Virus 2b Protein Research Articles

Strain-specific differences in the interactions of the cucumber mosaic virus 2b protein with the viral 1a and host Argonaute 1 proteins.

The cucumber mosaic virus (CMV) 2b protein was among the first discovered viral suppressors of RNA silencing. It has additional pro-viral functions through effects on plant defensive signaling pathways mediated by salicylic acid and jasmonic acid, the abscisic acid pathway and virus-induced drought resistance, and on host plant interactions with insect vectors. Many of these effects occur due to interaction with the important host RNA silencing component Argonaute 1 (AGO1). It was thought that only 2b proteins of "severe" CMV strains interacted with AGO1 and inhibited its microRNA-mediated "slicing" of cellular mRNAs and that the lack of interaction with AGO1 explained the moderate symptoms typically seen in plants infected with mild CMV strains. Our work overthrows this paradigm by showing that mild strain CMV 2b proteins can interact with AGO1, but their in vivo localization prevents them from interacting with AGO1 molecules present in the infected cell cytoplasm.

Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

A selective autophagy receptor VISP1 induces symptom recovery by targeting viral silencing suppressors

Selective autophagy is a double-edged sword in antiviral immunity and regulated by various autophagy receptors. However, it remains unclear how to balance the opposite roles by one autophagy receptor. We previously identified a virus-induced small peptide called VISP1 as a selective autophagy receptor that facilitates virus infections by targeting components of antiviral RNA silencing. However, we show here that VISP1 can also inhibit virus infections by mediating autophagic degradation of viral suppressors of RNA silencing (VSRs). VISP1 targets the cucumber mosaic virus (CMV) 2b protein for degradation and attenuates its suppression activity on RNA silencing. Knockout and overexpression of VISP1 exhibit compromised and enhanced resistance against late infection of CMV, respectively. Consequently, VISP1 induces symptom recovery from CMV infection by triggering 2b turnover. VISP1 also targets the C2/AC2 VSRs of two geminiviruses and enhances antiviral immunity. Together, VISP1 induces symptom recovery from severe infections of plant viruses through controlling VSR accumulation.

Myricetin Derivative LP11 Targets Cucumber Mosaic Virus 2b Protein to Achieve In Vivo Antiviral Activity in Plants.

Cucumber mosaic virus (CMV) 2b protein plays a key role in the process of CMV infecting plants and symptom formation and is a potential molecular target for the control of this important plant virus. The exploitation of antiviral compounds is one of the strategies with the highest input: output ratio in plant protection. In this study, the CMV 2b recombinant protein was cloned, purified, and identified as the target protein by mass spectrometry. Subsequently, we carried out preliminary functional screening of the LP series of myricetin derivatives designed and synthesized in our laboratory and commercial antiviral compounds by microscale thermophoresis (MST), which showed that LP compounds LP4, LP11, LP13, and LP20 interacted well with CMV 2b, with dissociation constant (Kd) values of 1.39, 0.88, 1.52, and 1.77 μM, respectively. Among the commercially available antiviral compounds, ningnanmycin (NNM) was the most active, with a Kd value of 4.09 μM. Then, the strongest binding force to CMV 2b was identified to be from LP11 by isothermal titration calorimetry (ITC) experiments, with a Kd of 1.19 μM. Among the commercial compounds, NNM had the strongest binding force with CMV 2b, with a Kd of 4.62 μM. Through the screening of commercial compounds and LP series compounds by MST and ITC, LP11, NNM (positive control), LP16 (negative control), and the blank control group were selected to test the in vivo impact of LP11 on CMV. Specifically, the screened compounds were sprayed onto CMV-inoculated Nicotiana benthamiana plants to determine their impact on the regulation of CMV pathogenic gene expression, symptoms, and virus titer. The results showed that LP11 had a strong ability to inhibit CMV infection of tobacco at the transcriptional and translational levels. By mutating the CMV 2b protein, the 15th amino acid leucine and the 18th amino acid methionine at the N-terminal region were shown to be potential sites for binding to compound LP11. This finding provided a theoretical basis for screening and developing anti-CMV agents.

