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 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 The Benyvirus RNA Silencing Suppressor Is Essential for Long-Distance Movement, Requires Both Zinc-Finger and NoLS Basic Residues but Not a Nucleolar Localization for Its Silencing-Suppression Activity

2013 ◽  
Vol 26 (2) ◽  
pp. 168-181 ◽  
Author(s):  
Sotaro Chiba ◽  
Kamal Hleibieh ◽  
Alice Delbianco ◽  
Elodie Klein ◽  
Claudio Ratti ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Rna Silencing ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nucleolar Localization ◽  
Silencing Suppressor ◽  
Long Distance ◽  
Microscopy Imaging ◽  
Silencing Suppression ◽  
Long Distance Movement

The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.

scholarly journals The Cucumber vein yellowing virus Silencing Suppressor P1b Can Functionally Replace HCPro in Plum pox virus Infection in a Host-Specific Manner

2012 ◽  
Vol 25 (2) ◽  
pp. 151-164 ◽  
Author(s):  
Alberto Carbonell ◽  
Gabriela Dujovny ◽  
Juan Antonio García ◽  
Adrian Valli
Keyword(s):  
Rna Silencing ◽  
Deleterious Effect ◽  
Prunus Persica ◽  
Plant Viruses ◽  
Natural Host ◽  
Plum Pox Virus ◽  
Silencing Suppressor ◽  
Specific Manner ◽  
Silencing Suppression ◽  
Yellowing Virus

Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silencing suppressors (RSS) to counteract antiviral RNA silencing responses. HCPro is the RSS of Potyvirus spp., and its activity is enhanced by the upstream P1 protein. Distinctively, the ipomovirus Cucumber vein yellowing virus (CVYV) lacks HCPro but contains two P1 copies in tandem (P1aP1b), the second of which functions as RSS. Using chimeras based on the potyvirus Plum pox virus (PPV), we found that P1b can functionally replace HCPro in potyviral infections of Nicotiana plants. Interestingly, P1a, the CVYV protein homologous to potyviral P1, disrupted the silencing suppression activity of P1b and reduced the infection efficiency of PPV in Nicotiana benthamiana. Testing the influence of RSS in host specificity, we found that a P1b-expressing chimera poorly infected PPV's natural host, Prunus persica. Conversely, P1b conferred on PPV chimeras the ability to replicate locally in cucumber, CVYV's natural host. The deleterious effect of P1a on PPV infection is host dependent, because the P1aP1b-expressing PPV chimera accumulated in cucumber to higher levels than PPV expressing P1b alone. These results demonstrate that a potyvirus can use different RSS, and that particular RSS and upstream P1-like proteins contribute to defining the virus host range.

scholarly journals Role of RNA Silencing Suppression in the Pathogenicity and Host Specificity of the Grapevine Virus A

2010 ◽  
Author(s):  
Munir Mawassi ◽  
Valerian Dolja
Keyword(s):  
Rna Silencing ◽  
Defense Mechanism ◽  
Plant Viruses ◽  
Small Interfering Rnas ◽  
Grapevine Virus ◽  
Virus Transport ◽  
Systematic Analysis ◽  
Grapevine Virus A ◽  
Silencing Suppression ◽  
And Function

