Birnaviridae is a family of non-enveloped icosahedral viruses that infect birds, fishes, insects or rotifers [47]. Two members of the family, namely Infectious bursal disease virus (IBDV) and Infectious pancreatic necrosis virus (IPNV), are etiological agents of diseases imposing a heavy economical burden on the poultry and aquaculture industries, respectively. IBDV causes a highly acute immunosuppressive disease affecting juvenile domestic chickens [48]. IPNV is responsible for an acute systemic disease affecting different species of freshwater and marine fishes, mollusks, and crustaceans [49]. Birnaviruses contain a polyploid bipartite dsRNA genome that is packaged into a single virus particle [50]. Structural units are derived from a polyprotein precursor that is translationally self-cleaved to release three polypeptides, pVP2 (the capsid protein precursor), VP4 (the protease) and VP3 [51]. VP3 is a multitasking protein that has several activities throughout the viral life cycle. In addition to being a self-interacting protein [52], VP3 interacts with pVP2 during particle morphogenesis [53], with VP1 acting as a transcriptional activator [54], and with the dsRNA to make ribonucleoprotein complexes [55], a unique feature among dsRNA viruses. The atomic structure of VP3 was partially solved by X-ray crystallography to 2.3 A˚ [56]. The genome of most dsRNA viruses is contained within a specialized icosahedral capsid involved in transcription and replication of the dsRNA genome [57]. The structural integrity of these functional cores remains undisturbed after virus entry in the infected cell [58], protecting the dsRNA and replicative inter- mediates from host defense mechanisms. In contrast, birnaviruses lack this replicative core [59,60], but riboncleoprotein complexes might have acquired some of its defensive functions. Here, we report that different birnaviral VP3 proteins suppress the RNA silencing of a GFP reporter gene and that, despite their ability to bind ds-siRNAs, they appear to use a different mechanism than that of the well-studied short interfering (si)RNA-hijacking suppressors. In addition, we show that IBDV VP3 is able to functionally replace the well-characterized HCPro VSR in the plant-infecting Plum pox virus (PPV), which highlights the silencing suppression activity of VP3 in the context of a highly sensitive infection process. Results IBDV VP3 Suppresses Both Sense RNA and dsRNA- triggered RNA Silencing in Plants The high affinity displayed by IBDV VP3 to bind long dsRNA molecules as well as to protect them from RNase III-mediated cleavage in vitro [55], prompted us to test whether IBDV VP3 could suppress RNA silencing in vivo. For this, we decided to use an heterologous transient expression system in Nicotiana benthamiana [15], as it provides readout of RNA silencing without the confounding induction of other immune and interferon-responsive pathways [61,62]. Different plasmids expressing untagged and N- terminal TAP (NTAP)-tagged versions of IBDV VP3 were constructed and transferred to Agrobacterium tumefaciens. Equivalent constructs expressing Influenza A virus NS1 and the well- characterized CVYV P1b silencing suppressor [63,64] were used as positive controls. A GFP-expressing plasmid (p35S:GFP) was used as the inducer, target and reporter of RNA silencing. For simplicity, we will refer to Agrobacterium strains by the name of the plasmid they carry. Leaf patches infiltrated with p35S:GFP plus either pMDC32 or pNTAPi empty vectors displayed the highest intensity of GFP fluorescence at 2–3 days post-agroinfiltration (dpa) (not shown). GFP fluorescence in these leaves dropped to hardly detectable levels by 6 dpa (Fig. 1A and not shown) and, consistently with this fact, Northern blot analysis showed very low VP3 Proteins Suppress RNA Silencing accumulation of GFP mRNA in them (Fig. 1A and data not shown). In contrast, leaves infiltrated with p35S:GFP plus pMDC32-NS1, p35S-NTAP-NS1 or p35S-NTAP-P1b, expressing untagged or TAP-tagged NS1, or TAP-tagged P1b, respectively, showed bright fluorescence at 6 dpa given the protection of GFP mRNAs from degradation by post-transcriptional gene silencing (Fig. 1A and data not shown). Interestingly, those leaf patches infiltrated with p35S:GFP plus either pMDC32-VP3 or p35S- NTAP-NS1 expressing untagged or TAP-tagged VP3, respective- ly, displayed GFP fluorescence as bright as positive controls at 6 dpa. Northern blot analysis revealed that the expression of IBDV VP3 inhibited GFP mRNA degradation very efficiently (Fig. 1A and data not shown). To induce RNA silencing, the GFP mRNA is firstly converted to dsRNA. In order to better understand the mechanism by which IBDV VP3 suppresses RNA silencing, a third Agrobacterium strain expressing an inverted repeat (IR) that directly generate dsRNA from the 59 region of the GFP gene (p35S:GF-IR) was added to the infiltrated mixes. Given that GF-IR directed a fast and strong silencing of the GFP mRNA reporter, no fluorescence was detected in the infiltrated patches at 6 dpa in the absence of silencing suppressors, and consistent with this observation, a Northern blot assay showed no accumulation of GFP mRNA (Fig. 1B). In contrast, those patches expressing NTAP-VP3, NTAP-NS1 or NTAP-P1b displayed strong fluorescence and high accumulation of GFP mRNA as consequence of their ability to protect the reporter mRNA from silencing also in this assay (Fig. 1B). IBDV VP3 and Influenza Virus NS1 Strongly Reduce the Generation of Primary siRNAs Accumulation of specific siRNAs is a main hallmark of RNA silencing induction, and it can be observed in the Northern blot analyses of leaf patches infiltrated with either p35S:GFP or p35S:GFP plus p35S:GF-IR, together with the empty vector (Fig. 1A and B). In the sense RNA-triggered silencing assay, NTAP-P1b had, as previously reported [63], very little effect on GFP siRNA accumulation (Fig. 1A). In contrast, both NTAP-NS1 and NTAP-VP3 proteins appeared to abolish the generation of GFP siRNAs (Fig. 1A). Similarly, in the dsRNA-triggered silencing assay, NTAP-P1b had no effect on the generation of siRNAs from the 59 terminal two thirds of the GFP gene (siRNA GF), which derive mainly from the dsRNA trigger (p35S:GF-IR). However, the accumulation of siRNAs from the 39 terminal region of the GFP sequence, which are not encoded by p35S:GF-IR and, thus, are secondarily generated in the amplification phase of the RNA silencing process (siRNA P), is strongly inhibited by NTAP-P1b (Fig. 1B and [64]). Interestingly, expression of either NTAP-VP3 or NTAP-NS1 produced a strong reduction in the generation not only of P-derived siRNAs, but also of those siRNAs deriving from the GF dsRNA region (Fig. 1B), indicating that these proteins use a mechanism different from that of CVYV P1b to suppress the RNA silencing. VP3 Efficiently Binds ds-small RNAs It was previously reported that birnaviral VP3 proteins, including IBDV VP3, are able to bind dsRNAs molecules [65– 67]. In order to test the ability of IBDV VP3 to interact with typical siRNA duplexes, EMSAs using 32P-labelled synthetic ds- siRNAs carrying 2-nt overhand at their 39 ends were carried out. For these experiments, a 6xHis-tagged version of IBDV VP3 was produced and purified from insect cell cultures making use of recombinant baculoviruses. Purified VP3 samples were incubated with different dsRNA molecules, and complexes were then PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45957