RNA Silencing Suppressor Proteins Research Articles

Probing the Nucleic Acid Flexibility to Disarm the Viral Counter-Defense Machinery: Design and Characterization of Potent p19 Inhibitors.

Plant viruses are highly destructive and significant contributors to several global pandemics and epidemics in plants. A viral disease outbreak in plants can cause a scarcity of food supply and is a severe concern to humanity. The siRNA (small interfering RNA)-mediated RNA-induced silencing complex (RISC) formation is a primary defense mechanism in plants against viruses, where the RISC binds and degrades viral mRNAs. As a counter-defense, many viruses encode RNA-silencing suppressor proteins (e.g., the p19 protein from the Tombusviridae family) for viral proliferation in plants. The functional form of p19 (homodimer) binds to plant siRNA with high affinities, thereby interrupting the RISC formation and thus preventing the viral mRNA silencing in plants. By altering the RISC formation, the p19 protein helps the virus invasion in the plant and ultimately stunts host growth. In this study, we designed several modified siRNA-based molecules for p19 inhibition. The viral p19 protein is known to interact predominantly through H-bonds with 2'-OH and phosphates of the plant siRNA. We utilized this information and in silico-designed flexible substituents of siRNA, where we removed the C2'-C3' bond in each nucleotide unit. We performed all-atom explicit-solvent molecular dynamics simulations (400 ns, 3 replicates each) for control/modified siRNA─p19 complexes (8 in total) followed by energetic estimations. Strikingly, in a few modified complexes, the siRNA not only retained the double-helical structural integrity but also displayed remarkably enhanced p19 binding compared to the control siRNA; hence, we consider it important to perform biological and chemical in vitro and in vivo studies on proposed flexible nucleic acids as p19 inhibitors for crop protection.

Coat protein of rice stripe virus enhances autophagy activity through interaction with cytosolic glyceraldehyde-3-phosphate dehydrogenases, a negative regulator of plant autophagy

Viral infection commonly induces autophagy, leading to antiviral responses or conversely, promoting viral infection or replication. In this study, using the experimental plant Nicotiana benthamiana, we demonstrated that the rice stripe virus (RSV) coat protein (CP) enhanced autophagic activity through interaction with cytosolic glyceraldehyde-3-phosphate dehydrogenase 2 (GAPC2), a negative regulator of plant autophagy that binds to an autophagy key factor, autophagy-related protein 3 (ATG3). Competitive pull-down and co-immunoprecipitation (Co-IP)assays showed that RSV CP activated autophagy by disrupting the interaction between GAPC2 and ATG3. An RSV CP mutant that was unable to bind GAPC2 failed to disrupt the interaction between GAPC2 and ATG3 and therefore lost its ability to induce autophagy. RSV CP enhanced the autophagic degradation of a viral movement protein (MP) encoded by a heterologous virus, citrus leaf blotch virus (CLBV). However, the autophagic degradation of RSV-encoded MP and RNA-silencing suppressor (NS3) proteins was inhibited in the presence of CP, suggesting that RSV CP can protect MP and NS3 against autophagic degradation. Moreover, in the presence of MP, RSV CP could induce the autophagic degradation of a remorin protein (NbREM1), which negatively regulates RSV infection through the inhibition of viral cell-to-cell movement. Overall, our results suggest that RSV CP induces a selective autophagy to suppress the antiviral factors while protecting RSV-encoded viral proteins against autophagic degradation through an as-yet-unknown mechanism. This study showed that RSV CP plays dual roles in the autophagy-related interaction between plants and viruses.

Complete genome sequence of a novel ophiovirus associated with chlorotic disease of pepper (Capsicum annuum L.) in Japan

In July 2018, pepper plants (Capsicum annuum L.) with chlorotic leaves and fruits were observed in Kochi prefecture, Japan. High-throughput sequencing (HTS) identified the possible presence of an ophiovirus-like virus possessing three RNA segments in a chlorotic leaf. Using Sanger sequencing with primers designed based on the HTS results and a different source of RNA from the one used for HTS, the complete nucleotide sequences of three RNA segments encoding four proteins on their complementary strand were determined. The amino acid sequences of these four proteins showed similarity to those of the RNA-dependent RNA polymerase, RNA-silencing suppressor protein, movement protein, and coat protein, respectively, of ophioviruses, which are negative-sense single-stranded RNA viruses. However, the coat protein amino acid sequence of the virus found on pepper plants was no more than 61.9% identical to those of any known ophioviruses, which is lower than the species demarcation threshold of 85 %. Hence, we suggest that this virus, which we have named "pepper chlorosis associated virus" (PepCaV) should be considered a member of a new species in the genus Ophiovirus, for which we propose the name "Ophiovirus capsici". The results of phylogenetic analysis using coat protein amino acid sequences of PepCaV and other ophioviruses also supported this conclusion. PepCaV RNA was found to have conserved nucleotide sequences at both the 5' and 3' termini of the different RNA segments, and the conserved terminal nucleotide sequences were predicted to form a self-complementary double-stranded region, resulting in a panhandle structure in each of the genomic RNAs.

