VP3 Proteins Suppress RNA Silencing Figure 4. VP3 proteins from fish- and insect-infecting birnaviruses suppress RNA silencing in a dsRNA-triggered test. (A) (Upper panels) purified DXV VP3 (final concentration of 8.5, 17, 34 and 68 nM) and IPNV VP3 (final concentration of 10, 20, 40 and 80 nM) were incubated with IBDV genomic dsRNAs. Protein-dsRNA complexes were resolved in agarose gels, stained with EtBr, and photographed under UV light. (Lower panels) purified DXV VP3 (final concentration of 70, 140, 280, 560 and 1120 nM) and IPNV VP3 (final concentration of 80, 160, 320, 640 and 1280 nM) were incubated with 32P-labelled 21-nt ds small RNAs. Protein-small RNA complexes were resolved in polyacrylamide gels and revealed by autoradiography. (B) GFP fluorescence pictures taken under an epifluorescence microscope using the same exposure time, and (C) Northern blot analysis of GFP mRNA in agroinfiltrated leaf patches from two N. benthamiana plants expressing the indicated constructs, and collected at 6 days post agroinfiltration. EtBr-stained rRNA is shown as loading control. doi:10.1371/journal.pone.0045957.g004 IBDV VP3 is able to Support PPV Infection in the Absence of its Natural Silencing Suppressor HCPro To assess the ability of IBDV VP3 to counteract an RNA silencing-based antiviral mechanism, we made use of a plant/virus pathosystem consisting of N. benthamiana as host and the potyvirus PPV as pathogen. A PPV full-length cDNA clone was engineered to obtain a chimera expressing IBDV VP3 instead of its natural VSR HCPro (PPV-VP3, Fig. 5A). HCPro is expressed as part of a genomic-length polyprotein, from which it is exscinded by the serine proteinase activity of the upstream protein P1 and its own cisteine proteinase activity [68]. To allow the excision of VP3, which lacks auto-proteolytic activity, from the viral polyprotein, both ends of the VP3 coding sequence cloned in the PPV chimera were slightly modified to generate cleavage sites recognized by P1 and NIa, the third PPV proteinase. Thus, the VP3 produced by PPV-VP3 is expected to have two extra aa (SD) at its N-end and a QVVVHQ tail at its C-end. Plants were biolistically inoculated with cDNA of PPV-VP3, as well as wild type PPV and HCPro deletion mutant (PPV-DHC) clones, which were used as positive and negative control, respectively. The infection processes were monitored by visual inspection under visible light and under a fluorescence micro- scope, taking advantage of the GFP tag expressed by the three viruses. As expected, at 7 days post-inoculation (dpi), GFP foci were observed in all the leaves that had been inoculated with wild type PPV, whereas no foci appeared in leaves of plants inoculated with PPV-DHC. Interestingly, GFP foci also appeared at the same time in leaves of all plants inoculated with PPV-VP3 (Fig. 5B, upper panel). Later in time, virus-like symptoms and GFP fluorescence were detected in the upper leaves of plants inoculated with PPV and PPV-VP3 chimera (Fig. 5B, lower panels), supporting the idea that IBDV VP3 is able to replace HCPro in a potyviral infection. Western blot analysis of inoculated and systemically infected tissues confirmed the visual observations (Fig. 5C). Similar levels of accumulation of PPV-VP3 and wild type PPV were detected in the inoculated leaves. Viral CP was also detected in upper non- inoculated leaves of plants infected with PPV-VP3, albeit in somewhat lower levels than in wild type PPV-infected plants (Fig. 5C, lower panel). Further observations at later time of infection (38 dpi) showed that plant infected with PPV-VP3, but not those infected with wild type PPV, recovered from viral infection, displaying neither GFP fluorescence nor viral symptoms in new growing leaves (data not shown). The recovery process was confirmed by Western blot analysis of samples collected at 38 dpi, which showed an obvious drop in PPV CP accumulation in older leaves when compared with younger leaves in PPV-VP3-infected plants, a phenomenon that did not occur in plants infected with wild type PPV (Fig. 5D). Discussion Viral RNAs, particularly in double-stranded conformation, are specifically recognized by a diverse battalion of sentinels that form part of the innate immune system of eukaryotes [10]. In many viral infections, dsRNA molecules are produced from their single- stranded counterparts as replication-intermediates or fold-back structures, or by action of RdRps, being then recognized by host factors for further activation of appropriate defence cascades. Interestingly, several viral families whose members have dsRNA PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45957