RNA silencing is a defense mechanism that functions against virus infection and involves sequence-specific degradation of viral RNA. Diverse RNA and DNA viruses of plants encode RNA silencing suppressors (RSSs), which, in addition to their role in viral counterdefense, were implicated in the efficient accumulation of viral RNAs, virus transport, pathogenesis, and determination of the virus host range. Despite rapidly growing understanding of the mechanisms of RNA silencing suppression, systematic analysis of the roles played by diverse RSSs in virus biology and pathology is yet to be completed. Our research was aimed at conducting such analysis for two grapevine viruses, Grapevine virus A (GVA) and Grapevine leafroll-associated virus-2 (GLRaV- 2). Our major achievements on the previous cycle of BARD funding are as follows. 1. GVA and GLRaV-2 were engineered into efficient gene expression and silencing vectors for grapevine. The efficient techniques for grapevine infection resulting in systemic expression or silencing of the recombinant genes were developed. Therefore, GVA and GLRaV-2 were rendered into powerful tools of grapevine virology and functional genomics. 2. The GVA and GLRaV-2 RSSs, p10 and p24, respectively, were identified, and their roles in viral pathogenesis were determined. In particular, we found that p10 functions in suppression and pathogenesis are genetically separable. 3. We revealed that p10 is a self-interactive protein that is targeted to the nucleus. In contrast, p24 mechanism involves binding small interfering RNAs in the cytoplasm. We have also demonstrated that p10 is relatively weak, whereas p24 is extremely strong enhancer of the viral agroinfection. 4. We found that, in addition to the dedicated RSSs, GVA and GLRaV-2 counterdefenses involve ORF1 product and leader proteases, respectively. 5. We have teamed up with Dr. Koonin and Dr. Falnes groups to study the evolution and function of the AlkB domain presents in GVA and many other plant viruses. It was demonstrated that viral AlkBs are RNA-specific demethylases thus providing critical support for the biological relevance of the novel process of AlkB-mediated RNA repair. 

scholarly journals Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors

2009 ◽  
Vol 90 (2) ◽  
pp. 519-525 ◽  
Author(s):  
Sandra Martínez-Turiño ◽  
Carmen Hernández
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Rna Silencing ◽  
Potato Virus X ◽  
Plant Viruses ◽  
Small Interfering Rnas ◽  
Silencing Suppression ◽  
Viral Suppressors

Viral-derived double-stranded RNAs (dsRNAs) activate RNA silencing, generating small interfering RNAs (siRNAs) which are incorporated into an RNA-induced silencing complex (RISC) that promotes homology-dependent degradation of cognate RNAs. To counteract this, plant viruses express RNA silencing suppressors. Here, we show that the coat protein (CP) of Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, is able to efficiently inhibit RNA silencing. Interestingly, PFBV CP blocked both sense RNA- and dsRNA-triggered RNA silencing and did not preclude generation of siRNAs, which is in contrast with the abilities that have been reported for other carmoviral CPs. We have also found that PFBV CP can bind siRNAs and that this ability correlates with silencing suppression activity and enhancement of potato virus X pathogenicity. Collectively, the results indicate that PFBV CP inhibits RNA silencing by sequestering siRNAs and preventing their incorporation into a RISC, thus behaving similarly to unrelated viral suppressors but dissimilarly to orthologous ones.

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 Diverse and Newly Recognized Effects Associated with Short Interfering RNA Binding Site Modifications on the Tomato Bushy Stunt Virus P19 Silencing Suppressor

2008 ◽  
Vol 83 (5) ◽  
pp. 2188-2200 ◽  
Author(s):  
Yi-Cheng Hsieh ◽  
Rustem T. Omarov ◽  
Herman B. Scholthof
Keyword(s):  
Symptom Severity ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Rna Binding ◽  
Tomato Bushy Stunt Virus ◽  
Silencing Suppressor ◽  
Virus Spread ◽  
Systemic Spread ◽  
Specific Activities ◽  
Interfering Rna

ABSTRACT The Tomato bushy stunt virus-encoded P19 forms dimers that bind duplex short interfering RNAs (siRNAs) to suppress RNA silencing. P19 is also involved in multiple host-specific activities, including the elicitation of symptoms, and in local and/or systemic spread. To study the correlation between those various roles and the siRNA binding by P19, predicted siRNA-interacting sites were modified. Twenty-two mutants were generated and inoculated onto Nicotiana benthamiana plants, to reveal that (i) they were all infectious, (ii) symptom differences did not correlate strictly with mutation-associated variation in P19 accumulation, and (iii) substitutions affecting a central domain of P19 generally exhibited symptoms more severe than for mutations affecting peripheral regions. Three mutants selected to represent separate phenotypic categories all displayed a substantially reduced ability to sequester siRNA. Consequently, these three mutants were compromised for systemic virus spread in P19-dependent hosts but had differential plant species-dependent effects on the symptom severity. One mutant in particular caused relatively exacerbated symptoms, exemplified by extensive morphological leaf deformations in N. benthamiana; this was especially remarkable because P19 was undetectable. Another striking feature of this mutant was that only within a few days after infection, viral RNA was cleared by silencing. One more original property was that host RNAs and proteins (notably, the P19-interactive Hin19 protein) were also susceptible to degradation in these infected N. benthamiana plants but not in spinach. In conclusion, even though siRNA binding by P19 is a key functional property, compromised siRNA sequestration can result in novel and diverse host-dependent properties.

