Crystal structure of p19--a universal suppressor of RNA silencing

RNA silencing in plants has an antiviral role and, consequently, plant viruses encode counter-defensive suppressor proteins that block this process. The recently reported crystal structure of two Tombusvirus suppressor proteins reveals a novel RNA-binding structure and illustrates precisely how the silencing mechanism is blocked. These suppressor protein structures, combined with molecular analyses of their effects in animal and plant cells, are informative about RNA silencing mechanisms. They also suggest various ways that Tombusvirus suppressors can be used to investigate RNA silencing in plants and animals.     

Identification of an RNA silencing suppressor from a plant double-stranded RNA virus

RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of Rice dwarf phytoreovirus (RDV), exhibited RSS activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c carrying GFP. The other gene segments of the RDV genome did not have such a function. Pns10 suppressed local and systemic silencing induced by sense RNA but did not interfere with local and systemic silencing induced by dsRNA. Expression of Pns10 also increased the expression of beta-glucuronidase in transient assays and enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, our results establish Pns10 as an RSS encoded by a plant dsRNA virus and further suggest that Pns10 targets an upstream step of dsRNA formation in the RNA silencing pathway.     

pH‐sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability

Tombusviruses, such as Carnation Italian ringspot virus (CIRV), encode a protein homodimer called p19 that is capable of suppressing RNA silencing in their infected hosts by binding to and sequestering short‐interfering RNA (siRNA) away from the RNA silencing pathway. P19 binding stability has been shown to be sensitive to changes in pH but the specific amino acid residues involved have remained unclear. Using constant pH molecular dynamics simulations, we have identified key pH‐dependent residues that affect CIRV p19–siRNA binding stability at various pH ranges based on calculated changes in the free energy contribution from each titratable residue. At high pH, the deprotonation of Lys60, Lys67, Lys71, and Cys134 has the largest effect on the binding stability. Similarly, deprotonation of several acidic residues (Asp9, Glu12, Asp20, Glu35, and/or Glu41) at low pH results in a decrease in binding stability. At neutral pH, residues Glu17 and His132 provide a small increase in the binding stability and we find that the optimal pH range for siRNA binding is between 7.0 and 10.0. Overall, our findings further inform recent experiments and are in excellent agreement with data on the pH‐dependent binding profile.     

Size selective recognition of siRNA by an RNA silencing suppressor

RNA silencing in plants likely exists as a defense mechanism against molecular parasites such as RNA viruses, retrotransposons, and transgenes. As a result, many plant viruses have adapted mechanisms to evade and suppress gene silencing. Tombusviruses express a 19 kDa protein (p19), which has been shown to suppress RNA silencing in vivo and bind silencing-generated and synthetic small interfering RNAs (siRNAs) in vitro. Here we report the 2.5 A crystal structure of p19 from the Carnation Italian ringspot virus (CIRV) bound to a 21 nt siRNA and demonstrate in biochemical and in vivo assays that CIRV p19 protein acts as a molecular caliper to specifically select siRNAs based on the length of the duplex region of the RNA.     

The Ebola virus VP35 protein is a suppressor of RNA silencing

RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.     

Effects of pH and salt concentration on the siRNA binding activity of the RNA silencing suppressor protein p19

The RNA silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19kDa protein (p19) that is a suppressor of RNA silencing in infected plants. This protein acts as a dimer and binds specifically to short-interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV p19. Previously, we established a convenient fluorescence-based method for assaying CIRV p19:siRNA binding using Ni(2+)-NTA coated 96-well plates. Using this method, we observe that the CIRV p19 protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH-dissociation constant profile shows that CIRV p19:siRNA binding is dependent on three different apparent pK(a) values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV p19 occurs in the pH range from 6.2 to 7.6.     

Independent parallel functions of p19 plant viral suppressor of RNA silencing required for effective suppressor activity

Plant viruses ubiquitously mediate the induction of miR168 trough the activities of viral suppressors of RNA silencing (VSRs) controlling the accumulation of ARGONAUTE1 (AGO1), one of the main components of RNA silencing based host defence system. Here we used a mutant Tombusvirus p19 VSR (p19-3M) disabled in its main suppressor function, small interfering RNA (siRNA) binding, to investigate the biological role of VSR-mediated miR168 induction. Infection with the mutant virus carrying p19-3M VSR resulted in suppressed recovery phenotype despite the presence of free virus specific siRNAs. Analysis of the infected plants revealed that the mutant p19-3M VSR is able to induce miR168 level controlling the accumulation of the antiviral AGO1, and this activity is associated with the enhanced accumulation of viral RNAs. Moreover, saturation of the siRNA-binding capacity of p19 VSR mediated by defective interfering RNAs did not influence the miR168-inducing activity. Our data indicate that p19 VSR possesses two independent silencing suppressor functions, viral siRNA binding and the miR168-mediated AGO1 control, both of which are required to efficiently cope with the RNA-silencing based host defence. This finding suggests that p19 VSR protein evolved independent parallel capacities to block the host defence at multiple levels.     