The plant protein NbP3IP directs degradation of Rice stripe virus p3 silencing suppressor protein to limit virus infection through interaction with the autophagy-related protein NbATG8.

In plants, autophagy is involved in responses to viral infection. However, the role of host factors in mediating autophagy to suppress viruses is poorly understood. A previously uncharacterized plant protein, NbP3IP, was shown to interact with p3, an RNA-silencing suppressor protein encoded by Rice stripe virus (RSV), a negative-strand RNA virus. The potential roles of NbP3IP in RSV infection were examined. NbP3IP degraded p3 through the autophagy pathway, thereby affecting the silencing suppression activity of p3. Transgenic overexpression of NbP3IP conferred resistance to RSV infection in Nicotiana benthamiana. RSV infection was promoted in ATG5- or ATG7-silenced plants and was inhibited in GAPC-silenced plants where autophagy was activated, confirming the role of autophagy in suppressing RSV infection. NbP3IP interacted with NbATG8f, indicating a potential selective autophagosomal cargo receptor role for P3IP. Additionally, the rice NbP3IP homolog (OsP3IP) also mediated p3 degradation and interacted with OsATG8b and p3. Through identification of the involvement of P3IP in the autophagy-mediated degradation of RSV p3, we reveal a new mechanism to antagonize the infection of RSV, and thereby provide the first evidence that autophagy can play an antiviral role against negative-strand RNA viruses.

Elucidating micro RNAs role in different plant-pathogen interactions.

Bacteria, fungi, virus and nematode constitute the primary class of pathogens causing plant diseases. Plant-pathogen interactions are crucial for the identification of the host and pathogen and further establishments of a network of interaction that can cross regulate the gene expressions in both sides. After infection, the correct identification of pathogen through various molecular interactions elicit a defense response against the pathogen by alteration of gene expression by the host. Co-evolution of pathogen gives them the ability to counter the virulence response of the host and pathogen can also modulate the host gene expression pattern to make it more susceptible to the infection. Small non-coding RNA molecules (siRNAs and miRNAs) efficiently modulate gene expression at the transcriptional and post-transcriptional level and play a vital role in host defense. The pathogen can also use this double-sided sward in their defense by deregulating the plant immunity via transcriptional control of plant genes utilizing RNA interference or suppressing the host RNA interference response with the help of various RNA silencing suppressor proteins. This mini-review focused on the miRNAs involvement in host defense and how different families of these non-coding regulatory RNAs regulate the defense response against the pathogen.

Evaluating the strength of RNA silencing suppressor proteins encoded by two geminiviruses using assay based on reversal of GFP silencing

Virus Induced Gene Silencing (VIGS) is conserved and dynamic gene regulation machinery, which plays an active role in shielding plants against viral infections. In counter-defense, viruses encode proteins that can suppress RNA silencing to ensure successful systemic invasion. The AC2 protein of Tomato Leaf Curl New Delhi Virus (ToLCV-AC2) and Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are well-characterized suppressors of RNA silencing. In this study we have compared the structure and suppressor strength of ToLCV-AC2 and MYMIV-AC2, to gain insight on the conservation of the RNA silencing suppression activities. The relative suppressor strength was measured using an assay based on the reversal of GFP silencing and quantitated in terms of the amount of reversal of GFP expression and the sustenance of the reversal activity. The phenotypic observations in the reversal of silencing assay were further corroborated with molecular analysis. Our results indicate that ToLCV-AC2 exhibited weaker RNA silencing suppression than MYMIV-AC2. The suppression activity of ToLCV-AC2 is supplemented by related, though weak, activities encoded by ToLCV-AC4 and ToLCV-AV2 proteins. Collectively the three ToLCV proteins showed suppressor strength comparable to MYMIV-AC2. These results indicate that in all probability the ToLCV deploys more than one RNA silencing suppressor protein to synergistically counteract the host RNA silencing mechanism.

