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Robert H A Coutts - One of the best experts on this subject based on the ideXlab platform.
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investigating the biological relevance of in vitro identified putative packaging signals at the 5 terminus of satellite Tobacco Necrosis Virus 1 genomic rna
Journal of Virology, 2019Co-Authors: Ioly Kottaloizou, Robert H A Coutts, Hadrien Peyret, Keith Saunders, George P LomonossoffAbstract:Satellite Tobacco Necrosis Virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114), leading to the identification of degenerate packaging signals (PSs) proposed to be involved in the recognition of its genome by the capsid protein (CP). The aim of the present work was to investigate whether these putative PSs can confer selective packaging of STNV-1 RNA in vivo and to assess the prospects of using decoy RNAs in antiviral therapy. We have developed an in planta packaging assay based on the transient expression of STNV-1 CP and have assessed the ability of the resulting Virus-like particles (VLPs) to encapsidate mutant STNV-1 RNAs expected to have different encapsidation potential based on in vitro studies. The results revealed that >90% of the encapsidated RNAs are host derived, although there is some selectivity of packaging for STNV-1 RNA and certain host RNAs. Comparison of the packaging efficiencies of mutant STNV-1 RNAs showed that they are encapsidated mainly according to their abundance within the cells, rather than the presence or absence of the putative PSs previously identified from in vitro studies. In contrast, subsequent infection experiments demonstrated that host RNAs represent only <1% of virion content. Although selective encapsidation of certain host RNAs was noted, no direct correlation could be made between this preference and the presence of potential PSs in the host RNA sequences. Overall, the data illustrate that the differences in RNA packaging efficiency identified through in vitro studies are insufficient to explain the specific packaging of STNV-1 RNA.IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114) using satellite Tobacco Necrosis Virus 1 (STNV-1) as a model system for in vitro studies. It has been suggested that competing with these putative PSs may constitute a novel therapeutic approach against pathogenic single-stranded RNA Viruses. Our work demonstrates that the previously identified PSs have no discernible significance for the selective packaging of STNV-1 in vivo in the presence and absence of competition or replication: viral sequences are encapsidated mostly on the basis of their abundance within the cell, while encapsidation of host RNAs also occurs. Nevertheless, the putative PSs identified in STNV-1 RNA may still have applications in bionanotechnology, such as the in vitro selective packaging of RNA molecules.
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investigations on the Tobacco Necrosis Virus d p60 replicase protein
PLOS ONE, 2013Co-Authors: Liang Fang, Robert H A CouttsAbstract:Tobacco Necrosis Virus D (TNV-D), in the genus BetanecroVirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite Tobacco Necrosis Virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on Virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecroVirus polymerase.
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sequence specific rna protein interactions overcome electrostatic barriers preventing assembly of satellite Tobacco Necrosis Virus coat protein
Journal of Molecular Biology, 2013Co-Authors: Robert J Ford, Robert H A Coutts, Amy M Barker, Saskia E Bakker, Neil A Ranson, Simon E V Phillips, Arwen R Pearson, Peter G StockleyAbstract:We have examined the roles of RNA–coat protein (CP) interactions in the assembly of satellite Tobacco Necrosis Virus (STNV). The viral genomic RNA encodes only the CP, which comprises a β-barrel domain connected to a positively charged N-terminal extension. In the previous crystal structures of this system, the first 11 residues of the protein are disordered. Using variants of an RNA aptamer sequence isolated against the CP, B3, we have studied the sequence specificity of RNA-induced assembly. B3 consists of a stem–loop presenting the tetra-loop sequence ACAA. There is a clear preference for RNAs encompassing this loop sequence, as measured by the yield of T = 1 capsids, which is indifferent to sequences within the stem. The B3-containing Virus-like particle has been crystallised and its structure was determined to 2.3 A. A lower-resolution map encompassing density for the RNA has also been calculated. The presence of B3 results in increased ordering of the N-terminal helices located at the particle 3-fold axes, which extend by roughly one and a half turns to encompass residues 8–11, including R8 and K9. Under assembly conditions, STNV CP in the absence of RNA is monomeric and does not self-assemble. These facts suggest that a plausible model for assembly initiation is the specific RNA-induced stabilisation of a trimeric capsomere. The basic nature of the helical extension suggests that electrostatic repulsion between CPs prevents assembly in the absence of RNA and that this barrier is overcome by correct placement of appropriately orientated helical RNA stems. Such a mechanism would be consistent with the data shown here for assembly with longer RNA fragments, including an STNV genome. The results are discussed in light of a first stage of assembly involving compaction of the genomic RNA driven by multiple RNA packaging signal–CP interactions.
