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J. Virol. doi:10.1128/JVI.00272-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Structural and Genetic Requirements for the Biogenesis of Tobacco Rattle Virus-derived Small Interfering RNAs

Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain; Departamento de Biotecnología and Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain

^* To whom correspondence should be addressed. Email: cesarllave{at}cib.csic.es.

	Abstract

In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco Rattle Virus-derived siRNAs (TRV siRNAs). TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3 or DCL2 targets alone, or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNA reveals differential contribution throughout the TRV genome to siRNA formation. Indirect evidence suggests that DCL2 is responsible for production of a subset of siRNAs derived from the 3' end region of TRV. TRV siRNA biogenesis and antiviral silencing is strongly dependent on the combined activity of the host-encoded RNA-dependent RNA polymerases RDR1, RDR2 and RDR6, thus providing evidence that perfectly complementary double-stranded RNA serves as a substrate for siRNA production. We conclude that the overall composition of viral siRNAs in TRV-infected plants reflects the combined action of several interconnected pathways involving different DCL and RDR activities.