Switching on RNA silencing suppressor activity by restoring argonaute binding to a viral protein

We found that Sweet potato feathery mottle virus (SPFMV) P1, a close homologue of Sweet potato mild mottle virus P1, did not have any silencing suppressor activity. Remodeling the Argonaute (AGO) binding domain of SPFMV P1 by the introduction of two additional WG/GW motifs converted it to a silencing suppressor with AGO binding capacity. To our knowledge, this is the first instance of the transformation of a viral protein of unknown function to a functional silencing suppressor.     

Characterization of silencing suppressor p24 of Grapevine leafroll‐associated virus 2

Grapevine leafroll‐associated virus 2 (GLRaV‐2) p24 has been reported to be an RNA silencing suppressor (RSS). However, the mechanisms underlying p24's suppression of RNA silencing are unknown. Using Agrobacterium infiltration‐mediated RNA silencing assays, we showed that GLRaV‐2 p24 is a strong RSS triggered by positive‐sense green fluorescent protein (GFP) RNA, and that silencing suppression by p24 effectively blocks the accumulation of small interfering RNAs. Deletion analyses showed that the region of amino acids 1–188, which contains all predicted α‐helices and β‐strands, is required for the RSS activity of p24. Hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, previously shown to be critical for p24 self‐interaction, are also crucial for silencing suppression, and western blotting results suggested that a lack of self‐interaction ability results in decreased p24 accumulation in plants. The mutants showed greatly weakened or a lack of RSS activity. Substitution with two basic residues at positions 2 or 86, putatively involved in RNA binding, totally abolished the RSS activity of p24, suggesting that p24 uses an RNA‐binding strategy to suppress RNA silencing. Our results also showed that W54 in the WG/GW‐like motif (W54/G55) is crucial for the RSS activity of p24, whereas p24 does not physically interact with AGO1 of Nicotiana benthamiana. Furthermore, p24 did not promote AGO1 degradation, but significantly up‐regulated AGO1 mRNA expression, and this effect was correlated with the RSS activity of p24, indicating that p24 may interfere with microRNA‐directed processes. The presented results contribute to our understanding of viral suppression of RNA silencing and the molecular mechanisms underlying GLRaV‐2 infection.     

The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance

The Cucumber mosaic virus (CMV)-encoded 2b protein (Cmv2b) is a nuclear protein that suppresses transgene RNA silencing in Nicotiana benthamiana. Cmv2b is an important virulence determinant but nonessential for systemic spread in N. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. Here, we report that Cmv2b became essential for systemic infections in older N. glutinosa plants or in young seedlings pretreated with salicylic acid (SA). Expression of Cmv2b from the genome of either CMV or Tobacco mosaic virus significantly reduced the inhibitory effect of SA on virus accumulation in inoculated leaves and systemic leaves. A close correlation is demonstrated between Cmv2b expression and a reduced SA-dependent induction of the alternative oxidase gene, a component of the recently proposed SA-regulated antiviral defense. These results collectively reveal a novel activity of Cmv2b in the inhibition of SA-mediated virus resistance. We used a N. tabacum line expressing a bacterial nahG transgene that degrades SA to provide evidence for a Cmv2b-sensitive antiviral defense mechanism in tobacco in which SA acts as a positive modifier but not as an essential component. We propose that SA induces virus resistance by potentiating a RNA-silencing antiviral defense that is targeted by Cmv2b.     

Nucleotide sequence of beet western yellows virus RNA.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).     

Constrained peptides mimic a viral suppressor of RNA silencing

The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.     

Protease activity, self interaction, and small interfering RNA binding of the silencing suppressor p1b from cucumber vein yellowing ipomovirus

The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement of Potato virus X pathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNA-induced silencing complex.     

Size-independent and noncooperative recognition of dsRNA by the Rice stripe virus RNA silencing suppressor NS3

Plant and animal viruses employ diverse suppressor proteins to thwart the host antiviral reaction of RNA silencing. Many suppressors bind dsRNA with different size specificity. Here, we examine the dsRNA recognition mechanism of the Rice stripe virus NS3 suppressor using quantitative biochemical approaches, as well as mutagenesis and suppression activity analyses in plants. We show that dimeric NS3 is a size-independent, rather than small interfering RNA-specific, dsRNA-binding protein that recognizes a minimum of 9 bp and can bind to long dsRNA with two or more copies. Global analysis using a combinatorial approach reveals that NS3 dimer has an occluded site size of ∼ 13 bp on dsRNA, an intrinsic binding constant of 1 × 10⁸ M^− 1, and virtually no binding cooperativity. This lack of cooperativity suggests that NS3 is not geared to target long dsRNA. The larger site size of NS3, compared with its interacting size, indicates that the NS3 structure has a border region that has no direct contact with dsRNA but occludes a ∼ 4-bp region from binding. We also develop a method to correct the border effect of ligand by extending the lattice length. In addition, we find that NS3 recognizes the helical structure and 2′-hydroxyl group of dsRNA with moderate specificity. Analysis of dsRNA-binding mutants suggests that silencing of the suppression activity of NS3 is mechanistically related to its dsRNA binding ability.