Homeobox 27, a Homeodomain Transcription Factor, Confers Tolerances to CMV by Associating with Cucumber Mosaic Virus 2b Protein.

Transcription factors (TFs) play an important role in plant development; however, their role during viral infection largely remains unknown. The present study was designed to uncover the role transcription factors play in Cucumber mosaic virus (CMV) infection. During the screening of an Arabidopsis thaliana (Col-0) transcription factor library, using the CMV 2b protein as bait in the yeast two-hybrid system, the 2b protein interacted with Homeobox protein 27 (HB27). HB27 belongs to the zinc finger homeodomain family and is known to have a regulatory role in flower development, and responses to biotic and abiotic stress. The interaction between CMV 2b and HB27 proteins was further validated using in planta (bimolecular fluorescence complementation assay) and in vitro far-Western blotting (FWB) methods. In the bimolecular fluorescence complementation assay, these proteins reconstituted YFP fluorescence in the nucleus and the cytoplasmic region as small fluorescent dots. In FWB, positive interaction was detected using bait anti-MYC antibody on the target HB27-HA protein. During CMV infection, upregulation (~3-fold) of the HB27 transcript was observed at 14 days post-infection (dpi) in A. thaliana plants, and expression declined to the same as healthy plants at 21 dpi. To understand the role of the HB27 protein during CMV infection, virus accumulation was determined in HB27-overexpressing (HB27 OE) and knockout mutants. In HB27-overexpressing lines, infected plants developed mild symptoms, accumulating a lower virus titer at 21 dpi compared to wild-type plants. Additionally, knockout HB27 mutants had more severe symptoms and a higher viral accumulation than wild-type plants. These results indicate that HB27 plays an important role in the regulation of plant defense against plant virus infection.

Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b's efficient suppression of RNA silencing.

The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b’s nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b’s C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b’s nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b’s efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm.

NtRBP45, a nuclear RNA-binding protein of Nicotiana tabacum, facilitates post-transcriptional gene silencing.

The tobacco RBP45 is a nuclear RNA binding protein (RBP). In this study, we identified that the gene expression of NtRBP45 was significantly up‐regulated upon the Tobacco mosaic virus infection and the central region of the protein accounted for its nuclear localization. In particular, using a green fluorescent protein‐based transient suppression assay, we uncovered that the transiently overexpressed NtRBP45 was able to enhance local post‐transcriptional gene silencing (PTGS), facilitate siRNA accumulation, and compromise the RNA silencing suppression mediated by Tomato aspermy virus 2b protein. Deletion mutagenesis showed that both the N‐ and C‐terminal regions of NtRBP45 were necessary for enhancing PTGS. The data overall indicated a novel RNA silencing factor that might participate in antiviral defense.

Influenza A virus M2 protein triggers mitochondrial DNA-mediated antiviral immune responses

Cytosolic mitochondrial DNA (mtDNA) activates cGAS-mediated antiviral immune responses, but the mechanism by which RNA viruses stimulate mtDNA release remains unknown. Here we show that viroporin activity of influenza virus M2 or encephalomyocarditis virus (EMCV) 2B protein triggers translocation of mtDNA into the cytosol in a MAVS-dependent manner. Although influenza virus-induced cytosolic mtDNA stimulates cGAS- and DDX41-dependent innate immune responses, the nonstructural protein 1 (NS1) of influenza virus associates with mtDNA to evade the STING-dependent antiviral immunity. The STING-dependent antiviral signaling is amplified in neighboring cells through gap junctions. In addition, we find that STING-dependent recognition of influenza virus is essential for limiting virus replication in vivo. Our results show a mechanism by which influenza virus stimulates mtDNA release and highlight the importance of DNA sensing pathway in limiting influenza virus replication.

