Viruses Encode RNA Silencing Suppressors Research Articles

Diverse conserved domains and a positively selected site in the sugarcane streak mosaic virus P1 protein are essential for RNA silencing suppression and protein stability

AbstractRNA silencing is one of the conserved antiviral mechanisms in plants, and viruses encode RNA silencing suppressors (RSS) to overcome host RNA silencing and facilitate virus infection. Sugarcane streak mosaic virus (SCSMV; species Sugarcane streak mosaic virus, genus Poacevirus, family Potyviridae) is a major causal agent of sugarcane mosaic disease in many countries in Asia, including China. In this study, we used Agrobacterium co‐infiltration to show that the SCSMV P1 protein, rather than the helper component‐proteinase (HC‐Pro), functions as a strong RSS to suppress local RNA silencing in Nicotiana benthamiana. Mutational analysis indicated that the 15 amino acids (aa; aa 1–15) of the SCSMV P1 N‐terminus were not important for RNA silencing suppression, but rather another 15 aa domain (aa 108–122) containing a conserved motif (LFR/KNKQAYIST) was essential for efficient silencing suppression by P1. In addition to the 15 aa (aa 344–358) domain in the P1 N‐terminus, another 15 aa domain (aa 65–79) of P1, containing the LXKA motif and one conserved aa (D78), were associated with P1 protein stability. Furthermore, substituting the histidine (H263) residue in P1 with threonine (H263T) or alanine (H263A) also affected P1 protein stability. Notably, the H263 residue is both a positively selected site and part of the serine protease catalytic triad (HDS). Taken together, our data demonstrate that SCSMV P1, and not HC‐Pro, plays a functional role in suppressing RNA silencing, and also show that some conserved motifs and a positivelyselected site in the P1 protein are associated with RSS activity and protein stability.

Highly Abundant Small Interfering RNAs Derived from a Satellite RNA Contribute to Symptom Attenuation by Binding Helper Virus-Encoded RNA Silencing Suppressors.

Highly Abundant Small Interfering RNAs Derived from a Satellite RNA Contribute to Symptom Attenuation by Binding Helper Virus-Encoded RNA Silencing Suppressors.

Probing an optimal class distribution for enhancing prediction and feature characterization of plant virus-encoded RNA-silencing suppressors.

To counter the host RNA silencing defense mechanism, many plant viruses encode RNA silencing suppressor proteins. These groups of proteins share very low sequence and structural similarities among them, which consequently hamper their annotation using sequence similarity-based search methods. Alternatively the machine learning-based methods can become a suitable choice, but the optimal performance through machine learning-based methods is being affected by various factors such as class imbalance, incomplete learning, selection of inappropriate features, etc. In this paper, we have proposed a novel approach to deal with the class imbalance problem by finding the optimal class distribution for enhancing the prediction accuracy for the RNA silencing suppressors. The optimal class distribution was obtained using different resampling techniques with varying degrees of class distribution starting from natural distribution to ideal distribution, i.e., equal distribution. The experimental results support the fact that optimal class distribution plays an important role to achieve near perfect learning. The best prediction results are obtained with Sequential Minimal Optimization (SMO) learning algorithm. We could achieve a sensitivity of 98.5 %, specificity of 92.6 % with an overall accuracy of 95.3 % on a tenfold cross validation and is further validated using leave one out cross validation test. It was also observed that the machine learning models trained on oversampled training sets using synthetic minority oversampling technique (SMOTE) have relatively performed better than on both randomly undersampled and imbalanced training data sets. Further, we have characterized the important discriminatory sequence features of RNA-silencing suppressors which distinguish these groups of proteins from other protein families.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0410-1) contains supplementary material, which is available to authorized users.

Cytorhabdovirus phosphoprotein shows RNA silencing suppressor activity in plants, but not in insect cells

RNA silencing in plants and insects provides an antiviral defense and as a countermeasure most viruses encode RNA silencing suppressors (RSS). For the family Rhabdoviridae, no detailed functional RSS studies have been reported in plant hosts and insect vectors. In agroinfiltrated Nicotiana benthamiana leaves we show for the first time for a cytorhabdovirus, lettuce necrotic yellows virus (LNYV), that one of the nucleocapsid core proteins, phosphoprotein (P) has relatively weak local RSS activity and delays systemic silencing of a GFP reporter. Analysis of GFP small RNAs indicated that the P protein did not prevent siRNA accumulation. To explore RSS activity in insects, we used a Flock House virus replicon system in Drosophila S2 cells. In contrast to the plant host, LNYV P protein did not exhibit RSS activity in the insect cells. Taken together our results suggest that P protein may target plant-specific components of RNA silencing post siRNA biogenesis.

Broad bean wilt virus 2 encoded VP53, VP37 and large capsid protein orchestrate suppression of RNA silencing in plant

Viruses encode RNA silencing suppressors to counteract host RNA silencing-mediated defense responses. In this study, we demonstrate that VP53, VP37 and LCP encoded by RNA2 of broad bean wilt virus 2 (BBWV-2), a member of the genus Fabavirus, are strong suppressors of RNA silencing triggered by single-stranded sense RNA. They, however, had no effect on suppression of RNA silencing induced by double-stranded RNA. We provide evidence that these three suppressors can significantly limit the accumulation of small interfering RNAs (siRNAs) in tissues where the GFP gene has been silenced, and prevent the long distance spread of the induced silencing signal. Gel mobility shift assays showed that all three suppressors could bind ssRNA in a size-specific manner. Interestingly, VP37 and LCP, but not VP53, could reverse the silencing of a GFP gene in leaf tissue. Furthermore, these three proteins are capable of enhancing pathogenicity of potato virus X. Collectively, our findings indicate that viruses employ a more sophisticated strategy to overcome the host defense response mediated through suppression of RNA silencing during virus infection. As far as we are aware, this is the first report of RNA silencing suppressors encoded by a virus in the genus Fabavirus.

