Polerovirus protein P0 prevents the assembly of small RNA‐containing RISC complexes and leads to degradation of ARGONAUTE1

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double‐stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein–protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA‐induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre‐programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high‐molecular‐weight complex, suggesting the existence of a multi‐protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA–RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co‐expressed AGO1 in both the presence and absence of P0.     

Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.     

Lessons on RNA Silencing Mechanisms in Plants from Eukaryotic Argonaute Structures

RNA silencing refers to a collection of gene regulatory mechanisms that use small RNAs for sequence specific repression. These mechanisms rely on ARGONAUTE (AGO) proteins that directly bind small RNAs and thereby constitute the central component of the RNA-induced silencing complex (RISC). AGO protein function has been probed extensively by mutational analyses, particularly in plants where large allelic series of several AGO proteins have been isolated. Structures of entire human and yeast AGO proteins have only very recently been obtained, and they allow more precise analyses of functional consequences of mutations obtained by forward genetics. To a large extent, these analyses support current models of regions of particular functional importance of AGO proteins. Interestingly, they also identify previously unrecognized parts of AGO proteins with profound structural and functional importance and provide the first hints at structural elements that have important functions specific to individual AGO family members. A particularly important outcome of the analysis concerns the evidence for existence of Gly-Trp (GW) repeat interactors of AGO proteins acting in the plant microRNA pathway. The parallel analysis of AGO structures and plant AGO mutations also suggests that such interactions with GW proteins may be a determinant of whether an endonucleolytically competent RISC is formed.     

The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation

Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.     

Molecular insights into the function of the viral RNA silencing suppressor HCPro

Potyviral helper component proteinase (HCPro) is a well‐characterized suppressor of antiviral RNA silencing, but its mechanism of action is not yet fully understood. In this study, we used affinity purification coupled with mass spectrometry to identify binding partners of HCPro in potyvirus‐infected plant cells. This approach led to identification of various HCPro interactors, including two key enzymes of the methionine cycle, S–adenosyl‐l –methionine synthase and S–adenosyl‐l –homocysteine hydrolase. This finding, together with the results of enzymatic activity and gene knockdown experiments, suggests a mechanism in which HCPro complexes containing viral and host proteins act to suppress antiviral RNA silencing through local disruption of the methionine cycle. Another group of HCPro interactors identified in this study comprised ribosomal proteins. Immunoaffinity purification of ribosomes demonstrated that HCPro is associated with ribosomes in virus‐infected cells. Furthermore, we show that HCPro and ARGONAUTE1 (AGO1), the core component of the RNA‐induced silencing complex (RISC), interact with each other and are both associated with ribosomes in planta. These results, together with the fact that AGO1 association with ribosomes is a hallmark of RISC‐mediated translational repression, suggest a second mechanism of HCPro action, whereby ribosome‐associated multiprotein complexes containing HCPro relieve viral RNA translational repression through interaction with AGO1.     

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.