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mutation analysis of cis elements in the 3 and 5 untranslated regions of satellite Tobacco Necrosis Virus strain c rna
Virology, 1999Co-Authors: David H Bringloe, C W A Pleij, Robert H A CouttsAbstract:The putative, 3'-terminal stem-loop structure in satellite Tobacco Necrosis Virus strain C (STNV-C) RNA constitutes an essential cis-acting structure for the promotion of negative-strand RNA synthesis and a single-stranded tail is also important. The putative, 5'-terminal stem-loop structure in STNV-C RNA is not essential for productive, plus-strand RNA accumulation but is required for optimal accumulation. Residues 2 and 3 are the minimal cis-acting sequences required for RNA synthesis. The RNA of chimeric mutants, which exchanged 3'- and 5'-untranslated regions between STNV-C and helper Tobacco Necrosis Virus strain D RNAs, accumulated in protoplasts, implying similar replication mechanisms for both RNAs.
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the nucleotide sequence of satellite Tobacco Necrosis Virus strain c and helper assisted replication of wild type and mutant clones of the Virus
Journal of General Virology, 1998Co-Authors: D H Bringloe, C W A Pleij, Alexander P Gultyaev, M Pelpel, Robert H A CouttsAbstract:The complete nucleotide sequence of satellite Tobacco Necrosis Virus strain C (STNV-C) was determined. The genome has a similar overall organization to two STNV isolates studied previously but differs significantly from them in the secondary structure of the translated and untranslated regions (UTRs). STNV-C RNA is naturally uncapped and contains 1221 nt: 101 nt in the 5' UTR, 606 nt in the capsid protein (CP) coding region and 514 nt in the 3' UTR. Using the known sequences of STNV-C and Tobacco Necrosis Virus strain D (TNV-D) RNAs, full-length cDNA clones of both RNAs were constructed. Synthetic transcripts derived from STNV-C cDNA clones only replicated in plants and protoplasts when co-inoculated with TNV-D transcripts. A number of mutant clones in both the 3' and the 5' STNV-C RNA UTRs were constructed which disrupted putative cis-acting elements recognized by helper Virus polymerase. Deletion analysis revealed an essential requirement of all 3' and 5' proximal sequences in the STNV-C UTRs for replication. However, an internal region in the 3' UTR could be deleted without loss of infectivity. Likewise, the entire STNV-C CP-encoding region could be deleted and replaced with a marker gene of a similar size without loss of transcript accumulation in plants.
Genevieve Defago - One of the best experts on this subject based on the ideXlab platform.
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salicylic acid biosynthetic genes expressed in pseudomonas fluorescens strain p3 improve the induction of systemic resistance in Tobacco against Tobacco Necrosis Virus
Phytopathology, 1998Co-Authors: M Maurhofer, Cornelia Reimmann, P Schmidlisacherer, Stephan Heeb, Dieter Haas, Genevieve DefagoAbstract:ABSTRACT Application of salicylic acid induces systemic acquired resistance in Tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in Tobacco against Tobacco Necrosis Virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of Tobacco, but did not improve the ability of CHA0 to induce systemic resistance in Tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobac...
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induction of systemic resistance of Tobacco to Tobacco Necrosis Virus by the root colonizing pseudomonas fluorescens strain cha0 influence of the gaca gene and of pyoverdine production
Phytopathology, 1994Co-Authors: M Maurhofer, Jean-pierre Métraux, Carsten Hase, P Meuwly, Genevieve DefagoAbstract:Pseudomonas fluorescens strain CHA0, which suppresses various plant diseases caused by soilborne pathogens, also can restrict leaf disease. Plants of Nicotiana glutinosa and of two cultivars of N. tabacum were grown in autoclaved natural soil previously inoculated with strain CHA0. After 6 wk, all the plants tested showed resistance in leaves to infection with Tobacco Necrosis Virus (TNY) to the same extent as plants previously immunized with TNY (induced resistance control). Polyacrylamide gel electrophoresis and enzyme assays showed that the same amount of PR proteins (PR-1 group proteins, β-1,3-glucanases, and endochitinases) was induced in the intercellular fluid of leaves of plants grown in the presence of strain CHA0 as in the intercellular fluid of leaves of plants immunized by a previous TNV inoculation on a lower leaf [...]