Supervised Learning Classification Models for Prediction of Plant Virus Encoded RNA Silencing Suppressors

Viral encoded RNA silencing suppressor proteins interfere with the host RNA silencing machinery, facilitating viral infection by evading host immunity. In plant hosts, the viral proteins have several basic science implications and biotechnology applications. However in silico identification of these proteins is limited by their high sequence diversity. In this study we developed supervised learning based classification models for plant viral RNA silencing suppressor proteins in plant viruses. We developed four classifiers based on supervised learning algorithms: J48, Random Forest, LibSVM and Naïve Bayes algorithms, with enriched model learning by correlation based feature selection. Structural and physicochemical features calculated for experimentally verified primary protein sequences were used to train the classifiers. The training features include amino acid composition; auto correlation coefficients; composition, transition, and distribution of various physicochemical properties; and pseudo amino acid composition. Performance analysis of predictive models based on 10 fold cross-validation and independent data testing revealed that the Random Forest based model was the best and achieved 86.11% overall accuracy and 86.22% balanced accuracy with a remarkably high area under the Receivers Operating Characteristic curve of 0.95 to predict viral RNA silencing suppressor proteins. The prediction models for plant viral RNA silencing suppressors can potentially aid identification of novel viral RNA silencing suppressors, which will provide valuable insights into the mechanism of RNA silencing and could be further explored as potential targets for designing novel antiviral therapeutics. Also, the key subset of identified optimal features may help in determining compositional patterns in the viral proteins which are important determinants for RNA silencing suppressor activities. The best prediction model developed in the study is available as a freely accessible web server pVsupPred at http://bioinfo.icgeb.res.in/pvsup/.

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.

Suppressors of RNA silencing encoded by tomato leaf curl betasatellites

Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF beta C1 in satellite DNA beta which are predicted to function as silencing suppressors. In the present study suppressor function of ORF beta C1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB-[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing betaC1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with betaC1 gene from ToLCBB. However, in the case of 35S-beta C1 CLCuMB and 35S- beta C1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which beta C1 interfere in RNA-silencing pathway is different.

Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.

Identification of Pns6, a putative movement protein of RRSV, as a silencing suppressor

RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of Rice ragged stunt virus (RRSV), exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus.

Identification of two RNA silencing suppressors from banana bunchy top virus

In order to suppress RNA silencing, many plant and some animal viruses encode RNA silencing suppressors to achieve infection. In this study, we report that B3 and B4, encoded by DNA3 and DNA4 of banana bunchy top virus (BBTV), exhibit RNA silencing suppression activity. B3 and B4 were able to increase the transient expression of green fluorescent protein (GFP) and dramatically enhanced the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana. B4 was able to reverse established gene silencing on an inoculated leaf or on an upper leaf. B3, however, was only active during infection of an inoculated leaf. Furthermore, B4, but not B3, was able to enhance GFP expression in the transgenic N. benthamiana line 16c. In conclusion, B3 and B4 are the RNA silencing suppressors of BBTV, and they may act at different steps in the RNA silencing pathways.

Variability in the level of RNA silencing suppression caused by triple gene block protein 1 (TGBp1) from various potexviruses during infection

RNA silencing is an important defence mechanism against virus infection, and many plant viruses encode RNA silencing suppressors as a counter defence. In this study, we analysed the RNA silencing suppression ability of multiple virus species of the genus Potexvirus. Nicotiana benthamiana plants exhibiting RNA silencing of a green fluorescent protein (GFP) transgene showed reversal of GFP fluorescence when systemically infected with potexviruses. However, the degree of GFP fluorescence varied among potexviruses. Agrobacterium-mediated transient expression assay in N. benthamiana leaves demonstrated that the triple gene block protein 1 (TGBp1) encoded by these potexviruses has drastically different levels of silencing suppressor activity, and these differences were directly related to variations in the silencing suppression ability during virus infection. These results suggest that suppressor activities differ even among homologous proteins encoded by viruses of the same genus, and that TGBp1 contributes to the variation in the level of RNA silencing suppression by potexviruses. Moreover, we investigated the effect of TGBp1 encoded by Plantago asiatica mosaic virus (PlAMV), which exhibited a strong suppressor activity, on the accumulation of microRNA, virus genomic RNA and virus-derived small interfering RNAs.

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.

Two virus-encoded RNA silencing suppressors, P14 ofBeet necrotic yellow vein virus and S6 ofRice black streak dwarf virus

Functional analysis for gene silencing suppressor of P14 geneof Beet necrotic yellow vein virus and S6 gene ofRice black streak dwarf virus was carried out by agro-infiltration with recombinant vectors ofPotato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing ofgfp transgenicNicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the suppressed plants, thegfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially thegfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.

RNA silencing suppressors encoded by viruses of the family Tombusviridae

RNA silencing is a sequence-specific gene-inactivation mechanism conserved among eukaryotes that functions as an antiviral defense in plants and animals. To counteract this defense, viruses encode RNA silencing suppressors. Over 30 RNA silencing suppressors have been identified, but the mechanisms by which they suppress RNA silencing is unclear for most of them. The best-characterized suppressor is P19, encoded by viruses of the genus Tombusvirus, which belongs to the family Tombusviridae. Three suppressors have also been identified in the genera Carmovirus, Aureusvirus, and Dianthovirus in the family Tombusviridae. In this review, we summarize recent findings regarding these four RNA silencing suppressors, focusing on their modes of action in RNA silencing suppression and their roles in virus infection.

Two virus-encoded RNA si-lencing suppressors, P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus

Two virus-encoded RNA si-lencing suppressors, P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus