Absence of a coding region for the helper component-proteinase in the genome of cucumber vein yellowing virus, a whitefly-transmitted member of the Potyviridae

Janssen, Dirk; Martín, G.; Velasco, L.; Gómez, Pedro Sánchez; Segundo, E.; Ruiz, Leticia; Cuadrado, I. M.

doi:10.1007/s00705-005-0515-z

Cited by 53 publications

(57 citation statements)

References 28 publications

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“…Primers 7-CVYV and VYVC-1667rev were used in one of the reactions to amplify a fragment including CVYV nt 7 to 1667 (numbering is according to the sequence published by Janssen et al [20]). This fragment was cloned into pCRII by the TOPO cloning system (Invitrogen) to create pCRII-5ЈP1.…”

Section: Methodsmentioning

confidence: 99%

“…However, CVYV differed from another ipomovirus, Sweet potato mild mottle virus (SPMMV) and from the remaining sequenced monopartite members of the family Potyviridae, in that it lacks a sequence coding for a putative HCPro. CVYV showed an exceptionally large P1 protein, with a C-terminal serine protease domain similar to those of the P1s of members of the genus Potyvirus (20). The putative cleavage site separating CVYV P1 and P3 was also similar to the P1-HCPro junction of potyviruses.…”

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confidence: 91%

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RNA Silencing Suppression by a Second Copy of the P1 Serine Protease ofCucumber Vein Yellowing Ipomovirus, a Member of the FamilyPotyviridaeThat Lacks the Cysteine Protease HCPro

Vallí

Martín-Hernández

López‐Moya

et al. 2006

J Virol

112

123

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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.The regulatory systems of gene expression mediated by sequence-specific RNA silencing are mechanisms conserved in a wide variety of eukaryotic organisms (50). These pathways are triggered by double-stranded RNA (dsRNA), which is recognized by Dicer-like RNases to produce short dsRNAs of 21 to 26 nucleotides (nt) in length (small interfering RNAs [siRNAs]) (3, 32). One strand of these small RNAs is then incorporated into different silencing effector complexes, guiding them, by sequence complementarity, to degrade mRNA, inhibit RNA translation, or interfere with transcription by chromatin rearrangements (47).Different RNA silencing pathways have been identified, and one of them, active in the cytoplasm, has been shown to play an antiviral role in eukaryotes, where viruses generate dsRNA tracts in replicative intermediates, highly structured mRNAs, or other RNA molecules as a consequence of the action of cellular RNA-dependent RNA polymerases (10, 56). In plants, fungi, and Caenorhabditis elegans at least, this is a non-cellautonomous process, which spreads to remote cells or tissues (57, 59). The systemic silencing signal has still not been identified, although the sequence specificity of this pathway suggests the involvement of a nucleic acid component (19,34).To cou...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 91%

RNA Silencing Suppression by a Second Copy of the P1 Serine Protease ofCucumber Vein Yellowing Ipomovirus, a Member of the FamilyPotyviridaeThat Lacks the Cysteine Protease HCPro

Vallí

Martín-Hernández

López‐Moya

et al. 2006

J Virol

112

123

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“…However, the sequence encoding this protein is absent from the genome of some members of the Ipomovirus genus, such as Cucumber vein yellowing virus (CVYV), Squash vein yellowing virus (SqVYV), and Cassava brown streak virus (CBSV) (Janssen et al 2005;Li et al 2008;Mbanzibwa et al 2009). Whereas in typical potyviruses the 59 terminal region of the genome codes for a single serine proteinase (P1), the 59 terminal region of the genome of CVYV and SqVYV codes for two similar serine proteinases arranged in tandem (P1a and P1b), and the P1b has been shown to have RNA silencing suppression activity (Valli et al 2006;Li et al 2008).…”

Section: Introductionmentioning

confidence: 99%

The specific binding to 21-nt double-stranded RNAs is crucial for the anti-silencing activity of Cucumber vein yellowing virus P1b and perturbs endogenous small RNA populations

Vallí¹,

Oliveros²,

Molnár³

et al. 2011

RNA

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RNA silencing mediated by siRNAs plays an important role as an anti-viral defense mechanism in plants and other eukaryotic organisms, which is usually counteracted by viral RNA silencing suppressors (RSSs). The ipomovirus Cucumber vein yellowing virus (CVYV) lacks the typical RSS of members of the family Potyviridae, HCPro, which is replaced by an unrelated RSS, P1b. CVYV P1b resembles potyviral HCPro in forming complexes with synthetic siRNAs in vitro. Electrophoretic mobility shift assays showed that P1b, like potyviral HCPro, interacts with double-stranded siRNAs, but is not able to bind single-stranded small RNAs or small DNAs. These assays also showed a preference of CVYV P1b for binding to 21-nt siRNAs, a feature also reported for HCPro. However, these two potyvirid RSSs differ in their requirements of 2-nucleotide (nt) 39 overhangs and 59 terminal phosphoryl groups for siRNA binding. Copurification assays confirmed in vivo P1b-siRNA interactions. We have demonstrated by deep sequencing of small RNA populations interacting in vivo with CVYV P1b that the size preference of P1b for small RNAs of 21 nt also takes place in the plant, and that expression of this RSS causes drastic changes in the endogenous small RNA populations. In addition, a site-directed mutagenesis analysis strongly supported the assumption that P1b-siRNA binding is decisive for the anti-silencing activity of P1b and localized a basic domain involved in the siRNA-binding activity of this protein.

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“…This family consists of five genera with monopartite plus-strand RNA genomes, namely, Potyvirus, Rymovirus, Macluravirus, Ipomovirus, and Tritimovirus, and the genus Bymovirus, which has a bipartite genome (41). It was recently reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks HCPro (32), has a duplicated P1 coding sequence, and the downstream P1 copy, P1b, is a second RNA silencing suppressor in the Potyviridae family (76). In silico analysis suggested that both P1 copies, P1a and P1b, are serine proteinases (75), and the enzymatic activity of P1a was verified experimentally (76).…”

mentioning

confidence: 99%

Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b fromCucumber Vein Yellowing Ipomovirus

Vallí

Dujovny

Garcı́a

2008

J Virol

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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 RNAinduced silencing complex.

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Absence of a coding region for the helper component-proteinase in the genome of cucumber vein yellowing virus, a whitefly-transmitted member of the Potyviridae

Cited by 53 publications

References 28 publications

RNA Silencing Suppression by a Second Copy of the P1 Serine Protease ofCucumber Vein Yellowing Ipomovirus, a Member of the FamilyPotyviridaeThat Lacks the Cysteine Protease HCPro

RNA Silencing Suppression by a Second Copy of the P1 Serine Protease ofCucumber Vein Yellowing Ipomovirus, a Member of the FamilyPotyviridaeThat Lacks the Cysteine Protease HCPro

The specific binding to 21-nt double-stranded RNAs is crucial for the anti-silencing activity of Cucumber vein yellowing virus P1b and perturbs endogenous small RNA populations

Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b fromCucumber Vein Yellowing Ipomovirus

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