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J. Virol. doi:10.1128/JVI.02383-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Molecular bases of viral RNA targeting by viral siRNA programmed RISC

Agricultural Biotechnology Center, P.O. Box 411, H-2101 Gödöll, Hungary; Istituto di Virologia Vegetale del CNR, 70126, Bari, Italy

^* To whom correspondence should be addressed. Email: burgyan{at}abc.hu.

	Abstract

RNA silencing is conserved in a broad range of eukaryotes and operates in the development and in the maintenance of genome integrity in many organisms. Plants have adapted this system for antiviral defense and plant viruses have in turn developed mechanisms to suppress RNA silencing. RNA silencing related RNA inactivation is likely based on target RNA cleavage or translational arrest. Although, it is widely assumed that virus induced gene silencing (VIGS) promotes the endonucleolytic cleavage of viral RNA genome this popular assumption has never been tested experimentally. Here we analyzed the viral RNA targeting by VIGS in tombusvirus infected plants and we show evidence that antiviral response of VIGS is based on viral RNA cleavage by RNA-induced silencing effector complex (RISC) programmed by virus specific siRNAs. In addition, we found that the RISC mediated cleavages do not occur randomly on the viral genome. Indeed, sequence analysis of cloned cleavage products identified hotspots for target RNA cleavage and the regions of specific RISC mediated cleavages are asymmetrically distributed along the positive and the negative sense viral RNA strands. In addition, we identified viral siRNAs containing large molecular weight protein complexes purified from the recovery leaves of the silencing suppressor mutant virus infected plants. Strikingly these large nucleoproteins co-fractionated with miRNAs-containing complexes, suggesting that these nucleoproteins are silencing related effector complexes.

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