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Gilmer David - One of the best experts on this subject based on the ideXlab platform.

  • Biological properties of Beet soil-borne mosaic virus and Beet necrotic yellow vein virus cDNA clones produced by isothermal in vitro recombination: Insights for reassortant appearance
    2018
    Co-Authors: Laufer Marlene, Liebe Sebastian, Gilmer David, Mohammad Hamza, Maiss Edgar, Richert-pöggeler Katja, Dall'ara Mattia, Ratti Claudio, Varrelmann Mark
    Abstract:

    Two members of the Benyviridae family and genus Benyvirus, Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV), possess identical genome organization, host range and high sequence similarity; they infect Beta vulgaris with variable symptom expression. In the US, mixed infections are described with limited information about viral interactions. Vectors suitable for agroinoculation of all genome components of both viruses were constructed by isothermal in vitro recombination. All 35S promoter-driven cDNA clones allowed production of recombinant viruses competent for Nicotiana benthamiana and Beta macrocarpa systemic infection and Polymyxa betae transmission and were compared to available BNYVV B-type clone. BNYVV and BSBMV RNA1 + 2 reassortants were viable and spread long-distance in N. benthamiana with symptoms dependent on the BNYVV type. Small genomic RNAs were exchangeable and systemically infected B. macrocarpa. These infectious clones represent a powerful tool for the identification of specific molecular host-pathogen determinants

  • Mécanismes moléculaires à l'origine de la pathogenicité de phytovirus de betterave sucrière transmis par un vecteur tellurique
    Université de Strasbourg Strasbourg, 2013
    Co-Authors: Delbianco Alice, Gilmer David, Rubies Autonell Concepcion
    Abstract:

    Le virus des nervures jaunes et nécrotiques de la betterave (Beet necrotic yellow vein virus, BNYVV) est l agent infectieux responsable de la rhizomanie de la betterave sucrière, une maladie caractérisée par une prolifération anarchique du chevelu racinaire. Le Beet soil-borne mosaic virus (BSBMV) appartient également au genre Benyvirus mais n est retrouvé qu en Amérique du Nord. Ce virus, identifié pour la première fois au Texas, est morphologiquement et génétiquement semblable au BNYVV mais sérologiquement éloigné. Compte tenu des différences moléculaires existant, le BSBMV et BNYVV correspondent à deux espèces virales distinctes. Mon projet de thèse a consisté à étudier les interactions moléculaires entre le BNYVV et le BSBMV et rechercher les mécanismes impliqués dans la pathogénicité de ces deux virus. Des clones complets cDNA infectieux du BNYVV étaient disponibles, tout comme ceux de BSBMV. Compte tenu de l aspect versatile de l obtention de transcrits infectieux de ces différents clones, j ai entrepris de produire des clones cDNA de chacun des ARN viraux sous contrôle d un promoteur constitutive végétal pour initier l infection par agroinfiltration. Les plantes hôtes Chenopodium quinoa et Nicotiana benthamiana ont été inoculées par des transcrits et agroinfiltrées pour initier l infection virale et étudier l interaction entre les ARN génomiques 1 et 2 des deux virus et étudier les propriétés de constructions chimères. En parallèle à ce travail, j ai réalisé la caractérisation du suppresseur de RNA silencing du BSBMV en le comparant à celui du BNYVV.The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

  • PRODUCTION AND INVESTIGATION OF BIOLOGICAL PROPERTIES OF BEET NECROTIC YELLOW VEIN VIRUS AND BEET SOIL-BORNE MOSAIC VIRUS CHIMERAS
    U. Merz, 2013
    Co-Authors: Delbianco Alice, Gilmer David, Dall’ara Mattia, Autonell, Concepcion Rubies, Ratti Claudio
    Abstract:

    The genus Benyvirus (Benyvidiae) includes the most important and widespread sugar beet viruses. In particular, Beet necrotic yellow vein virus (BNYVV) is worldwide distributed and causes an abnormal rootlet proliferation known as rhizomania, whereas Beet soil-borne mosaic virus (BSBMV) is present only in the United States of America. These two viruses have the same genomic organization, infect the same host range and are both transmitted by Polymyxa betae. However, no chimeric forms have been described in nature so far. Thus, we investigate the behavior of artificial viral chimeras

