scholarly journals The p122 Subunit of Tobacco Mosaic Virus Replicase Is a Potent Silencing Suppressor and Compromises both Small Interfering RNA- and MicroRNA-Mediated Pathways

2007 ◽  
Vol 81 (21) ◽  
pp. 11768-11780 ◽  
Author(s):  
Tibor Csorba ◽  
Aurelie Bovi ◽  
Tamás Dalmay ◽  
József Burgyán
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nuclear Export ◽  
Rna Silencing ◽  
Plant Viruses ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
In Planta ◽  
Interfering Rna

ABSTRACT One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3′ overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised.

scholarly journals Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b from Cucumber Vein Yellowing Ipomovirus

2007 ◽  
Vol 82 (2) ◽  
pp. 974-986 ◽  
Author(s):  
Adrian Valli ◽  
Gabriela Dujovny ◽  
Juan Antonio García
Keyword(s):  
Zinc Finger ◽  
Rna Silencing ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Plant Viruses ◽  
Bimolecular Fluorescence Complementation ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
Silencing Suppression ◽  
Interfering Rna

ABSTRACT The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement of Potato virus X pathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNA-induced silencing complex.

scholarly journals The Potyvirus Silencing Suppressor Protein VPg Mediates Degradation of SGS3 via Ubiquitination and Autophagy Pathways

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Xiaofei Cheng ◽  
Aiming Wang
Keyword(s):  
Mosaic Virus ◽  
Rna Silencing ◽  
Plant Viruses ◽  
Turnip Mosaic Virus ◽  
Host Protein ◽  
Ubiquitin Proteasome ◽  
Cellular Pathways ◽  
Viral Suppressors ◽  
Interfering Rna ◽  
Innate Antiviral Immunity

ABSTRACT RNA silencing is an innate antiviral immunity response of plants and animals. To counteract this host immune response, viruses have evolved an effective strategy to protect themselves by the expression of viral suppressors of RNA silencing (VSRs). Most potyviruses encode two VSRs, helper component-proteinase (HC-Pro) and viral genome-linked protein (VPg). The molecular biology of the former has been well characterized, whereas how VPg exerts its function in the suppression of RNA silencing is yet to be understood. In this study, we show that infection by Turnip mosaic virus (TuMV) causes reduced levels of suppressor of gene silencing 3 (SGS3), a key component of the RNA silencing pathway that functions in double-stranded RNA synthesis for virus-derived small interfering RNA (vsiRNA) production. We also demonstrate that among 11 TuMV-encoded viral proteins, VPg is the only one that interacts with SGS3. We furthermore present evidence that the expression of VPg alone, independent of viral infection, is sufficient to induce the degradation of SGS3 and its intimate partner RNA-dependent RNA polymerase 6 (RDR6). Moreover, we discover that the VPg-mediated degradation of SGS3 occurs via both the 20S ubiquitin-proteasome and autophagy pathways. Taken together, our data suggest a role for VPg-mediated degradation of SGS3 in suppression of silencing by VPg. IMPORTANCE Potyviruses represent the largest group of known plant viruses and cause significant losses of many agriculturally important crops in the world. In order to establish infection, potyviruses must overcome the host antiviral silencing response. A viral protein called VPg has been shown to play a role in this process, but how it works is unclear. In this paper, we found that the VPg protein of Turnip mosaic virus (TuMV), which is a potyvirus, interacts with a host protein named SGS3, a key protein in the RNA silencing pathway. Moreover, this interaction leads to the degradation of SGS3 and its interacting and functional partner RDR6, which is another essential component of the RNA silencing pathway. We also identified the cellular pathways that are recruited for the VPg-mediated degradation of SGS3. Therefore, this work reveals a possible mechanism by which VPg sabotages host antiviral RNA silencing to promote virus infection.