Artificially induced phased siRNAs promote virus resistance in transgenic plants

We previously developed transgenic tobacco plants that were resistant to two geminiviruses. We generated resistance using RNAi constructs that produced trans-acting siRNA (tasiRNA) like secondary siRNAs known as phased siRNA (phasiRNA) that targeted several regions of Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Tomato Leaf Curl Gujarat Virus (ToLCGV) transcripts encoding the RNA silencing suppressor proteins AC2 and AC4. Here, we performed degradome analysis to determine the precise cleavage sites of RNA-RNA interaction between phasiRNA and viral transcripts. We then applied our RNAi technology in tomato, which is the natural host for ToLCNDV and ToLCGV. The relative ease of developing and using phasiRNA constructs represents a significant technical advance in imparting virus resistance in crops and/or important model systems.

A sensitive and rapid RNA silencing suppressor activity assay based on alfalfa mosaic virus expression vector

Plant viruses express RNA silencing suppressor (RSS) proteins to counteract plant defence mechanisms. Here, we describe a method to assess the RSS activity based on an alfalfa mosaic virus (AMV) RNA 3 expression vector and transgenic Nicotiana tabacum plants that express the P1 and P2 subunits of the AMV replicase (P12 plants). Inoculation of P12 plants with different AMV RNA 3 constructs expressing different HC-Pro mutants that differ in their RSS capabilities, revealed a perfect correlation between necrotic lesions on inoculated leaves and RSS activity. Protoplast analysis showed that the RSS activity correlated with the accumulation of the AMV RNAs. A direct comparison between three RSS activity assays and the AMV-P12 system revealed that the coat protein of carnation mottle virus displays RSS activity with the four assays and reduced the accumulation of the siRNAs.

Plant defense systems against cucumber mosaic virus: lessons learned from CMV–Arabidopsis interactions

To protect against infection, plants have evolved multi-layered defense systems such as RNA silencing, salicylic acid (SA)-mediated resistance, resistance (R) protein-conferred resistance and defense priming. Antiviral defense systems seem to act in a coordinated manner in response to viral infection, but the underlying mechanisms are unknown except in a limited number of specific virus–plant combinations. Cucumber mosaic virus (CMV) infection of Arabidopsis thaliana is a well-known sophisticated pathosystem often used to investigate antiviral defense reactions. On the basis of extensive studies on the interaction between CMV and A. thaliana, RNA silencing plays a key role in suppressing CMV infection in the host plant. However, several strains of CMV are able to overcome the RNA silencing system by producing an RNA silencing suppressor protein. If the virulent CMV strain is indeed able to overcome basal antiviral RNA silencing, host plants carrying the R gene exhibit a resistance response, generally accompanied by necrotic local lesions at the primary infection sites. RCY1, which encodes an NB-LRR class R protein, was isolated from A. thaliana and identified as the first R gene conferring resistance to CMV. SA-dependent and -independent signaling pathways also seem to be associated with CMV resistance, while in A. thaliana, resistance induced by a plant activator treatment is thought to activate defense priming against a broad range of pathogens. In this review, we summarize current knowledge on the multiple defense systems of A. thaliana against CMV infection.

Expression profiling across wild and cultivated tomatoes supports the relevance of early miR482/2118 suppression for Phytophthora resistance.

Plants possess a battery of specific pathogen resistance (R-)genes. Precise R-gene regulation is important in the presence and absence of a pathogen. Recently, a microRNA family, miR482/2118, was shown to regulate the expression of a major class of R-genes, nucleotide-binding site leucine-rich repeats (NBS-LRRs). Furthermore, RNA silencing suppressor proteins, secreted by pathogens, prevent the accumulation of miR482/2118, leading to an upregulation of R-genes. Despite this transcriptional release of R-genes, RNA silencing suppressors positively contribute to the virulence of some pathogens. To investigate this paradox, we analysed how the regulation of NBS-LRRs by miR482/2118 has been shaped by the coevolution between Phytophthora infestans and cultivated and wild tomatoes. We used degradome analyses and qRT-PCR to evaluate and quantify the co-expression of miR482/2118 and their NBS-LRR targets. Our data show that miR482/2118-mediated targeting contributes to the regulation of NBS-LRRs in Solanum lycopersicum. Based on miR482/2118 expression profiling in two additional tomato species—with different coevolutionary histories with P. infestans—we hypothesize that pathogen-mediated RNA silencing suppression is most effective in the interaction between S. lycopersicum and P. infestans. Furthermore, an upregulation of miR482/2118 early in the infection may increase susceptibility to P. infestans.