Phosphorylation regulates the subcellular localization of Cucumber Mosaic Virus 2b protein

The 2b protein of Cucumber mosaic virus has a role in nearly all steps of the viral cycle including cell-to-cell movement, symptom induction and suppression of antiviral RNA silencing. Previous studies demonstrated the presence of 2b protein in the nucleus and in cytoplasm as well. Phosphorylation site of 2b protein is conserved in all CMV isolates, including proposed constitute motifs for casein kinase II and cyclin-dependent kinase 2. To discern the impact of 2b protein phosphorylation, we created eight different mutants to mimic the non-phosporylated (serine to alanine) as well as the phosphorylated state (serine to aspartic acid) of the protein. We compared these mutants to the wild-type (Rs-CMV) virus in terms of symptom induction, gene silencing suppressor activity as well as in cellular localization. Here, in this study we confirmed the phosphorylation of 2b protein in vivo, both in infected N. benthamiana and in infiltrated patches. Mutants containing aspartic acid in the phosphorylation site accumulated only in the cytoplasm indicating that phosphorylated 2b protein could not enter the nucleus. We identified a conserved dual phosphorylation switch in CMV 2b protein, which equilibrates the shuttling of the 2b protein between the nucleus and the cytoplasm, and regulates the suppressor activity of the 2b protein.

The subcellular distribution of Cucumber mosaic virus LS2b protein, correlation between its nuclear localization and predicted nuclear localization signals

Cucumber mosaic virus (CMV) 2b protein, a gene silencing suppressor, shows cytoplasmic localization and predominantly nuclear localization due to its two nuclear localization signals (NLSs) regions. We used a series of fluorescent organelle markers and two cytoskeleton markers co-localized with GFP tagged Fny2b (the 2b protein encoded by the CMV Fny strain) fusion protein transiently expressed in leaf epidermal cells of Nicotiana benthamiana by agroinfiltration. Subcellular localization of Fny2b on microtubules was confirmed by perfectly merging with mCherry-MAP (Microtubule-associated protein) marker. High consistency of de-polymerization effect on GFP-Fny2b and microtubules after oryzalin treatment imaged by confocal scanning microscopy was an evidence of Fny2b microtubule localization. Western blot analysis showed that drug treatment affected solubility characteristics of Fny2b, however, oryzalin treatment did not affect its gene silencing suppression function. We need further research to investigate the effect of 2b protein subcellular distribution on its function. We also investigated the nuclear localization ability of transiently expressed GFP-GUS protein fused with various strains of 2b protein. Nuclear localization ability analysis of each 2b protein was obtained in this paper, which is expected to provide an experimental basis to further analyze the correlation between nuclear localization and its predicted nuclear localization signals function. Finally, we successfully verified the great capability of PGsNLS oligo nucleotides as an effective nuclear localization signal could localize macromolecular protein in nucleus.

Effects of the 2b Protein of Cucumber mosaic virus Subgroup IB Strain IA on Different Transgene-Induced RNA Silencing Pathways

The Cucumber mosaic virus (CMV) 2b protein has multiple activities as a viral suppressor of RNA silencing (VSR). Here, we characterized the 2b protein (IA2b) of a CMV-IA isolate. This IA2b protein has two nuclear localization signals, and when it was fused with GFP, the protein was found to be preferentially localized in the nucleus. A transgenic tobacco line, 2b8, expressing the IA2b gene showed an enhanced expression of an agroinfiltrated sense transgene, which indicated that IA2b has VSR activity. When 2b8 plants were crossed with plants showing sense-transgene-induced post-transcriptional gene silencing (S-PTGS), the IA2b protein caused the disappearance of small interfering RNAs (siRNAs) and the S-PTGS phenotype was converted to the overexpression phenotype. When 2b8 plants were crossed with plants showing inverted repeat-induced post-transcriptional gene silencing (IR-PTGS), the IA2b protein did not suppress the accumulation of siRNAs processed from hairpin RNAs and the silencing phenotype was maintained. When a tobacco plant showing an overexpressed phenotype of a sense transgene was crossed with IR-PTGS plants targeting the same transgene, the primary siRNAs that were produced by the IR-PTGS construct triggered the production of the secondary siRNAs through the RNA-dependent RNA polymerase6 (RDR6)-dependent pathway. The IA2b protein suppressed the secondary siRNA formation but not the primary siRNA production from hairpin RNAs, resulting in the silencing phenotype from IR-PTGS being maintained. These results indicate that IA2b suppresses RDR6-dependent siRNA production and does not interfere with the silencing step by siRNA-guided RNA degradation.

