The VP3 Factor from Viruses of Birnaviridae Family Suppresses RNA Silencing by Binding Both Long and Small RNA Duplexes Adrian Valli.¤a, Idoia Busnadiego . , Varvara Maliogka¤b, Diego Ferrero, Jose´ R. Casto´ n, Jose´ Francisco Rodrı´guez*, Juan Antonio Garcı´a* Centro Nacional de Biotecnolog´ıa-CSIC, Madrid, Spain Abstract RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery. Citation: Valli A, Busnadiego I, Maliogka V, Ferrero D, Casto´ n JR, et al. (2012) The VP3 Factor from Viruses of Birnaviridae Family Suppresses RNA Silencing by Binding Both Long and Small RNA Duplexes. PLoS ONE 7(9): e45957. doi:10.1371/journal.pone.0045957 Editor: Y. Adam Yuan, National University of Singapore, Singapore Received June 2, 2012; Accepted August 23, 2012; Published September 25, 2012 Copyright: 2012 Valli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants BIO2010-18541, BIO2009-12443 and BFU2011-25902 from the Spanish MICINN and KBBE-204429 from European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jfrodrig@cnb.csic.es (JFR); jagarcia@cnb.uam.es (JAG) ¤a Current address: Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom ¤b Current address: Plant Pathology Laboratory, School of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece . These authors contributed equally to this work. Introduction RNA silencing collectively refers to diverse gene expression regulatory pathways that control a large number of cellular processes, such as developmental patterning, responses to biotic and abiotic stresses, and maintenance of genome stability. It is conserved in animals, plants, most fungi and some protists [1–3]. RNA silencing pathways involve small RNAs of 20–30 nucleo- tides, which are mainly derived from digestion of RNA duplexes by the action of RNase III–like nucleases called Dicer or Dicer-like (DCL) proteins [4–6]. These small RNAs are then associated with effector complexes, containing a protein belonging to the Argonaute (AGO) family, to guide sequence specific mRNA degradation, translational inhibition, or epigenetic modifications [7]. Interestingly, it has been well-established that RNA silencing plays a key antiviral role in plants, fungi and invertebrate animals, where infecting viruses induce the production of viral-derived small interfering (si)RNAs from their genomes, replication-in- termediate double-stranded (ds)RNAs, fold-back structures within viral mRNAs, or dsRNA molecules produced by the action of RNA-dependent RNA polymerases (RdRps) on a single stranded viral RNA template [8–11]. Although the role of RNA silencing as an antiviral mechanism in vertebrate animals is controversial, the fact that bona fide viral-derived small RNAs from different mammal-infecting viruses have recently been identified suggests that RNA silencing could also be an antiviral strategy in these organisms [12–14]. Viruses have evolved to overcome RNA silencing-based defences by encoding viral suppressors of RNA silencing (VSR) [15–18]. The key relevance of these proteins for the infection life cycle has been established in multiple virus/host combinations, showing that the absence of an active VSR results in greatly reduced viral infection [19,20–25]. Interestingly, it has been found that several vertebrate-infecting viruses also encode proteins with the ability to counteract RNA silencing, such as NS1 from Influenza A virus [26–29], VP30, VP35 and VP40 from Ebola virus [28,30,31], NSs from La Crosse virus [32], s3 from Mammalian orthoreovirus type 3 [33], E3L from Vaccinia virus [28,29], Tas from Primate foamy virus type 1 [34], Tat from Human immunodeficiency virus [35], Rex from Human T-cell lymphotropic virus [36], C and E2 proteins from Hepatitis C virus [37–39], 7a from Severe acute respiratory syndrome virus [40], and HVT063 from Turkey herpesvirus [41], presumably as part of the attack-defense-counterdefense arms race between viruses and their hosts (some of these RSSs were recently reviewed [42,43–46]). PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45957