Viral suppressors of RNA silencing

The suppression of RNA silencing by plant viruses represents a viral adaptation to a novel host antiviral defense. Three types of viral suppressors have been identified through the use of a variety of silencing suppression assays. The first two types of suppressor are capable of a complete or partial reversal of pre-existing RNA silencing; the third type does not reverse RNA silencing but can instead prevent its systemic signaling.     

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.     

Effects and side-effects of viral RNA silencing suppressors on short RNAs

In eukaryotes, short RNAs play a crucial regulatory role in many processes including development, maintenance of genome stability and antiviral responses. These different but overlapping RNA-guided pathways are collectively termed 'RNA silencing'. To counteract an antiviral RNA silencing response, plant viruses express silencing suppressor proteins. Recent results have shown that silencing suppressors operate by modifying the accumulation and/or activity of short RNAs involved in the antiviral response. Because RNA silencing pathways intersect, silencing suppressors can also inhibit other short-RNA-regulated pathways. Thus, suppressors contribute to viral symptoms. These findings fuel further research to test whether certain symptoms caused by animal viruses are also manifestations of altered RNA regulatory pathways.     

Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level

Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defence against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defence. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. We showed that RNA silencing against a green fluorescent protein (GFP) reporter gene in the protoplasts could be induced rapidly and specifically by co-transfection with the reporter gene and various silencing inducers [i.e. siRNA, double-stranded RNA (dsRNA) or plasmid encoding dsRNA]. Using this system, we uncovered novel roles of some viral suppressors. Notably, the Cucumber mosaic virus 2b protein, shown previously to function predominantly by preventing the long-distance transmission of systemic silencing signals, was a very strong silencing suppressor in the protoplasts. Some suppressors thought to interfere with upstream steps of siRNA production appeared to also act downstream. Therefore, a viral suppressor can affect multiple steps of the RNA silencing pathway. Our analyses suggest that protoplast-based transient RNA silencing is a useful experimental system to investigate the functions of viral suppressors and further dissect the mechanistic details of the RNA silencing pathway in single cells.     

A new cell-to-cell transport model for Potexviruses

In the last five years, we have gained significant insight into the role of the Potexvirus proteins in virus movement and RNA silencing. Potexviruses require three movement proteins, named triple gene block (TGB)p1, TGBp2, and TGBp3, and the viral coat protein (CP) to facilitate viral cell-to-cell and vascular transport. TGBp1 is a multifunctional protein that has RNA helicase activity, promotes translation of viral RNAs, increases plasmodesmal size exclusion limits, and suppresses RNA silencing. TGBp2 and TGBp3 are membrane-binding proteins. CP is required for genome encapsidation and forms ribonucleoprotein complexes along with TGBp1 and viral RNA. This review considers the functions of the TGB proteins, how they interact with each other and CP, and how silencing suppression might be linked to viral transport. A new model of the mechanism for Potexvirus transport is proposed.     

Suppressors of RNA silencing encoded by plant viruses and their role in viral infections

RNA silencing as a robust host defense mechanism against plant viruses is generally countered by virus-encoded silencing suppressors. This strategy is now increasingly recognized to be used by animal viruses as well. We present here an overview of the common features shared by some of the better studied plant viral silencing suppressors. We then briefly describe the characteristics of the few reported animal viral suppressors, notably their extraordinary ability of cross-kingdom suppression. We next discuss the basis for biased protection of viral RNA and subviral parasites by silencing suppressors, the link between movement and silencing suppression, the influence of temperature on the outcome of viral infection and the effect of viral silencing suppressors on the microRNA pathway.     

Multiple domains of the tobacco mosaic virus p126 protein can independently suppress local and systemic RNA silencing

Small RNA-mediated RNA silencing is a widespread antiviral mechanism in plants and other organisms. Many viruses encode suppressors of RNA silencing for counter-defense. The p126 protein encoded by Tobacco mosaic virus (TMV) has been reported to be a suppressor of RNA silencing but the mechanism of its function remains unclear. This protein is unique among the known plant viral silencing suppressors because of its large size and multiple domains. Here, we report that the methyltransferase, helicase, and nonconserved region II (NONII) of p126 each has silencing-suppressor function. The silencing-suppression activities of methyltransferase and helicase can be uncoupled from their enzyme activities. Specific amino acids in NONII previously shown to be crucial for viral accumulation and symptom development are also crucial for silencing suppression. These results suggest that some viral proteins have evolved to possess modular structural domains that can independently interfere with host silencing, and that this may be an effective mechanism of increasing the robustness of a virus.     