Polyacrylamide–agarose gel electrophoretic analysis of tobacco mosaic virus disassembly intermediates

1984 ◽  
Vol 62 (11) ◽  
pp. 2336-2339 ◽  
Author(s):  
Richard Hogue ◽  
Alain Asselin
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Gel Electrophoresis ◽  
Electrophoretic Analysis ◽  
Sucrose Density Gradient ◽  
Plant Viruses ◽  
Buffer System ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis

The intermediates of disassembly of the U1, U4, U6, and U7 strains of tobacco mosaic virus (TMV) induced by alkali and urea were directly analysed by electrophoresis in composite polyacrylamide (2.0 to 2.5% (w/v)) – agarose (0.5% (w/v)) gels, using a discontinuous buffer system with Tris, borate, EDTA, and sodium chloride (pH 8.3). The results show no difference between the disassembly patterns of the four strains. In the case of alkaline stripping, two new major partially stripped virus (PSV) particles (PSV 5a and PSV 5c) and several minor intermediates were characterized. The overall results indicate that polyacrylamide–agarose gel electrophoresis is superior to agarose gel electrophoresis and to sucrose density gradient ccntrifugation for the analytical separation of PSVs. Polyacrylamide–agarose gel electrophoresis is thus a rather simple, rapid, and efficient method of studying in vitro disassembly of plant viruses.

scholarly journals Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity

2005 ◽  
Vol 79 (11) ◽  
pp. 7217-7226 ◽  
Author(s):  
Zsuzsanna Mérai ◽  
Zoltán Kerényi ◽  
Attila Molnár ◽  
Endre Barta ◽  
Anna Válóczi ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Regulatory System ◽  
Sequence Specificity ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
Reading Frame ◽  
Dsrna Binding ◽  
Silencing Suppression ◽  
Orf 5

ABSTRACT RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.

scholarly journals Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein

2007 ◽  
Vol 81 (19) ◽  
pp. 10379-10388 ◽  
Author(s):  
Hannes Vogler ◽  
Rashid Akbergenov ◽  
Padubidri V. Shivaprasad ◽  
Vy Dang ◽  
Monika Fasler ◽  
...  
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Plant Viruses ◽  
Micro Rna ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
Suppressor Activity ◽  
Virus Family ◽  
Green Fluorescent

ABSTRACT Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.

scholarly journals Production of Platinum Atom Nanoclusters at One End of Helical Plant Viruses

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Yuri Drygin ◽  
Olga Kondakova ◽  
Joseph Atabekov
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Pt Nanoparticles ◽  
Potato Virus X ◽  
Plant Viruses ◽  
Platinum Atom ◽  
Protein Subunits ◽  
Virus Particles ◽  
Atom Clusters

Platinum atom clusters (Pt nanoparticles, Pt-NPs) were produced selectively at one end of helical plant viruses, tobacco mosaic virus (TMV) and potato virus X (PVX), when platinum coordinate compounds were reduced chemically by borohydrides. Size of the platinum NPs depends on conditions of the electroless deposition of platinum atoms on the virus. Results suggest that the Pt-NPs are bound concurrently to the terminal protein subunits and the 5′ end of encapsidated TMV RNA. Thus, a special structure of tobacco mosaic virus and potato X virus particles with nanoparticles of platinum, which looks like a push-pin with platinum head and virus needle, was obtained. Similar results were obtained with ultrasonically fragmented TMV particles. By contrast, the Pt-NPs fully filled the central axial hole ofin vitroassembled RNA-free TMV-like particles. We believe that the results presented here will be valuable in the fundamental understanding of interaction of viral platforms with ionic metals and in a mechanism of nanoparticles formation.

scholarly journals Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity

2010 ◽  
Vol 23 (3) ◽  
pp. 294-303 ◽  
Author(s):  
Inmaculada González ◽  
Llucia Martínez ◽  
Daria V. Rakitina ◽  
Mathew G. Lewsey ◽  
Félix A. Atencio ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Small Rnas ◽  
Rna Silencing ◽  
Silencing Suppressor ◽  
Suppressor Activity ◽  
Rna Silencing Suppressor ◽  
2B Protein

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.

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