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.     

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.     

Extreme Resistance as a Host Counter-counter Defense against Viral Suppression of RNA Silencing

RNA silencing mediated by small RNAs (sRNAs) is a conserved regulatory process with key antiviral and antimicrobial roles in eukaryotes. A widespread counter-defensive strategy of viruses against RNA silencing is to deploy viral suppressors of RNA silencing (VSRs), epitomized by the P19 protein of tombusviruses, which sequesters sRNAs and compromises their downstream action. Here, we provide evidence that specific Nicotiana species are able to sense and, in turn, antagonize the effects of P19 by activating a highly potent immune response that protects tissues against Tomato bushy stunt virus infection. This immunity is salicylate- and ethylene-dependent, and occurs without microscopic cell death, providing an example of “extreme resistance” (ER). We show that the capacity of P19 to bind sRNA, which is mandatory for its VSR function, is also necessary to induce ER, and that effects downstream of P19-sRNA complex formation are the likely determinants of the induced resistance. Accordingly, VSRs unrelated to P19 that also bind sRNA compromise the onset of P19-elicited defense, but do not alter a resistance phenotype conferred by a viral protein without VSR activity. These results show that plants have evolved specific responses against the damages incurred by VSRs to the cellular silencing machinery, a likely necessary step in the never-ending molecular arms race opposing pathogens to their hosts.     

Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-alpha-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function.     

A non-structural protein encoded by Rice Dwarf Virus targets to the nucleus and chloroplast and inhibits local RNA silencing

RNA silencing is a potent antiviral mechanism in plants and animals. As a counter-defense, many viruses studied to date encode one or more viral suppressors of RNA silencing (VSR). In the latter case, how different VSRs encoded by a virus function in silencing remains to be fully understood. We previously showed that the nonstructural protein Pns10 of a Phytoreovirus, Rice dwarf virus (RDV), functions as a VSR. Here we present evidence that another nonstructural protein, Pns11, also functions as a VSR. While Pns10 was localized in the cytoplasm, Pns11 was localized both in the nucleus and chloroplasts. Pns11 has two bipartite nuclear localization signals (NLSs), which were required for nuclear as well as chloroplastic localization. The NLSs were also required for the silencing activities of Pns11. This is the first report that multiple VSRs encoded by a virus are localized in different subcellular compartments, and that a viral protein can be targeted to both the nucleus and chloroplast. These findings may have broad significance in studying the subcellular targeting of VSRs and other viral proteins in viral-host interactions.

     

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.     

Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection

Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well-characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant-encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two-hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co-localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N-terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP-interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3-overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans-acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA-binding protein 4) and AGO2 (ARGONAUTE protein 2), to achieve efficient suppression of RNA silencing-mediated antiviral defence.     

Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses

Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0^Tu), PLRV (P0^PL) and Cucurbit aphid‐borne yellows virus (P0^CA) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0^PL only. Genetic analysis showed that recognition of P0^Tu by a resistance gene designated RPO1 (R esistance to PO leroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0^Tu mutants with substitutions in the F‐box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F‐box protein motif could indicate detection of P0‐induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance.     

The Cricket Paralysis Virus Suppressor Inhibits microRNA Silencing Mediated by the Drosophila Argonaute-2 Protein

Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.     

Multiple functions of Rice dwarf phytoreovirus Pns10 in suppressing systemic RNA silencing

RNA silencing is a potent mechanism of antiviral defense response in plants and other organisms. For counterdefense, viruses have evolved a variety of suppressors of RNA silencing (VSRs) that can inhibit distinct steps of a silencing pathway. We previously identified Pns10 encoded by Rice dwarf phytoreovirus (RDV) as a VSR, the first of its kind from double-stranded RNA (dsRNA) viruses. In this study we investigated the mechanisms of Pns10 function in suppressing systemic RNA silencing in the widely used Nicotiana benthamiana model plant. We report that Pns10 suppresses local and systemic RNA silencing triggered by sense mRNA, enhances viral replication and/or viral RNA stability in inoculated leaves, accelerates the systemic spread of viral infection, and enables viral invasion of shoot apices. Mechanistically, Pns10 interferes with the perception of silencing signals in recipient tissues, binds double-stranded small interfering RNA (siRNAs) with two-nucleotide 3' overhangs, and causes the downregulated expression of RDR6. These results significantly deepen our mechanistic understanding of the VSR functions encoded by a dsRNA virus and contribute additional evidence that binding siRNAs and interfering with RDR6 expression are broad mechanisms of VSR functions encoded by diverse groups of viruses.     

RNA沉默在植物生物逆境反应中的作用

RNA沉默是真核生物共有的基因表达调节机制和防御机制。在植物RNA沉默中, 一些小RNAs, 如微小 RNAs和小干扰RNAs, 在植物防御病毒、细菌或食草动物的反应中具有重要作用。为了抑制宿主的RNA沉默系统, 植物病毒或细菌进化出了在RNA沉默不同阶段起作用的病毒沉默抑制子或细菌沉默抑制子, 来克服寄主的RNA沉默反应。文章就植物RNA沉默、病毒沉默抑制子、细菌沉默抑制子及其相关防御反应的一些新进展做一概述。     

Structural insights into the arms race between host and virus along RNA silencing pathways in Arabidopsis thaliana

RNA silencing refers to a conserved sequence‐specific gene‐regulation mechanism mediated by small RNA molecules. In plants, microRNA (miRNA) and small interfering RNA (siRNA) represent two major types of small RNA molecules which play pivotal roles in plant developmental control and antiviral defences. To escape these plant defences, plant viruses have encoded a vast array of viral suppressors of RNA silencing (VSRs) to attack the host antiviral silencing pathway by interfering with small RNA processing, RNA‐induced silencing complex (RISC) assembly, viral mRNA cleavage etc. Transgenic plants expressing distinct VSRs often show developmental aberrations that resemble the phenotype of miRNA‐deficient mutants, implying a potential intrinsic link between VSRs and the miRNA pathway (at least in Arabidopsis thaliana) even though their pathogenic mechanisms remain largely unknown. In this review, we summarise our current structural understandings of the arms race between the host and virus along the RNA silencing pathway in A. thaliana by focusing on several important ribonucleoprotein (RNP) structures involved in RNA silencing and unique structural features adopted by VSRs.     

Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2

RNA interference (RNAi) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. Viruses have also gained counter‐defensive measures by producing virulence determinants called viral‐suppressors‐of‐RNAi (VSRs). Interestingly, in spite of dominance of interferon‐based immunity over RNAi in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of RNAi in mammalian cells. The present study focuses on the modulation of intracellular RNAi during infection with rotavirus (RV), an enteric virus with double‐stranded RNA genome. Intriguingly, a time point‐dependent bimodal regulation of RNAi was observed in RV‐infected cells, where short interfering RNA (siRNA)‐based RNAi was rendered non‐functional during early hours of infection only to be reinstated fully beyond that early infection stage. Subsequent investigations revealed RV nonstructural protein 1 to serve as a putative VSR by associating with and triggering degradation of Argonaute2 (AGO2), the prime effector of siRNA‐mediated RNAi, via ubiquitin–proteasome pathway. The proviral significance of AGO2 degradation was further confirmed when ectopic overexpression of AGO2 significantly reduced RV infection. Cumulatively, the current study presents a unique modulation of host RNAi during RV infection, highlighting the importance of antiviral RNAi in mammalian cells.     

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

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

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.