Interaction between Cucumber mosaic virus 2b protein and plant catalase induces a specific necrosis in association with proteasome activity

Key messageCucumber mosaic virus(CMV) can induce a specific necrosis onArabidopsisthrough the interaction between the CMV 2b protein and host catalase, in which the ubiquitin–proteasome pathway may be involved.We previously reported that the CMV 2b protein, the viral RNA silencing suppressor, interacted with the H2O2 scavenger catalase (CAT3), leading to necrosis on CMV-inoculated Arabidopsis leaves. We here confirmed that CMV could more abundantly accumulate in the CAT3-knockout mutant (cat3), and that CAT3 makes host plants a little more tolerant to CMV. We also found that the necrosis severity is not simply explained by a high level of H2O2 given by the lack of CAT3, because the recombinant CMV, CMV-N, induced much milder necrosis in cat3 than in the wild type, suggesting some specific mechanism for the necrosis induction. To further characterize the 2b-inducing necrosis in relation to its binding to CAT3, we conducted the agroinfiltration experiments to overexpress CAT3 and 2b in N. benthamiana leaves. The accumulation levels of CAT3 were higher when co-expressed with the CMV-N 2b (N2b) than with CMV-Y 2b (Y2b). We infer that N2b made a more stable complex with CAT3 than Y2b did, and the longevity of the 2b–CAT3 complex seemed to be important to induce necrosis. By immunoprecipitation (IP) with an anti-ubiquitin antibody followed by the detection with anti-CAT3 antibodies, we detected a higher molecular-weight smear and several breakdown products of CAT3 among the IP-proteins. In addition, the proteasome inhibitor MG132 treatment could actually increase the accumulation levels of CAT3. This study suggests that the host proteasome pathway is, at least partially, responsible for the degradation of CAT3, which is manifested in CMV-infected tissues.

CMV2b-AGO Interaction Is Required for the Suppression of RDR-Dependent Antiviral Silencing in Arabidopsis.

Using a transient plant system, it was previously found that the suppression of Cucumber mosaic virus (CMV) 2b protein relies on its double-strand (ds) RNA binding capacity, but it is independent of its interaction with ARGONAUTE (AGO) proteins. Thus, the biological meaning of the 2b-AGO interaction in the context of virus infection remains elusive. In this study, we created infectious clones of CMV mutants that expressed the 2b functional domains of dsRNA or AGO binding and tested the effect of these CMV mutants on viral pathogenicity. We found that the mutant CMV2b(1–76) expressing the 2b dsRNA-binding domain exhibited the same virulence as wild-type CMV in infection with either wild-type Arabidopsis or rdr1/6 plants with RDR1- and RDR6-deficient mutations. However, remarkably reduced viral RNA levels and increased virus (v)siRNAs were detected in CMV2b(1–76)-infected Arabidopsis in comparison to CMV infection, which demonstrated that the 2b(1–76) deleted AGO-binding domain failed to suppress the RDR1/RDR6-dependent degradation of viral RNAs. The mutant CMV2b(8–111) expressing mutant 2b, in which the N-terminal 7 amino acid (aa) was deleted, exhibited slightly reduced virulence, but not viral RNA levels, in both wild-type and rdr1/6 plants, which indicated that 2b retained the AGO-binding activity acquired the counter-RDRs degradation of viral RNAs. The deletion of the N-terminal 7 aa of 2b affected virulence due to the reduced affinity for long dsRNA. The mutant CMV2b(18–111) expressing mutant 2b lacked the N-terminal 17 aa but retained its AGO-binding activity greatly reduced virulence and viral RNA level. Together with the instability of both 2b(18–111)-EGFP and RFP-AGO4 proteins when co-expressed in Nicotiana benthamiana leaves, our data demonstrates that the effect of 2b-AGO interaction on counter-RDRs antiviral defense required the presence of 2b dsRNA-binding activity. Taken together, our findings demonstrate that the dsRNA-binding activity of the 2b was essential for virulence, whereas the 2b-AGO interaction was necessary for interference with RDR1/6-dependent antiviral silencing in Arabidopsis.