Biochemical analysis of NSs from different tospoviruses

Tospoviruses suppress antiviral RNA interference by coding for an RNA silencing suppressor (NSs) protein. Previously, using NSs-containing crude plant and insect cell extracts, the affinity of NSs for double-stranded (ds)RNA molecules was demonstrated by electrophoretic mobility shifts assays (EMSAs). While NSs from tomato spotted wilt virus (TSWV) and groundnut ringspot virus (GRSV) were able to bind small and long dsRNA molecules, the one from tomato yellow ring virus (TYRV), a distinct Asian tospovirus, only bound small dsRNA. Here, using bacterially expressed and purified NSs from GRSV and TYRV, it is shown that they are both able to bind to small and long dsRNA. Binding of siRNAs by NSs revealed two consecutive shifts, i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When NSs of TSWV resistance inducing (RI) and resistance breaking (RB) isolates were analyzed using extracts from infected plants only a major siRNA shift was observed. In contrast, plant extracts containing the respective transiently expressed NSs proteins showed only the lower shift with NSsRI but no shift with NSsRB. The observed affinity for RNA duplexes, as well as the two-stepwise shift pattern, is discussed in light of NSs as a suppressor of silencing and its importance for tospovirus infection.

Identification and characterization of two RNA silencing suppressors encoded by ophioviruses

Citrus psorosis virus and Mirafiori lettuce big-vein virus are two members of the genus Ophiovirus, family Ophioviridae. So far, how these viruses can interfere in the antiviral RNA silencing pathway is not known. In this study, using a local GFP silencing assay on Nicotiana benthamiana, the 24K–25K and the movement protein (MP) of both viruses were identified as RNA silencing suppressor proteins. Upon their co-expression with GFP in N. benthamiana 16c plants, the proteins also showed to suppress systemic RNA (GFP) silencing. The MPCPsV and 24KCPsV proteins bind long (114 nucleotides) but not short-interfering (21 nt) dsRNA, and upon transgenic expression, plants showed developmental abnormalities that coincided with an altered miRNA accumulation pattern. Furthermore, both proteins were able to suppress miRNA-induced silencing of a GFP-sensor construct and the co-expression of MPCPsV and 24KCPsV exhibited a stronger effect, suggesting they act at different stages of the RNAi pathway.

Functional analysis of a weak viral RNA silencing suppressor using two GFP variants as silencing inducers

RNA silencing in plants can be triggered by the introduction of an exogenous gene. Green fluorescent protein (GFP) has been widely used as a visual reporter to study RNA silencing and viral-mediated suppression of RNA silencing in the model plant Nicotiana benthamiana. In transgenic N. benthamiana plants expressing an endoplasmic reticulum targeted GFP variant (16c) known as mGFP5, RNA silencing can be induced by ectopic over-expression of mGFP5. However, other GFP variants can also be used to induce GFP silencing in these plants. We compared the efficiency to induce local and systemic silencing of two commonly used GFP variants: enhanced GFP (eGFP) and mGFP5. Using lettuce necrotic yellows virus (LNYV) P protein to suppress GFP silencing, we demonstrate that eGFP gene, which is 76% identical at the nucleotide level to the endogenously expressed mGFP5 in 16c plants, triggers silencing more slowly and concurrently prolongs detectable silencing suppressor activity of the weak LNYV P suppressor, compared to the homologous mGFP5 gene. The use of eGFP as RNA silencing inducer in wild type or 16c plants appears to be a useful tool in identifying and analysing weak viral RNA silencing suppressor proteins whose activity might otherwise have been masked when challenged by a stronger RNA silencing response. We also show that reducing the dosage of strong dsRNA silencing inducers in conjunction with their homologous GFP targets facilitates the discovery and analysis of “weaker” RNA silencing suppressor activities.

Transcriptomic changes in Nicotiana benthamiana plants inoculated with the wild-type or an attenuated mutant of Tobacco vein banding mosaic virus.

