrate organisms [23,24,72,83], and it has been recently demonstrated that antiviral silencing can terminate viral infection in adult insects [84]. Although a broad range of insect viruses are known targets of antiviral RNA silencing, VSRs have been identified only in positive sense strand RNA viruses [84,85]. Interestingly, it has also been reported that RNA silencing is an efficient antiviral immune response against the birnavirus DXV [86], but the mechanism used by the virus to counteract this antiviral defence remained uncharacterized. Here, we provide the first evidence that the VP3 protein from DXV has RNA silencing suppression activity (Fig. 4), which suggests that it plays this counter defensive role during insect infections. The best- studied VSR of an insect virus, the B2 protein of alphanodaviruses [23,71,79], has a functional counterpart in fish betanodaviruses [87,88]. Similarly, our data show that VP3 from IPNV, a fish infecting birnavirus, also has the ability to suppress the RNA silencing. It is worth to mention that even thought the primary amino acid sequence among these three VP3s is barely conserved, particularly remarkable in the case DXV VP3 [56], all of them interact with dsRNA without size specificity and counteract the RNA silencing (Fig. 4). Altogether, these results strongly supports the idea that silencing suppression is a conserved activity among different VP3 proteins and that RNA silencing could play a defensive role in viral infections of fishes and birds. In agreement VP3 Proteins Suppress RNA Silencing with this postulate, an VSR from an avian herpersvirus has also been recently characterized [41]. Conclusive evidence demonstrating that an antiviral silencing mechanism operates in vertebrates is still lacking. Although early tests failed to find viral-derived siRNAs, the hallmark of antiviral silencing, in different infected organisms (reviewed by [89]), the availability of deep sequencing technologies has recently allowed not only the identification of siRNAs derived from a wide set of mammal-infecting RNA viruses, but also revealed their association with silencing effector complexes [13,14]. These data, together with the observation that many mammal-infecting RNA viruses encode VSRs [42], suggest that RNA silencing of vertebrates is, to some extent, engaged in fighting against RNA viruses. It is important to note that the relationship between RNA silencing and viruses could be multifaceted in these organisms; hence, in addition to the possible cis-acting effect of viral-derived siRNAs, some of these molecules are able to target host genes involved in antiviral defense [90,91]. Moreover, several host miRNA have been shown to contribute to virus resistance [34,92–96], whereas others are essential for the replication of the virus [97]. In this intricate scenario, the contribution of VSRs is expected to be tightly regulated. In general, VSRs from mammal-infecting viruses also participate in the counter defense against other antiviral responses of the host, such as the interferon system, which add a further level of intricacy to the activity of these proteins [29,31]. Cross-talk between RNA silencing and IFN-dependent defense pathways have been reported [13,62,94] and consequently the dsRNA-binding abilities of these VSRs could contribute to interference with both IFN- and RNA silencing-mediated antiviral defences in tandem [29]. Therefore, the dsRNA binding activity of IBDV VP3 could prevent genomic dsRNAs to induce RNA silencing, as well as modulate other dsRNA-induced antiviral defences. In agreement with this hypothesis, recent results indicate that IBDV VP3 inhibits PKR-dependent apoptosis induced by the expression of the viral protein VP2 (Busnadiego et al., submitted). Materials and Methods Plant Hosts Agroinfiltration and viral infectivity assays were performed in Nicotiana benthamiana plants. All plants were grown in a greenhouse maintained at 16 hours light with supplementary illumination and a temperature range of 19–23uC. Plasmids Recombinant baculoviruses (rBVs) expressing 6 6 His-tagged versions of full-length IBDV VP3 as well as the VP3DC, which lacks the 36 C-terminal amino acid residues, were previously described [56,66]. The construction of the rBV expressing a 6 6 His-tagged version of VP3 Patch1 mutant (rBV hisVP3Patch1) was performed as follows. A 789 bp DNA fragment coding for a mutant version of VP3 with four aa substitutions (K99D, R102D, K105D and K106D, VP3MutPatch1) flanked by BamHI and EcoRI restriction sites, was generated by gene synthesis (Genscript) and inserted into the EcoRV site located in the multiple cloning site (MCS) of the pUC57 plasmid. This plasmid was digested with BamHI and EcoRI and the fragment containing the mutated VP3 ORF was purified and inserted into the MCS of the baculovirus transfer vector pFastBacHtb (Invitrogen), previously digested with the same restriction enzymes. The resulting plasmid (pFBhisVP3Patch1) was then used to generate the corresponding rBV making use of the Bac-to-Bac system, following the manufacturer’s instructions (Invitrogen). The generation of the rBV expressing a 6 6 His-tagged version of VP3 Patch2 mutant PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45957