RNA silencing refers to small regulatory RNA-mediated processes that repress endogenous gene expression and defend hosts from offending viruses. As an anti-host defense mechanism, viruses encode suppressors that can block RNA silencing pathways. Cucumber mosaic virus (CMV)-encoded 2b protein was among the first suppressors identified that could inhibit post-transcriptional gene silencing (PTGS), but with little or no effect on miRNA functions. The mechanisms underlying 2b suppression of RNA silencing are unknown. Here, we demonstrate that the CMV 2b protein also interferes with miRNA pathways, eliciting developmental anomalies partially phenocopying ago1 mutant alleles. In contrast to most characterized suppressors, 2b directly interacts with Argonaute1 (AGO1) in vitro and in vivo, and this interaction occurs primarily on one surface of the PAZ-containing module and part of the PIWI-box of AGO1. Consistent with this interaction, 2b specifically inhibits AGO1 cleavage activity in RISC reconstitution assays. In addition, AGO1 recruits virus-derived small interfering RNAs (siRNAs) in vivo, suggesting that AGO1 is a major factor in defense against CMV infection. We conclude that 2b blocks AGO1 cleavage activity to inhibit miRNA pathways, attenuate RNA silencing, and counter host defense. These findings provide insight on the molecular arms race between host antiviral RNA silencing and virus counterdefense.[Keywords: Viral suppressor; Cucumber mosaic virus 2b; RNA silencing; AtAGO1; cleavage activity; counter defense] Supplemental material is available at http://www.genesdev.org.

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism 1-3 mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold 4,5 critical for the endonuclease cleavage activity of RISC 4-6 . Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5′-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere 7 , provide direct support for the 5′ region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the guide RNA.Small interfering RNAs (siRNAs), the cleavage products of RNase III enzyme Dicer, consist of RNA duplexes that contain two-nucleotide 3′ overhangs and 5′-phosphorylated ends. These unique features at the termini of 19-23-base-pair duplexes, which distinguish siRNAs from other endogenous RNAs, are also required for their functional role in RNAi-mediated processes. Extensive structural and biochemical studies on PAZ (for PIWI/Argonaute/ Zwille)-RNA recognition [8][9][10] , culminating in the structures of PAZ bound to singlestranded RNA 11 and siRNA-like duplexes 12 , have established that the 3′ overhang isCorrespondence and requests for materials should be addressed to D.J.P. (pateld@mskcc.org).. Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests.Coordinates have been deposited in the Protein Data Bank under accession code 1YTU. Here we describe the 2.5-Å crystal structure of the A. fulgidus Piwi protein (Fig. 1a) bound to a 5′-phosphate-containing 21-mer RNA, capable of self-complementary duplex formation (Fig. 1b). The crystallographic asymmetric unit contains two Piwi proteins, each bound to a short four-base-pair segment of duplex, positioned adjacent to the 5′ phosphate and its attached unpaired nucleotide (Fig. 1c). The structure of the A. fulgidus Piwi protein, which consists of a short N-terminal element and two domains labelled A and B (Fig. 1a), is similar (root-mean-square deviation = 0.65Å for Cα) in the free state 5 and in the RNAbound complex. The RNA, whose sequence ...

Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

Small interfering RNAs (siRNAs) have been widely exploited for sequence-specific gene knockdown, predominantly to investigate gene function in cultured vertebrate cells, and also hold promise as therapeutic agents. Because not all siRNAs that are cognate to a given target mRNA are equally effective, computational tools have been developed based on experimental data to increase the likelihood of selecting effective siRNAs. Furthermore, because target-complementary siRNAs can also target other mRNAs containing sequence segments that are partially complementary to the siRNA, most computational tools include ways to reduce potential off-target effects in the siRNA selection process. Though these methods facilitate selection of functional siRNAs, they do not yet alleviate the need for experimental validation. This perspective provides a practical guide based on current wisdom for selecting siRNAs.