Tobacco vein banding mosaic virus (TVBMV) is a potyvirus which mainly infects solanaceous crops. The helper component proteinase (HCpro) of a potyvirus is an RNA silencing suppressor protein and determines the severity of disease symptoms caused by different potyviruses, including TVBMV. It has been shown that substitution mutations introduced into the HCpro open reading frame (ORF) in a TVBMV infectious clone result in changes of Asp189 to Lys or Ile250 -Gln251 to Asp-Glu (Asp, aspartic acid; Gln, glutamine; Glu, glutamic acid; Ile, isoleucine). These amino acid changes eliminate the RNA silencing suppression activity of the mutant HCpro (HCm) and attenuate the disease symptoms caused by the mutant TVBMV (T-HCm) in Nicotiana benthamiana plants. Here, we used RNA-sequencing technology to compare gene expression in plants inoculated with the wild-type TVBMV (T-WT) with that in plants inoculated with T-HCm at 1, 2 and 10 days post-agroinfiltration (dpai). At 1 and 2 dpai, N. benthamiana genes related to the translation machinery were up-regulated, whereas genes related to lipid biosynthesis and metabolism or to responses to extracellular/external stimuli were down-regulated in leaves inoculated with T-WT or T-HCm. At 10 dpai, T-WT infection repressed photosynthesis-related genes. T-WT and T-HCm infections differentially perturbed the genes involved in the RNA silencing pathway. The salicylic acid and ethylene signalling pathways were induced, but the jasmonic acid signalling pathway was repressed after T-WT infection. Infections of T-WT and T-HCm also differentially regulated the genes involved in auxin signalling transduction, which is known to associate with the stunting phenotypes caused by TVBMV. These results illustrate the dynamic nature of TVBMV infection in N. benthamiana at the transcriptomic level.

Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing

Plants employ RNA silencing as an innate defense mechanism against viruses. As a counter-defense, plant viruses have evolved to express RNA silencing suppressor proteins (RSS), which target one or more steps of the silencing pathway. In this study, we show that the phosphoprotein (P) encoded by the negative-sense RNA virus alfalfa dwarf virus (ADV), a species of the genus Cytorhabdovirus, family Rhabdoviridae, is a suppressor of RNA silencing. ADV P has a relatively weak local RSS activity, and does not prevent siRNA accumulation. On the other hand, ADV P strongly suppresses systemic RNA silencing, but does not interfere with the short-distance spread of silencing, which is consistent with its lack of inhibition of siRNA accumulation. The mechanism of suppression appears to involve ADV P binding to RNA-induced silencing complex proteins AGO1 and AGO4 as shown in protein–protein interaction assays when ectopically expressed. In planta, we demonstrate that ADV P likely functions by inhibiting miRNA-guided AGO1 cleavage and prevents transitive amplification by repressing the production of secondary siRNAs. As recently described for lettuce necrotic yellows cytorhabdovirus P, but in contrast to other viral RSS known to disrupt AGO activity, ADV P sequence does not contain any recognizable GW/WG or F-box motifs, which suggests that cytorhabdovirus P proteins may use alternative motifs to bind to AGO proteins.

Virus Infection of Plants Alters Pollinator Preference: A Payback for Susceptible Hosts?

Plant volatiles play important roles in attraction of certain pollinators and in host location by herbivorous insects. Virus infection induces changes in plant volatile emission profiles, and this can make plants more attractive to insect herbivores, such as aphids, that act as viral vectors. However, it is unknown if virus-induced alterations in volatile production affect plant-pollinator interactions. We found that volatiles emitted by cucumber mosaic virus (CMV)-infected tomato (Solanum lycopersicum) and Arabidopsis thaliana plants altered the foraging behaviour of bumblebees (Bombus terrestris). Virus-induced quantitative and qualitative changes in blends of volatile organic compounds emitted by tomato plants were identified by gas chromatography-coupled mass spectrometry. Experiments with a CMV mutant unable to express the 2b RNA silencing suppressor protein and with Arabidopsis silencing mutants implicate microRNAs in regulating emission of pollinator-perceivable volatiles. In tomato, CMV infection made plants emit volatiles attractive to bumblebees. Bumblebees pollinate tomato by ‘buzzing’ (sonicating) the flowers, which releases pollen and enhances self-fertilization and seed production as well as pollen export. Without buzz-pollination, CMV infection decreased seed yield, but when flowers of mock-inoculated and CMV-infected plants were buzz-pollinated, the increased seed yield for CMV-infected plants was similar to that for mock-inoculated plants. Increased pollinator preference can potentially increase plant reproductive success in two ways: i) as female parents, by increasing the probability that ovules are fertilized; ii) as male parents, by increasing pollen export. Mathematical modeling suggested that over a wide range of conditions in the wild, these increases to the number of offspring of infected susceptible plants resulting from increased pollinator preference could outweigh underlying strong selection pressures favoring pathogen resistance, allowing genes for disease susceptibility to persist in plant populations. We speculate that enhanced pollinator service for infected individuals in wild plant populations might provide mutual benefits to the virus and its susceptible hosts.

