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 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 RNA Silencing Suppression by a Second Copy of the P1 Serine Protease of Cucumber Vein Yellowing Ipomovirus, a Member of the Family Potyviridae That Lacks the Cysteine Protease HCPro

2006 ◽  
Vol 80 (20) ◽  
pp. 10055-10063 ◽  
Author(s):  
Adrian Valli ◽  
Ana Montserrat Martín-Hernández ◽  
Juan José López-Moya ◽  
Juan Antonio García
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Serine Proteinase ◽  
Plum Pox Virus ◽  
Coding Region ◽  
Long Distance ◽  
Systemic Spread ◽  
The Family ◽  
Silencing Suppression ◽  
Green Fluorescent

ABSTRACT The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.

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.

scholarly journals Deciphering the RNA Silencing Suppressor Function in the Potyvirus SPV2

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 26
Author(s):  
Ornela Chase ◽  
Giannina Bambaren ◽  
Juan José López-Moya
Keyword(s):  
Rna Silencing ◽  
Ipomoea Batatas ◽  
Control Strategies ◽  
Regulation Of Gene Expression ◽  
Plant Viruses ◽  
High Yield ◽  
Gene Products ◽  
Silencing Suppressor ◽  
Sweet Potatoes ◽  
Rna Silencing Suppressor

In most eukaryotes, RNA silencing is a key element in the regulation of gene expression and defense against pathogens. Plants have developed a defensive barrier against exogenous microorganisms, such as plant-infecting viruses, by specifically targeting and degrading the viral RNAs and thus limiting the negative effects of the diseases caused by them. On the other hand, plant viruses encode for suppressor proteins that repress the host-silencing machinery, hence allowing viral replication and infection establishment. Our current project focuses on the characterization of gene products contributing to the RNA silencing suppressor (RSS) function of Sweet potato virus 2 (SPV2), genus Potyvirus, family Potyviridae. SPV2 infects sweet potatoes (Ipomoea batatas, family Convolvulaceae), one of the most important staple food crops worldwide. Infections by potyvirids result in the high yield losses of sweet potatoes, especially from coinfection with unrelated viruses, and our final goal is to develop efficient control strategies. Our preliminary results analyzing the P1 and HCPro proteases of SPV2, transiently expressed in N. benthamiana together with a reporter GFP construct, revealed that HCPro constitutes a strong RSS. This is a novel finding, and we are currently characterizing the functions of other gene products.

scholarly journals A Single Amino Acid Mutation in the Plum pox virus Helper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities

2005 ◽  
Vol 95 (8) ◽  
pp. 894-901 ◽  
Author(s):  
Pablo González-Jara ◽  
Felix A. Atencio ◽  
Belén Martínez-García ◽  
Daniel Barajas ◽  
Francisco Tenllado ◽  
...  
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Rna Silencing ◽  
Recombinant Virus ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Plum Pox Virus ◽  
Helper Component ◽  
Silencing Suppression ◽  
Helper Component Proteinase

The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL134H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL134H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.

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 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 The P1N-PISPOtrans-Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor

2016 ◽  
Vol 90 (7) ◽  
pp. 3543-3557 ◽  
Author(s):  
Ares Mingot ◽  
Adrián Valli ◽  
Bernardo Rodamilans ◽  
David San León ◽  
David C. Baulcombe ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sweet Potato ◽  
Rna Silencing ◽  
Mottle Virus ◽  
Gene Products ◽  
Silencing Suppressor ◽  
Content Type ◽  
Rna Silencing Suppressor ◽  
Silencing Suppression ◽  
Polymerase Slippage

ABSTRACTThe positive-sense RNA genome ofSweet potato feathery mottle virus(SPFMV) (genusPotyvirus, familyPotyviridae) contains a large open reading frame (ORF) of 3,494 codons translatable as a polyprotein and two embedded shorter ORFs in the −1 frame: PISPO, of 230 codons, and PIPO, of 66 codons, located in the P1 and P3 regions, respectively. PISPO is specific to some sweet potato-infecting potyviruses, while PIPO is present in all potyvirids. In SPFMV these two extra ORFs are preceded by conserved G2A6motifs. We have shown recently that a polymerase slippage mechanism at these sites could produce transcripts bringing these ORFs in frame with the upstream polyprotein, thus leading to P1N-PISPO and P3N-PIPO products (B. Rodamilans, A. Valli, A. Mingot, D. San Leon, D. B. Baulcombe, J. J. Lopez-Moya, and J.A. Garcia, J Virol 89:6965–6967, 2015, doi:10.1128/JVI.00337-15). Here, we demonstrate by liquid chromatography coupled to mass spectrometry that both P1 and P1N-PISPO are produced during viral infection and coexist in SPFMV-infectedIpomoea batatasplants. Interestingly, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene inNicotiana benthamianarevealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, our results reveal a further layer of complexity of the RNA silencing suppression activity within thePotyviridaefamily.IMPORTANCEGene products of potyviruses include P1, HCPro, P3, 6K1, CI, 6K2, VPg/NIaPro, NIb, and CP, all derived from the proteolytic processing of a large polyprotein, and an additional P3N-PIPO product, with the PIPO segment encoded in a different frame within the P3 cistron. In sweet potato feathery mottle virus (SPFMV), another out-of-frame element (PISPO) was predicted within the P1 region. We have shown recently that a polymerase slippage mechanism can generate the transcript variants with extra nucleotides that could be translated into P1N-PISPO and P3N-PIPO. Now, we demonstrate by mass spectrometry analysis that P1N-PISPO is indeed produced in SPFMV-infected plants, in addition to P1. Interestingly, while in other potyviruses the suppressor of RNA silencing is HCPro, we show here that P1N-PISPO exhibited this activity in SPFMV, revealing how the complexity of the gene content could contribute to supply this essential function in members of thePotyviridaefamily.

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.

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