Genetically modified Tomato aspermy virus 2b protein as a tumor-targeting siRNA delivery carrier

In nature, there exist a wide range of dsRNA-binding proteins that have different binding modes for small interfering RNA (siRNA) as well as structural differences, and some of these proteins have potential as effective siRNA delivery carriers. In order to deliver siRNA into cancer cells, a dsRNA-binding 2b protein derived from Tomato aspermy virus was genetically modified by fusing the integrin-targeting RGD peptide to its C-terminus, and biosynthesized. The resulting 2b-RGD protein possesses distinct characteristics favorable for biomedical applications of siRNA: (i) high affinity for siRNA, (ii) siRNA protection against RNases in serum, (iii) low cytotoxicity compared to the polycationic polymers often employed in conventional siRNA carriers, (iv) specific binding to integrins on cancer cells, and the ability to pass through the cell membrane via endocytosis, and (v) the ability to facilitate cytosolic release of siRNA. Here, we demonstrate that the 2b-RGD/siRNA complexes have great potential as a tumor-targeting siRNA delivery carrier and suggest their possible therapeutic applications for cancer treatment.

A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions

Localized expression of genes in plants from T-DNAs delivered into plant cells by Agrobacterium tumefaciens is an important tool in plant research. The technique, known as agroinfiltration, provides fast, efficient ways to transiently express or silence a desired gene without resorting to the time-consuming, challenging stable transformation of the host, the use of less efficient means of delivery, such as bombardment, or the use of viral vectors, which multiply and spread within the host causing physiological alterations themselves. A drawback of the agroinfiltration technique is its temperature dependence: early studies have shown that temperatures above 29 °C are nonpermissive to tumour induction by the bacterium as a result of failure in pilus formation. However, research in plant sciences is interested in studying processes at these temperatures, above the 25 °C experimental standard, common to many host-environment and host-pathogen interactions in nature, and agroinfiltration is an excellent tool for this purpose. Here, we measured the efficiency of agroinfiltration for the expression of reporter genes in plants from T-DNAs at the nonpermissive temperature of 30 °C, either transiently or as part of viral amplicons, and envisaged procedures that allow and optimize its use for gene expression at this temperature. We applied this technical advance to assess the performance at 30 °C of two viral suppressors of silencing in agropatch assays [Potato virus Y helper component proteinase (HCPro) and Cucumber mosaic virus 2b protein] and, within the context of infection by a Potato virus X (PVX) vector, also assessed indirectly their effect on the overall response of the host Nicotiana benthamiana to the virus.

Indirect immunofluorescence images of poliovirus‐infected cells. Blue, BODIPY‐cholesterol; Green, virus 2B protein; Red, phosphatidylinositol 4‐phosphate

Indirect immunofluorescence images of poliovirus‐infected cells. Blue, BODIPY‐cholesterol; Green, virus 2B protein; Red, phosphatidylinositol 4‐phosphate

Nuclear-Cytoplasmic Partitioning of Cucumber Mosaic Virus Protein 2b Determines the Balance between Its Roles as a Virulence Determinant and an RNA-Silencing Suppressor

ABSTRACTThe Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.IMPORTANCE In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.

Self‐interaction of the cucumber mosaic virus 2b protein plays a vital role in the suppression of RNA silencing and the induction of viral symptoms