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.

Mungbean yellow mosaic Indian virus encoded AC2 protein suppresses RNA silencing by inhibiting Arabidopsis RDR6 and AGO1 activities

RNA silencing refers to a conserved RNA-directed gene regulatory mechanism in a wide range of eukaryotes. It plays an important role in many processes including growth, development, genome stability, and antiviral defense in the plants. Geminivirus encoded AC2 is identified as an RNA silencing suppressor protein, however, the mechanism of action has not been characterized. In this paper, we elucidate another mechanism of AC2-mediated suppression activity of Mungbean Yellow Mosaic India Virus (MYMIV). The AC2 protein, unlike many other suppressors, does not bind to siRNA or dsRNA species and its suppression activity is mediated through interaction with key components of the RNA silencing pathway, viz., RDR6 and AGO1. AC2 interaction inhibits the RDR6 activity, an essential component of siRNA and tasi-RNA biogenesis and AGO1, the major slicing factor of RISC. Thus the study identifies dual sites of MYMIV-AC2 interference and probably accounts for its strong RNA silencing suppression activity.

Function and diversity of P0 proteins among cotton leafroll dwarf virus isolates.

BackgroundThe RNA silencing pathway is an important anti-viral defense mechanism in plants. As a counter defense, some members of the viral family Luteoviridae are able to evade host immunity by encoding the P0 RNA silencing suppressor protein. Here we explored the functional diversity of P0 proteins among eight cotton leafroll dwarf virus (CLRDV) isolates, a virus associated with a worldwide cotton disease known as cotton blue disease (CBD).MethodsCLRDV-infected cotton plants of different varieties were collected from five growing fields in Brazil and their P0 sequences compared to three previously obtained isolates. P0’s silencing suppression activities were scored based on transient expression experiments in Nicotiana benthamiana leaves.ResultsHigh sequence diversity was observed among CLRDV P0 proteins, indicating that some isolates found in cotton varieties formerly resistant to CLRDV should be regarded as new genotypes within the species. All tested proteins were able to suppress local and systemic silencing, but with significantly variable degrees. All P0 proteins were able to mediate the decay of ARGONAUTE proteins, a key component of the RNA silencing machinery.ConclusionsThe sequence diversity observed in CLRDV P0s is also reflected in their silencing suppression capabilities. However, the strength of local and systemic silencing suppression was not correlated for some proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0356-7) contains supplementary material, which is available to authorized users.

Key elements of the RNAi pathway are regulated by hepatitis B virus replication and HBx acts as a viral suppressor of RNA silencing.

The host-mediated RNAi pathways restrict replication of viruses in plant, invertebrate and vertebrate systems. However, comparatively little is known about the interplay between RNAi and various viral infections in mammalian hosts. We show in the present study that the siRNA-mediated silencing of Drosha, Dicer and Ago2 [argonaute RISC (RNA-induced silencing complex) catalytic component 2] transcripts in Huh7 cells resulted in elevated levels of HBV (hepatitis B virus)-specific RNAs and, conversely, we observed a decrease in mRNA and protein levels of same RNAi components in HepG2 cells infected with HBV. Similar reductions were also detectable in CHB (chronic hepatitis B) patients. Analysis of CHB liver biopsy samples, with high serum HBV DNA load (>log108 IU/ml), revealed a reduced mRNA and protein levels of Drosha, Dicer and Ago2. The low expression levels of key RNAi pathway components in CHB patient samples as well as hepatic cells established a link between HBV replication and RNAi components. The HBV proteins were also examined for RSS (RNA-silencing suppressor) properties. Using GFP-based reversion of silencing assays, in the present study we found that HBx is an RSS protein. Through a series of deletions and substitution mutants, we found that the full-length HBx protein is required for optimum RSS activity. The in vitro dicing assays revealed that the HBx protein inhibited the human Dicer-mediated processing of dsRNAs into siRNAs. Together, our results suggest that the HBx protein might function as RSS to manipulate host RNAi defence, in particular by abrogating the function of Dicer. The present study may have implications in the development of newer strategies to combat HBV infection.