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Journal of Virology, April 2007, p. 3797-3806, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02383-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Molecular Bases of Viral RNA Targeting by Viral Small Interfering RNA-Programmed RISC

Agricultural Biotechnology Center, P.O. Box 411, H-2101 Gödöll, Hungary,¹ Istituto di Virologia Vegetale del CNR, 70126 Bari, Italy²

Received 31 October 2006/ Accepted 24 January 2007

RNA silencing is conserved in a broad range of eukaryotes and operates in the development and 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 the 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 small interfering RNAs (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 hot spots for target RNA cleavage, and the regions of specific RISC-mediated cleavages are asymmetrically distributed along the positive- and negative-sense viral RNA strands. In addition, we identified viral siRNAs containing high-molecular-mass protein complexes purified from the recovery leaves of the silencing suppressor mutant virus-infected plants. Strikingly, these large nucleoproteins cofractionated with microRNA-containing complexes, suggesting that these nucleoproteins are silencing related effector complexes.

Published ahead of print on 31 January 2007.

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