植物病毒基因沉默抑制子研究进展

RNAi普遍存在于真核生物中,是植物应对外来病毒入侵的一种防御机制。但是植物病毒能通过产生不同的抑制子蛋白来抑制寄主基因沉默的发生。病毒抑制子通过干扰基因沉默的起始、siRNA的积累或干扰系统性基因沉默等方式抑制寄主的基因沉默。有的病毒抑制子蛋白还能促进病毒的积累和胞间移动,加强侵染组织的病毒病症状表现。主要阐述了RNAi的机制、病毒抑制PTGS的作用方式、几种常见的沉默抑制子以及抑制子与病毒侵染的关系。     

Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing

Viruses express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing-based host defenses. Although virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, small RNAs (sRNAs) and Argonaute (AGO) proteins seem to be the most frequent targets. Recently, GW/WG motifs of some VSRs have been proposed to dictate their suppressor function by mediating interaction with AGO(s). Here we have studied the VSR encoded by Pelargonium line pattern virus (family Tombusviridae). The results show that p37, the viral coat protein, blocks RNA silencing. Site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed generation of suppressor-competent and -incompetent molecules and uncoupling of the VSR and particle assembly capacities. The engineered mutants were used to assess the importance of p37 functions for viral infection and the relative contribution of diverse molecular interactions to suppressor activity. Two main conclusions can be drawn: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic Pelargonium line pattern virus infection, and (ii) the suppressor activity of p37 relies on the ability to bind sRNAs rather than on interaction with AGOs. The data also caution against potential misinterpretations of results due to overlap of sequence signals related to distinct protein properties. This is well illustrated by mutation of the GW motif in p37 that concurrently affects nucleolar localization, efficient interaction with AGO1, and sRNA binding capability. These concomitant effects could have been overlooked in other GW motif-containing suppressors, as we exemplify with the orthologous p38 of turnip crinkle virus.     

RNA silencing and antiviral defense in plants

Much progress has been made recently in identifying the molecular components of RNA silencing in plants, and in understanding their roles in the biogenesis of small interfering RNAs and microRNAs, in RNA-directed DNA methylation, and in RNA-mediated antiviral defense. However, many crucial questions remain unanswered. What are the molecular bases of sense and antisense transgene-mediated silencing? Why does silencing only appear to spread through transgenes? Plant viruses encode silencing suppressors to counteract host RNA silencing, and some of these suppressors affect microRNA accumulation and function and hence normal plant development. Is viral pathogenicity determined, partly or entirely, by their silencing suppressor activity?     

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.     

Cell-to-cell movement of potato potexvirus X is dependent on suppression of RNA silencing

RNA silencing in transgenic and virus-infected plants involves a mobile silencing signal that can move cell-to-cell and systemically through the plant. It is thought that this signal can influence long-distance movement of viruses because protein suppressors of silencing encoded in viral genomes are required for long-distance virus movement. However, until now, it was not known whether the mobile signal could also influence short-range virus movement between cells. Here, through random mutation analysis of the Potato Potexvirus X (PVX) silencing suppressor P25, we provide evidence that it does. All mutants that were defective for silencing suppression were also non-functional in viral cell-to-cell movement. However, we identified mutant P25 proteins that were functional as silencing suppressors but not as movement proteins and we conclude that suppression of silencing is not sufficient to allow virus movement between cells: there must be a second P25 function that is independent of silencing but also required for cell-to-cell movement. Consistent with this hypothesis, we identified two classes of suppressor-inactive P25 mutants. One class of these mutants is proposed to be functional for the accessory function because their failure to support PVX movement could be complemented by heterologous suppressors of silencing. The second class of P25 mutants is considered defective for both the suppressor and second functions because the heterologous silencing suppressors did not restore virus movement. It is possible, based on analyses of short interfering RNA accumulation, that P25 suppresses silencing by interfering with either assembly or function of the effector complexes of RNA silencing.     

Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.     

RNA silencing and plant viral diseases

RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.     

RNA silencing suppressor activity of Velvet bean severe mosaic begomovirus DNA-A encoded proteins

Velvet bean severe mosaic virus (VbSMV) is a bipartite DNA virus infecting Mucuna pruriens (Velvet bean), belongs to the genus Begomovirus in the family Geminiviridae. Velvet bean is a medicinal plant of enormous medicinal value. In the present study, it was delineated that proteins encoded by VbSMV viz. AV2 (pre-coat protein), AC2 (TrAP), AV1 (coat protein) are suppressors of RNA silencing as identified through Agrobacterium co-infiltration assays using Nicotiana benthamiana as a host plant. AV2 showed strong suppressor activity whereas AC1 and AV1 were found to be weak suppressors. To the best of our knowledge, this is the first report on identification of suppressor of RNA silencing encoded by VbSMV infecting a medicinal plant.     

Sterol isomerase HYDRA1 interacts with RNA silencing suppressor P1b and restricts potyviral infection

Plants use RNA silencing as a strong defensive barrier against virus challenges, and viruses counteract this defence by using RNA silencing suppressors (RSSs). With the objective of identifying host factors helping either the plant or the virus in this interaction, we have performed a yeast two‐hybrid screen using P1b, the RSS protein of the ipomovirus Cucumber vein yellowing virus (CVYV, family Potyviridae), as a bait. The C‐8 sterol isomerase HYDRA1 (HYD1), an enzyme involved in isoprenoid biosynthesis and cell membrane biology, and required for RNA silencing, was isolated in this screen. The interaction between CVYV P1b and HYD1 was confirmed in planta by Bimolecular Fluorescence Complementation assays. We demonstrated that HYD1 negatively impacts the accumulation of CVYV P1b in an agroinfiltration assay. Moreover, expression of HYD1 inhibited the infection of the potyvirus Plum pox virus, especially when antiviral RNA silencing was boosted by high temperature or by coexpression of homologous sequences. Our results reinforce previous evidence highlighting the relevance of particular composition and structure of cellular membranes for RNA silencing and viral infection. We report a new interaction of an RSS protein from the Potyviridae family with a member of the isoprenoid biosynthetic pathway.     

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/.