Suppression of both intra- and intercellular RNA silencing by the tombusviral P19 protein requires its small RNA binding property.
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ABSTRACT:
SUBMITTER: Brioudes F
PROVIDER: S-EPMC9328354 | biostudies-literature | 2022 Aug
REPOSITORIES: biostudies-literature
Publications
Suppression of both intra- and intercellular RNA silencing by the tombusviral P19 protein requires its small RNA binding property.
Brioudes Florian F Jay Florence F Voinnet Olivier O
The New phytologist 20220610 3
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Project description:RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19-siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing.
| S-EPMC381004 | biostudies-other
Project description:Transient transgenic expression accelerates pharming and facilitates protein studies in plants. One embodiment of the approach involves leaf infiltration of Agrobacterium strains whose T-DNA is engineered with the gene(s) of interest. However, gene expression during 'agro-infiltration' is intrinsically and universally impeded by the onset of post-transcriptional gene silencing (PTGS). Nearly 20 years ago, a simple method was developed, whereby co-expression of the tombusvirus-encoded P19 protein suppresses PTGS and thus enhances transient gene expression. Yet, how PTGS is activated and suppressed by P19 during the process has remained unclear to date. Here, we address these intertwined questions in a manner also rationalizing how vastly increased protein yields are achieved using a minimal viral replicon as a transient gene expression vector. We also explore, in side-by-side analyses, why some proteins do not accumulate to the expected high levels in the assay, despite vastly increased mRNA levels. We validate that enhanced co-expression of multiple constructs is achieved within the same transformed cells, and illustrate how the P19 system allows rapid protein purification for optimized downstream in vitro applications. Finally, we assess the suitability of the P19 system for subcellular localization studies - an originally unanticipated, yet increasingly popular application - and uncover shortcomings of this specific implement. In revisiting the P19 system using contemporary knowledge, this study sheds light onto its hitherto poorly understood mechanisms while further illustrating its versatility but also some of its limits.
| S-EPMC10107623 | biostudies-literature
Project description:Viral suppressors of RNA silencing, VSRs, counteract the antiviral RNA silencing of host plants by sequestration of virus-derived siRNAs. A central question concerns whether and how VSRs associate cellular miRNAs and thus modulate plant gene expression during a viral infection. In this study we characterize the binding behaviour of the tombusviral p19 protein to miRNAs by performing an RNA-pull down experiment with bead-associated p19 protein from carnation italian ringspot virus. For this, we used cytoplasmatic extracs of Nicotiana tabacum protoplasts as an RNA source. By applying Next Generation Sequencing (NGS) to the precipitated small RNAs, we were able to identify miRNAs specifically associating with the protein and other that were not efficiently bound by p19.
2018-05-09 | GSE98956 | GEO
Project description:RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5' nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination.
| S-EPMC5053613 | biostudies-literature
Project description:To control plant viruses, viral RNA silencing suppressors are important drug targets due to their key roles in interfering antiviral RNA silencing. Here we have presented a strategy, combining virtual and experimental screening, to discover the inhibitors of viral suppressor. By docking 157 026 compounds from a natural product library into P19 model, eighteen candidate compounds were selected. Candidates VS2, VS12, VS14 and VS15 displayed strong binding ability to P19 in the surface plasmon resonance imaging assay with K D values of 136.2, 111.6, 81.2 and 124.5 nM, respectively. Then the inhibition activities of these inhibitors on the association between P19 and siRNA were also affirmed by electrophoretic mobility shift assay. Moreover, the antiviral effects on plants showed that compounds VS14 and VS15 both exhibited antiviral activities against Tomato bushy stunt virus (TBSV) in vivo with inhibition rates of 32.35% and 16.61% in 11 dpi, respectively. This strategy would be a powerful tool for the discovery of novel antiviral agents and provide new insights into the control of plant viruses.
| S-EPMC9078884 | biostudies-literature
Project description:The p19 protein (p19) encoded from Tombusvirus is involved in various activities such as pathogenicity and virus transport. Recent studies have found that p19 is a plant suppressor of RNA silencing, which binds to short interfering RNAs (siRNAs) with high affinity. We use molecular dynamics (MD) simulations of the wild-type and mutant p19 protein (W39 and W42G) binding with a 21-nt siRNA duplex to study the p19-siRNA recognition mechanism and mutation effects. Our simulations with standard MD and steered molecular dynamics have shown that the double mutant structure is indeed much less stable than the wild-type, consistent with the recent experimental findings. Comprehensive structural analysis also shows that the W39/42G mutations first induce the loss of stacking interactions between p19 and siRNA, Trp(42)-Cyt1 (Cyt1 from the 5' to 3' strand) and Trp(39)-Gua'19 (Gua19 from the 3' to 5' strand), and then breaks the hydrophobic core formed by W39-W42 with nucleotide basepairs in the wild-type. The steered molecular dynamics simulations also show that the mutant p19 complex is "decompounded" very fast under a constant separation force, whereas the wild-type remains largely intact under the same steering force. Moreover, we have used the free energy perturbation to predict a binding affinity loss of 6.98 +/- 0.95 kcal/mol for the single mutation W39G, and 12.8 +/- 1.0 kcal/mol loss for the double mutation W39/42G, with the van der Waals interactions dominating the contribution ( approximately 90%). These results indicate that the W39/42G mutations essentially destroy the important p19-siRNA recognition by breaking the strong stacking interaction between Cyt1 and Gua'19 with end-capping tryptophans. These large scale simulations might provide new insights to the interactions and co-evolution relationship between RNA virus proteins and their hosts.
| S-EPMC2717360 | biostudies-literature
Project description:In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection.
| S-EPMC3916407 | biostudies-literature
Project description:In plants, non-cell autonomous RNA silencing spreads between cells and over long distances. Recent work has revealed insight on the genetic and molecular components essential for cell-to-cell movement of RNA silencing in Arabidopsis. Using a local RNA silencing assay, we report on a distinct mechanism that may govern the short-range (6-10 cell) trafficking of virus-induced RNA silencing from epidermal to neighbouring palisade and spongy parenchyma cells in Nicotiana benthamiana. This process involves a previously unrecognised function of the RNA-dependent RNA polymerase 6 (RDR6) gene. Our data suggest that plants may have evolved distinct genetic controls in intercellular RNA silencing among different types of cells.
| S-EPMC3381291 | biostudies-literature
Project description:Viral suppressors of RNA silencing (VSRSs) are a diverse group of viral proteins that have evolved to disrupt eukaryotic RNA silencing pathways, thereby contributing to viral pathogenicity. The p19 protein is a VSRS that selectively binds to short interfering RNAs (siRNAs) over microRNAs (miRNAs). Mutational analysis has identified single amino acid substitutions that reverse this selectivity through new high-affinity interactions with human miR-122. Herein, we report crystal structures of complexed p19-T111S (2.6 Å), p19-T111H (2.3 Å) and wild-type p19 protein (2.2 Å) from the Carnation Italian ringspot virus with small interfering RNA (siRNA) ligands. Structural comparisons reveal that these mutations do not lead to major changes in p19 architecture, but instead promote subtle rearrangement of residues and solvent molecules along the p19 midline. These observations suggest p19 uses many small interactions to distinguish siRNAs from miRNAs and perturbing these interactions can create p19 variants with novel RNA-recognition properties. DATABASE: Model data are deposited in the PDB database under the accession numbers 6BJG, 6BJH and 6BJV.
| S-EPMC6551489 | biostudies-literature
Project description: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.
| S-EPMC3874164 | biostudies-literature