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 Cymbidium Ringspot Virus Harnesses RNA Silencing To Control the Accumulation of Virus Parasite Satellite RNA

2008 ◽  
Vol 82 (23) ◽  
pp. 11851-11858 ◽  
Author(s):  
Vitantonio Pantaleo ◽  
József Burgyán
Keyword(s):  
Rna Silencing ◽  
Sequence Similarity ◽  
Helper Virus ◽  
Ringspot Virus ◽  
Silencing Suppressor ◽  
Short Interfering Rna ◽  
Satellite Rna ◽  
The Cost ◽  
Interfering Rna ◽  
Rna Replicon

ABSTRACT Cymbidium ringspot virus (CymRSV) satellite RNA (satRNA) is a parasitic subviral RNA replicon that replicates and accumulates at the cost of its helper virus. This 621-nucleotide (nt) satRNA species has no sequence similarity to the helper virus, except for a 51-nt-long region termed the helper-satellite homology (HSH) region, which is essential for satRNA replication. We show that the accumulation of satRNA strongly depends on temperature and on the presence of the helper virus p19 silencing suppressor protein, suggesting that RNA silencing plays a crucial role in satRNA accumulation. We also demonstrate that another member of the Tombusvirus genus, Carnation Italian ringspot virus (CIRV), supports satRNA accumulation at a higher level than CymRSV. Our results suggest that short interfering RNA (siRNA) derived from CymRSV targets satRNA more efficiently than siRNA from CIRV, possibly because of the higher sequence similarity between the HSH regions of the helper and CIRV satRNAs. RNA silencing sensor RNA carrying the putative satRNA target site in the HSH region was efficiently cleaved when transiently expressed in CymRSV-infected plants but not in CIRV-infected plants. Strikingly, replacing the CymRSV HSH box2 sequence with that of CIRV restores satRNA accumulation both at 24°C and in the absence of the p19 suppressor protein. These findings demonstrate the extraordinary adaptation of this virus to its host in terms of harnessing the antiviral silencing response of the plant to control the virus parasite satRNA.

scholarly journals Rapid screening of RNA silencing suppressors by using a recombinant virus derived from beet necrotic yellow vein virus

2009 ◽  
Vol 90 (10) ◽  
pp. 2536-2541 ◽  
Author(s):  
H. Guilley ◽  
D. Bortolamiol ◽  
G. Jonard ◽  
S. Bouzoubaa ◽  
V. Ziegler-Graff
Keyword(s):  
Rna Silencing ◽  
Plant Defence ◽  
Plant Viruses ◽  
Yellow Vein ◽  
Rapid Screening ◽  
Silencing Suppressor ◽  
Defence Mechanisms ◽  
Simple Method ◽  
Yellow Dwarf Virus ◽  
Plant Defence Mechanisms

To counteract plant defence mechanisms, plant viruses have evolved to encode RNA silencing suppressor (RSS) proteins. These proteins can be identified by a range of silencing suppressor assays. Here, we describe a simple method using beet necrotic yellow vein virus (BNYVV) that allows a rapid screening of RSS activity. The viral inoculum consisted of BNYVV RNA1, which encodes proteins involved in viral replication, and two BNYVV-derived replicons: rep3–P30, which expresses the movement protein P30 of tobacco mosaic virus, and rep5–X, which allows the expression of a putative RSS (X). This approach has been validated through the use of several known RSSs. Two potential candidates have been tested and we show that, in our system, the P13 protein of burdock mottle virus displays RSS activity while the P0 protein of cereal yellow dwarf virus-RPV does not.

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