The cucumber mosaic virus (CMV) 2b protein is an RNA silencing suppressor protein that can also play direct and indirect roles in symptom induction. Previous work has shown that a hybrid virus, FRad35(2b) -CMV (renamed here as CMV-FRad2b-Pro), generated by replacement of the 2b gene of strain Fny-CMV with that from Rad35-CMV, displays markedly lower pathogenicity than Fny-CMV on Nicotiana species. However, the replacement of proline with leucine at position 55 of the 2b protein of CMV-FRad2b-Pro (protein Rad2b-Pro) created a virus (CMV-FRad2b-Leu) that induced severe symptoms. Infection of Arabidopsis thaliana mutants defective in the expression of DICER-like (DCL) endoribonucleases 2 and 4, which mediate antiviral RNA silencing, as well as of dcl3 and dcl2/3/4 triple-mutant plants, indicated that Rad2b-Pro was a weaker RNA silencing suppressor than the protein Rad2b-Leu. This was confirmed in Nicotiana benthamiana using agroinfiltration assays, showing that, compared with either Rad2b-Leu or the Fny2b protein, Rad2b-Pro was ineffective at inhibiting local or systemic silencing of expression of a green fluorescent protein reporter gene. Transgenic expression of Rad2b-Leu, but not of Rad2b-Pro, in Arabidopsis induced symptom-like phenotypes and rescued the accumulation of the 2b-deletion mutant Fny-CMVΔ2b. Bimolecular fluorescent complementation indicated that, in planta, Rad2b-Leu, but not Rad2b-Pro, self-interacts. Thus, self-interaction is crucial to the ability of the 2b protein to suppress silencing and induce a symptom-like phenotype, and is dependent on the properties of the residue at position 55.

The C-terminal domain of the 2b protein of Cucumber mosaic virus is stabilized by divalent metal ion coordination

The main function of the 2b protein of Cucumber mosaic virus (CMV) is binding permanently the double stranded siRNA molecules in the suppression process of post-transcriptional gene silencing (PTGS). The crystal structure of the homologue Tomato aspermy virus (TAV) 2b protein is known, but without the C-terminal domain. The biologically active form is a tetramer: four 2b protein molecules and two siRNA duplexes. Regarding the complete 2b protein structure, we performed a molecular dynamics (MD) simulation of the whole siRNA–2b ribonucleoprotein complex. Unfortunately, the C-terminal domain is proved to be partially unstructured. Multiple sequence alignment showed a well conserved motif between residues 94 and 105. The negatively charged residues of the C-terminal domain are supposed to take part in coordination of a divalent metal ion and stabilize the three-dimensional structure of the C-terminal domain. MD simulations were performed on the detached C-terminal domains (aa 65–110). 0.15M MgCl2, CaCl2, FeCl2 and ZnCl2 salt concentrations were used in the screening simulations. Among the tested divalent metal ions Mg2+ proved to be very successful because Asp95, Asp96 and Asp98 forms a quasi-permanent Mg2+ binding site. However the control computations have resulted in any (at least) divalent metal ion remains in the binding site after replacement of the bound Mg2+ ion. A quadruple mutation (Rs2DDTD/95–98/AAAA) was introduced into the position of the putative divalent metal ion binding site to analyze the biological relevance of molecular modeling derived hypothesis. The plant inoculation experiments proved that the movement of the mutant virus is slower and the symptoms are milder comparing to the wild type virus. These results demonstrate that the quadruple mutation weakens the stability of the 2b protein tetramer–siRNA ribonucleoprotein complex.

Cucumber mosaic virus and its 2b RNA silencing suppressor modify plant-aphid interactions in tobacco.

The cucumber mosaic virus (CMV) 2b protein not only inhibits anti-viral RNA silencing but also quenches transcriptional responses of plant genes to jasmonic acid, a key signalling molecule in defence against insects. This suggested that it might affect interactions between infected plants and aphids, insects that transmit CMV. We found that infection of tobacco with a 2b gene deletion mutant (CMVΔ2b) induced strong resistance to aphids (Myzus persicae) while CMV infection fostered aphid survival. Using electrical penetration graph methodology we found that higher proportions of aphids showed sustained phloem ingestion on CMV-infected plants than on CMVΔ2b-infected or mock-inoculated plants although this did not increase the rate of growth of individual aphids. This indicates that while CMV infection or certain viral gene products might elicit aphid resistance, the 2b protein normally counteracts this during a wild-type CMV infection. Our findings suggest that the 2b protein could indirectly affect aphid-mediated virus transmission.