  • Étude des interactions Benyvirus-Polymyxa betae par génétique inverse (Rôle de la protéine codée par l'ARN4 dans la transmission)
    2011
    Co-Authors: D'alonzo Massimiliano, Gilmer David, Rubies Autonell Concepcion
    Abstract:

    Les virus Beet soil-borne mosaic virus (BSBMV) et Beet necrotic yellow vein virus (BNYVV, agent de la Rhizomanie) font partie du genre Benyvirus. Des clones d ADNc infectieux de BSBMV ont été obtenus et ont permis d entreprendre l étude des interactions moléculaires entre plante et Benyvirus sur les bases des différences biologiques des deux virus. Les transcrits infectieux de BSBMV peuvent substituer ceux du BNYVV et ainsi produire des recombinants chimériques.La transmission par Polymyxa betae est liée à la présence de l ARN4 et un ARN4 de 1730 nts de BSBMV, non encore décrit, a été caractérisé. Nous avons démontré que cet ARN4 pouvait remplacer celui du BNYVV lors de la transmission et que seule l expression de la protéine p32 suffit à la transmission du virus. La mutagenèse de la protéine p32 étiquetée a été réalisée et a permis de rechercher les domaines essentiels à ses fonctions.Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV, responsible for Rhizomania disease) belong to the Benyvirus genus. BSBMV full-length cDNA clones were produced to investigate molecular interactions between plant and Benyviruses exploiting BSBMV/BNYVV biological and molecular divergences. BSBMV Full-length infectious cDNA clones can substitute BNYVV RNA-1 or -2 to produce chimeric viral progenies. Virus transmission by Polymyxa betae is directly linked to the viral RNA4 and a 1,730 nts long BSBMV RNA4, not previously described, has been molecularly characterized. We demonstrated that BSBMV RNA4 could substitute BNYVV RNA4 for an efficient transmission. Replicon mediated expression of the BSBMV p32 protein complemented RNA4 defective strain demonstrating for the first time that the p32 protein but not full-length RNA4 is essential for Benyvirus transmission. Mutagenesis or Flag or GFP-tagged p32 has been to investigate the domains of BSBMV p32 protein essential for its properties.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

  • Etude de la variabilité moléculaire et du pouvoir pathogène d'isolats naturels du virus des nervures jaunes et nécrotiques de la betterave (caractérisation des protéines codées par les ARN-3 et -5 de différents isolats)
    2004
    Co-Authors: Link Didier, Gilmer David
    Abstract:

    Une étude phylogénétique réalisée sur des isolats viraux mondiaux de BNYVV a permis d affiner la classification de ce Benyvirus en 5 groupes distincts. Cette nouvelle classification repose sur le type de protéine de capside, la nature des acides aminés hypervariables en position 67 à 70 de la protéine P25 et enfin sur la présence d un ARN-5 génomique surnuméraire de type Européen ou Asiatique. L incidence des onze séquences hypervariables a été testée après mutagenèse dirigée de la protéine P25 provenant du clone infectieux disponible au laboratoire. Ainsi, nous avons démontré que ces quatre acides aminés influencent les capacités de la protéine P25 à dimériser et modulent ses fonctions biologiques sur plante hôte hypersensible.Quant à l ARN-5 de type Européen, un clone ADNc infectieux a pu être produit et son influence testée sur le pouvoir pathogène du virus. L étude de la protéine P26 codée par cet ARN-5 génomique a été initiée. Son action nécrogène sur plante hôte a été démontré. La localisation nucléaire de la protéine P26 a pu être observée à la fois en contexte viral (immuno-marquages et fusion GFP) et hors contexte viral (fusion GFP). La recherche de partenaires viraux et cellulaire en système double-hybride a révélé la forte capacité intrinsèque de la protéine à activer la transcription dans la levure. Le domaine responsable de cette activation de la transcription a pu être localisé par mutagenèse dirigée dans la partie N-terminale de la protéine, domaine également impliqué dans l adressage nucléaire de la protéine.L ensemble des résultats nous permet de proposer un nouveau modèle évolutif en se basant sur le postulat d'une pathogénicité décroissante des isolats de BNYVV, qu ils aient 4 ou 5 ARN génomiques de polarité positive.Based on the CP sequence, P25 variability at aa positions 67-70, and the presence of a fifth RNA, a new BNYVV classification into 5 groups has been proposed. The effect of the hypervariable sequence of P25 has been reproduced by mutagenesis and tested upon the biological properties of the infectious clone. We have demonstrated an important role of the hypervariable sequence upon dimerization of the P25 protein and the induced symptoms on plant leaves.A full-length cDNA clone of RNA-5 has been produced and the encoded P26 protein studied. P26 was located in the nuclear compartment of infected and transfected cells, by transmission electron microscopy and GFP fusion protein, respectively.A two-hybrid screen for viral and cellular partners revealed strong transcription activation activity located within the N-terminal part of the P26 protein. Such a domain was involved in the nuclear targeting of P26 protein.On the basis of our results and observations, we proposed a new evolutionary model for this multi-component positive-stranded RNA virus.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

C. Ratti - One of the best experts on this subject based on the ideXlab platform.

  • Beet soil-borne mosaic virus RNA-4 encodes a 32 kDa protein essential for virus transmission through Polymyxa betae.
    2012
    Co-Authors: D’alonzo M., Alice Delbianco, C Lanzoni, Rubies C. Autonell, D Gilmer, C. Ratti
    Abstract:

    Beet soil-borne mosaic virus (BSBMV), like Beet necrotic yellow vein virus (BNYVV), is a member of the Benyvirus genus and both are transmitted by Polymyxa betae. Both viruses possess a similar genomic organization: RNA-1 and -2 are essential for infection and replication while RNA-3 and -4 play important roles in disease development and vector-mediated infection in sugar beet roots. We characterized a new species of BSBMV RNA-4 that encodes a 32 kDa protein and a chimeric form of BSBMV RNA-3 and -4. We demonstrated that BSBMV RNA-4 can be amplified by BNYVV RNA-1 and -2 in planta, is involved in symptoms expression on Chenopodium quinoa plants and can also complement BNYVV RNA-4 for virus transmission through its vector P. betae in Beta vulgaris plants. Using replicon-mediated expression, we demonstrate for the first time that a correct expression of RNAs-4 encoded proteins is essential for Benyvirus transmission

  • Sugar beet infecting Benyviruses: mechanisms involved in the virus-host interaction
    Associazione Padre Monti, 2010
    Co-Authors: M. D&#8217, Rubies C. Autonell, D Gilmer, C. Weidemann, C. Ratti
    Abstract:

    Beet necrotic yellow vein virus (BNYVV), the causal agent of Rhizomania, is the model species of Benyvirus genus together with Beet soil-borne mosaic virus (BSBMV). Both viruses are vectored by Polymyxa betae. By the use of full-length clones from all genomic RNAs, molecular interactions between plant and Benyviruses are being investigated exploiting biological, epidemiological and molecular similarities/divergences between BNYVV and BSBMV. Results. cDNA copies from all BSBMV RNAs and BNYVV (type P) RNA-1 and 2 have been successfully synthesized and infectivity of the in vitro transcribed RNAs evaluated through rub-inoculation onto Chenopodium quinoa plant leaves. BSBMV RNA2 and BNYVV (type P) RNA-1 cDNA clones are not infectious and still need to be modified. A molecular characterization of chimeric strain consisting of B-type RNA-1 and P-type RNA-2 in comparison with B-type RNA-1 and RNA-2 will be reported. Difference on the expression level of proteins encoded by BNYVV P/B-type RNA-3 (p25), RNA-4 (p31) and RNA-5 (p26) have been evidenced

  • Reverse genetics study of the Beet soil-borne mosaic virus RNA4 role in the plant-vector-virus interaction
    'American Society of Nephrology (ASN)', 2010
    Co-Authors: M. D&#8217, Rubies C. Autonell, D Gilmer, C. Ratti
    Abstract:

    Beet soil-borne mosaic virus (BSBMV) belongs to the Benyvirus genus together with Beet necrotic yellow vein virus (BNYVV). Both viruses share similar genomic organization and are vectored by Polymyxa betae. We recently demonstrated the ability of a BNYVV helper strain to replicate and encapsidate BSBMV RNA-3 suggesting a common and conserved viral RNA selection mechanism for both viruses. A 1,733 nts long BSBMV RNA-4 has been molecularly and functionally characterized by our group and full-length cDNA-derived infectious transcripts obtained. Similarly to BSBMV RNA-3, these RNA-4 transcripts are amplified in planta by BNYVV machinery. We demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants. Two putative ORFs have been identified and could encode for a 32 kDa (p32) and a 13 kDa (p13) protein. P32 sequence is close to BNYVV p31 protein. Using BNYVV helper strain, BSBMV RNA4\u2019s role in the plant-virus-vector interaction investigation has been initiated using reverse genetics approaches. Mutated, deleted and Flag or GFP-tagged sequences of BSBMV RNA4 encoded proteins have been expressed in viral context in Chenopodium quinoa and Beta macrocarpa hosts. Necrotic local lesions phenotype has been associated specifically to the protein expression onto mechanically inoculated C. quinoa plants. Western blot analyses using FLAG-specific antibodies revealed a high molecular weight protein that suggest either a strong interaction of p32 protein with host protein(s) or post translational modifications that need to be better investigated. Infectious transcripts obtained from cDNA clones carrying deleted forms of BSBMV RNA-4 are being employed to identify the domains of the proteins involved in virus-vector interaction required for the efficient Benyviruses transmission

  • MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF BEET SOIL-BORNE MOSAIC VIRUS RNA-4
    place:BOLOGNA, 2009
    Co-Authors: D\u2019alonzo M., Rubies C. Autonell, D Gilmer, M. T. Renzi, V. Folli, C. Ratti
    Abstract:

    Beet soil-borne mosaic virus (BSBMV) is a member of Benyvirus genus together with Beet necrotic yellow vein virus (BNYVV), both vectored by Polymyxa betae. The described BSBMV genome consists of four RNAs capped at the 5\u2019 end and 3\u2019 polyadenylated. RNA-1, and 2 are, respectively, 6,683 and 4,615 nucleotides (nts) in length and contain putative ORFs similar to which identified on BNYVV RNA-1 and 2. The 1,720 nts BSBMV RNA-3 has a 29 kDa ORF that share 23% amino acid sequence identity with the 25 kDa ORF of BNYVV RNA3. A 1,203 nts long BSBMV RNA-4 form (GenBank accession number: NC_003508) has been so far described with a single putative ORF with a predicted mass (13 kDa) considerably smaller than the BNYVV 31 kDa RNA-4 product. During our research program, pointed to the studies of the molecular interactions between sugar beets and Benyviruses, unexpected 1,733 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil kindly supplied by Marc Richard-Molard (ITB, Paris)

  • Functional characterization of Beet soil-borne mosaic virus RNA-4-encoded protein
    IS-MPMI, 2009
    Co-Authors: D’alonzo M., Rubies C. Autonell, D Gilmer, M. T. Renzi, C. Ratti
    Abstract:

    Beet soil-borne mosaic virus (BSBMV) is a member of the Benyvirus genus together with Beet necrotic yellow vein virus (BNYVV), with similar genomic organization and both vectored by Polymyxa betae. The ability of BNYVV helper strain to replicate BSBMV RNA-3 suggests a common and conserved viral RNA selection mechanism for both viruses. We recently described a 1,733 nts long BSBMV RNA-4 (GenBank: FJ424610), which has been molecularly and functionally characterized. As for BSBMV RNA-3, fulllength BSBMV RNA-4 cDNA clone permitted the obtention of infectious transcripts that BNYVV viral machinery is able to replicate and to encapsidate in planta. Moreover, such BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants. Two putative ORFs have been identified and could encode for peptides of 32 kDa (383-1,231 nts) and 13 kDa (885-1,241 nts) respectively. Using BNYVV helper strain, BSBMV RNA4’s protein initiation codons have been studied by mutagenesis. We associated the local necrotic lesions phenotype to the protein expression onto mechanically inoculated Chenopodium quinoa plants. Flag or GFP-tagged sequences have been expressed in viral context. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications that need to be better investigated. GFP-fusion sequences permitted the sub-cellular localization of BSBMV RNA4’s proteins

David Gilmer - One of the best experts on this subject based on the ideXlab platform.

  • On the interaction and localization of the beet necrotic yellow vein virus replicase
    2015
    Co-Authors: Arezoo Pakdel, Kamal Hleibieh, Elodie Klein, Claudio Ratti, Salah Bouzoubaa, Claire Mounier, Baptiste Monsion, Jérôme Mutterer, Mathieu Erhardt, David Gilmer
    Abstract:

    Beet necrotic yellow vein virus (BNYVV) is a multipartite positive-strand RNA virus. BNYVV RNA-1 encodes a non-structural p237 polyprotein processed in two proteins (p150 and p66) by a cis-acting protease activity. BNYVV non-structural proteins are closely related to replication proteins of positive strand RNA viruses such as hepeviruses rather to other plant virus replicases. The p237 and dsRNA have been localized by TEM in ER structures of infected leaf cells whereas dsRNA was immunolabeled in infected protoplasts. The p150 contains domains with methyltransferase, protease, helicase and two domains of unknown function whereas p66 encompasses the RNA-dependent RNA-polymerase signature. We report the existing interactions between functional domains of the p150 and p66 proteins and the addressing of the Benyvirus replicase to the endoplasmic reticulum. Yeast two-hybrid approach, colocalization with FRET-FLIM analyses and co-immunoprecipitation highlighted existing interactions that suggest the presence of a multimeric complex at the vicinity of the cellular membranous we

  • 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
    Molecular Plant-microbe Interactions, 2013
    Co-Authors: Sotaro Chiba, Alice Delbianco, Kamal Hleibieh, Elodie Klein, Claudio Ratti, Veronique Zieglergraff, Salah Bouzoubaa, David Gilmer
    Abstract:

    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.

  • beet soil borne mosaic virus rna 3 is replicated and encapsidated in the presence of bnyvv rna 1 and 2 and allows long distance movement in beta macrocarpa
    Virology, 2009
    Co-Authors: Claudio Ratti, Kamal Hleibieh, Laura Bianchi, Audrey Schirmer, Concepcion Rubies Autonell, David Gilmer
    Abstract:

    Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) belong to the Benyvirus genus. BSBMV has been reported only in the United States, while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae and possess similar host ranges, particle number and morphology. BNYVV and BSBMV are not serologically related but they have similar genomic organizations. Field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles in plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed that BSBMV and BNYVV are sufficiently different to be classified as two species. Complementary base changes found within the BSBMV RNA-3 5' UTR made it resemble to BNYVV 5' RNA-3 structure whereas the 3' UTRs of both species were more conserved. cDNA clones were obtained, and allowed complete copies of BSBMV RNA-3 to be trans-replicated, trans-encapsidated by the BNYVV viral machinery. Long-distance movement was observed indicating that BSBMV RNA-3 could substitute BNYVV RNA-3 for systemic spread, even though the p29 encoded by BSBMV RNA-3 is much closer to the RNA-5-encoded p26 than to BNYVV RNA-3-encoded p25. Competition occurred when BSBMV RNA-3-derived replicons were used together with BNYVV-derived RNA-3 but not when the RNA-5-derived component was used. Exploitation of the similarities and divergences between BSBMV and BNYVV should lead to a better understanding of molecular interactions between Benyviruses and their hosts.

Joh F Atkins - One of the best experts on this subject based on the ideXlab platform.

  • stimulation of stop codon readthrough frequent presence of an extended 3 rna structural element
    Nucleic Acids Research, 2011
    Co-Authors: Andrew E Firth, Raymond F Gesteland, Norma M Wills, Joh F Atkins
    Abstract:

    In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3' RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.

  • Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element
    Nucleic acids research, 2011
    Co-Authors: Andrew E Firth, Raymond F Gesteland, Norma M Wills, Joh F Atkins
    Abstract:

    In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3'-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼ 150 nt 3'-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3' RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.

  • Stimulation of stop codon readthrough: frequent presence of an extended 39 RNA structural element
    2011
    Co-Authors: Andrew E Firth, Norma M Wills, Raymond F. Gestel, Joh F Atkins
    Abstract:

    In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 30-adjacent to the UGA. However, analysis of vari-ability at synonymous sites revealed strikingly enhanced conservation within the 150nt 30-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computa-tional analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 30 RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis

Ratti C. - One of the best experts on this subject based on the ideXlab platform.

  • Properties of post-transcriptional gene silencing suppression proteins of Benyviruses
    country:FRA, 2013
    Co-Authors: Delbianco A., Hleibieh K., Ratti C., Flobinus A., Klein E., Bouzoubaa S., Gilmer D.
    Abstract:

    Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) belong to the Benyvirus genus, possess a multipartite genome formed by four ssRNAs(+) and are both transmitted by the plasmodiophorid Polymyxa betae. BSBMV and BNYVV are closely related since they possess the same host range, vector and genome organization. The innate mechanism that plants use to protect themselves against viral infections is called Post Transcriptional Gene Silencing (PTGS). PTGS is triggered by the presence of aberrant RNA or dsRNA generated during the replication of viral genomes and leads to their degradation. To counteract this innate mechanism, viruses co-evolved with their hosts and express viral suppressors of RNA silencing (VSR) that inhibit the PTGS. In this work we investigated properties of the Benyvirus VSRs. Such VSR consist of cysteine-rich proteins (CRP) of 14kDa expressed from RNA2 of both BNYVV and BSBMV species used in this study. We demonstrated that P14s have a zinc-finger domain (Znf) able to bind nucleic acids. Agroinfection of Nicotiana benthamiana plants demonstrated that these proteins are able to suppress the PTGS downstream of the Dicer proteins action, without interfering with the transitivity. Sequence motifs essential for the nucleolus targeting of the protein (NoLS) and cysteine residues essential to the Znf structure folding, have been also identified. Both p14s localize in the nucleolus, form homodimers and bind the \u201ccoremin\u201d sequence, a stretch of 20 nucleotides present in the RNAs-3 sequence of Benyviruses and required for their systemic spread in the plant. Moreover, the coremin sequence is able to complement defective BNYVV P14 mutants in long distance movement illustrating an obvious link between P14, suppression of RNA silencing activity, RNAs-3 coremin sequence and long distance movement

  • Benyvirus chimeras evidence that silencing suppressor proteins specifically interact with viral RNAs
    2013
    Co-Authors: Delbianco A., Hleibieh K., Rubies Autonell C., Gilmer D., Flobinus A., Klein E., Dall’ara M., Ratti C.
    Abstract:

    Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) belong to the Benyvirus genus, possess a multipartite genome formed by four ssRNAs(+) and are both transmitted by the plasmodiophorid Polymyxa betae. BSBMV and BNYVV are closely related since they possess the same host range, vector and genome organization. Recent studies demonstrated a possible amplification and transmission of BSBMV RNAs by BNYVV helper strain. In the United States of America, both Benyviruses are frequently present in the same cultivated field, infecting the same plant but no chimeric forms have been described from field isolates so far. The possibility that BNYVV/BSBMV chimeras may be generated has been investigated. Chenopodium quinoa local infection has been carried out using in vitro infectious transcripts of BNYVV and BSBMV RNA-1 and -2 and the behavior of BSBMV/BNYVV chimeras and wild type isolates has been compared. The chimera BoStras12 (BSBMV RNA-1 + BNYVV RNA-2) induced severe necrotic lesions on the leaves, probably due to a hypersensitive response of the plant, in contrast to the typical chlorotic lesions of wild type combinations. The necrosis disappeared when the plant was co-inoculated with BoStras12 together with a viral replicon expressing BSBMV p14, a cysteine-rich protein acting as a suppressor of post-transcriptional gene silencing. Thus, properties of Benyvirus p14s have been investigated. P14s have a zinc-finger domain able to bind nucleic acids and agroinfection of Nicotiana benthamiana plants demonstrated that these proteins are able to suppress the PTGS downstream of the Dicer proteins, without interfering with the transitivity. Moreover, both p14s are localized in the nucleolus, forms homodimers and binds the “coremin” sequence, a stretch of 20 nucleotides present in the RNA-3 of Benyviruses and necessary for the systemic spread of viruses in the plant. Experiments performed to investigate relationships between BSBMV/BNYVV p14s and VSR activity, “coremin” sequence, long distance movement and absence of natural chimeras of Benyviruses will be presented

  • Post-transcriptional gene silencing suppression study of Beet soil-borne mosaic virus: characterization of p14 and production of chimeric isolates of Benyviruses
    country:FRA, 2013
    Co-Authors: Delbianco A., Hleibieh K., Rubies Autonell C., Gilmer D., Klein E., Dall\u2019ara M., Ratti C.
    Abstract:

    Beet soil-borne mosaic virus (BSBMV) belongs to the Benyvirus genus, together with Beet necrotic yellow vein virus (BNYVV). Both viruses possess a multipartite genome formed by four ssRNAs(+). BSBMV and BNYVV are closely related since they possess the same host range, vector and genome organization. Recent studies demonstrated a possible amplification and transmission of BSBMV RNAs by BNYVV. In the United States of America, both Benyviruses are frequently present in the same cultivated field, infecting the same plant but no chimeric forms have been described from field isolates so far. Chenopodium quinoa infection has been carried out using in vitro infectious transcripts of both BNYVV and BSBMV RNA-1 and -2 and the behavior of BSBMV/BNYVV combinations and wild type isolates has been compared. In parallel, the properties of the BSBMV VSR, a cysteine-rich protein (CRP) of 14 kDa expressed by RNA-2, have been investigated and compared to the BNYVV p14 RNA silencing suppressor. P14 has a zinc-finger domain able to bind nucleic acids and agroinfection of Nicotiana benthamiana plants demonstrated that p14 is able to suppress the PTGS downstream of the Dicer proteins, without interfering with the transitivity. Moreover, both p14 are localized in the nucleolus, forms homodimers and binds the \u201ccoremin\u201d sequence, a stretch of 20 nucleotides present in the RNA-3 of Benyviruses and necessary for the systemic spread of viruses in the plant. Experiments performed to investigate relationships between BSBMV/BNYVV p14s and VSR activity, \u201ccoremin\u201d sequence, long distance movement and absence of natural chimeras of Benyviruses will be presented

  • CHARACTERIZATION OF POST TRANSCRIPTIONAL GENE SILENCING SUPPRESSOR PROTEINS OF Benyvirus
    2012
    Co-Authors: Delbianco1 A. 2, Hleibieh K., Dall'ara M., Rubies Autonell C., Gilmer D., Ratti C.
    Abstract:

    An innate mechanism that plants use to protect themselves against viral infections is called Post Transcriptional Gene Silencing (PTGS). PTGS is triggered by the presence of aberrant RNA or dsRNA generated during the replication of viral genomes and leads to their degradation. To counteract this innate mechanism, viruses co-evolved with their hosts and express viral suppressors of RNA silencing (VSR) that inhibit the PTGS. . The properties of the Benyvirus VSRs have been investigated. Such VSR consist of cysteine-rich proteins (CRP) of 14kDa expressed from RNA2 of both Benyvirus species used in this study. The CRPs have a zinc-finger domain able to bind nucleic acids. Agroinfection of Nicotiana benthamiana plants demonstrated that these proteins are able to suppress the PTGS downstream of the Dicer proteins, without interfering with the transitivity. Both p14s are localized in the nucleolus and in the cytoplasm in and out of the viral context. Moreover yeast two-hybrid and three-hybrid tests proved that these proteins form homodimers and bind the “coremin” sequence, a stretch of 20 nucleotides present in the RNA-3 of both viruses and necessary for the systemic spread of the virus in the plant. Some of the investigation performed to elucidate the existing connection between the p14s and VSR activity, the “coremin” sequence and the long distance movement of Benyviruses will be presented

  • Beet soil-borne mosaic virus RNA4 encodes the p32 protein, essential for virus transmission.
    2011
    Co-Authors: D\u2019alonzo M., Rubies Autonell C., Gilmer D., Delbianco A., Lanzoni C., Ratti C.
    Abstract:

    Beet soil-borne mosaic virus (BSBMV) belongs to the Benyvirus genus together with Beet necrotic yellow vein virus (BNYVV), the causal agent of Rhizomania disease. Both viruses share similar genomic organization and are vectored by Polymyxa betae. Ability of BNYVV helper strain to replicate and encapsidate BSBMV RNA-3 has been recently described suggesting a common and conserved viral RNA selection mechanism for both viruses. Moreover a 1,730 nts long form of BSBMV RNA-4 can substitute for BNYVV RNA-4 for an efficient transmission to Beta vulgaris plants by the vector P. betae. Two putative ORFs have been identified in BSBMV RNA-4 that could code for a 32 kDa (p32) and a 13 kDa protein. We investigated the role of these proteins in plant-virus-vector interaction using reverse genetics approaches. Expression of BSBMV p32 using a BNYVV RNA-5- based viral vector (Rep5) complemented a RNA-4 defective strain and thus demonstrating for the first time that p32 but not full-length RNA-4 is essential for Benyvirus transmission. Mutated, deleted and FLAG- or GFP-tagged sequences of p32 have been expressed in Chenopodium quinoa and Beta macrocarpa. A necrotic local lesions phenotype was associated specifically to protein expression in mechanically inoculated C. quinoa plants. Western blot analyses using FLAG-specific antibodies revealed a high molecular weight protein that suggests either a strong interaction of p32 with host protein(s) or post translational modifications. Finally yeast two hybrid system was employed to search for a p32 viral partner among other viral proteins