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induction of systemic resistance of Tobacco to Tobacco Necrosis Virus by the root colonizing pseudomonas fluorescens strain cha0 influence of the gaca gene and of pyoverdine production
Phytopathology, 1994Co-Authors: M Maurhofer, Jean-pierre Métraux, Carsten Hase, P Meuwly, Genevieve DefagoAbstract:Pseudomonas fluorescens strain CHA0, which suppresses various plant diseases caused by soilborne pathogens, also can restrict leaf disease. Plants of Nicotiana glutinosa and of two cultivars of N. tabacum were grown in autoclaved natural soil previously inoculated with strain CHA0. After 6 wk, all the plants tested showed resistance in leaves to infection with Tobacco Necrosis Virus (TNV) to the same extent as plants previously immunized with TNV (induced resistance control). Polyacrylamide gel electrophoresis and enzyme assays showed that the same amount of PR proteins (Pr-1 group proteins, beta-1,3-glucanases, and endochitinases) was induced in the intercellular fluid of leaves of plants grown in the presence of strain CHA0 as in the intercellular fluid of leaves of plants immunized by a previous TNV inoculation on a lower leaf. Strain CHA0 was reisolated from the roots but could not be detected in stems or leaves. Strain CHA96, a gacA (global activator)-negative mutant of strain CHA0 defective in the production of antibiotics and in the suppression of black root rot of Tobacco, had the same capacity to induce PR proteins and resistance against TNV as did the wild-type strain. CHA400, a pyoverdine-negative mutant of strain CHA0 with the same capacity to suppress black root rot of Tobacco and take-all of wheat as the wild-type strain, was able to induce PR proteins but only partial resistance against TNV. P3, another P. fluorescens wild-type strain, does not suppress diseases caused by soilborne pathogens and induced neither resistance nor PR proteins in Tobacco leaves. Root colonization of Tobacco plants with strain CHA0 and its derivatives as well as leaf infection with TNV caused an increase in salicylic acid in leaves. These results show that colonization of Tobacco roots by strain CHA0 reduces TNV leaf Necrosis and induces physiological changes in the plant to the same extent as does induction of systemic resistance by leaf inoculation with TNV
M Maurhofer - One of the best experts on this subject based on the ideXlab platform.
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salicylic acid biosynthetic genes expressed in pseudomonas fluorescens strain p3 improve the induction of systemic resistance in Tobacco against Tobacco Necrosis Virus
Phytopathology, 1998Co-Authors: M Maurhofer, Cornelia Reimmann, P Schmidlisacherer, Stephan Heeb, Dieter Haas, Genevieve DefagoAbstract:ABSTRACT Application of salicylic acid induces systemic acquired resistance in Tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in Tobacco against Tobacco Necrosis Virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of Tobacco, but did not improve the ability of CHA0 to induce systemic resistance in Tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobac...
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induction of systemic resistance of Tobacco to Tobacco Necrosis Virus by the root colonizing pseudomonas fluorescens strain cha0 influence of the gaca gene and of pyoverdine production
Phytopathology, 1994Co-Authors: M Maurhofer, Jean-pierre Métraux, Carsten Hase, P Meuwly, Genevieve DefagoAbstract:Pseudomonas fluorescens strain CHA0, which suppresses various plant diseases caused by soilborne pathogens, also can restrict leaf disease. Plants of Nicotiana glutinosa and of two cultivars of N. tabacum were grown in autoclaved natural soil previously inoculated with strain CHA0. After 6 wk, all the plants tested showed resistance in leaves to infection with Tobacco Necrosis Virus (TNY) to the same extent as plants previously immunized with TNY (induced resistance control). Polyacrylamide gel electrophoresis and enzyme assays showed that the same amount of PR proteins (PR-1 group proteins, β-1,3-glucanases, and endochitinases) was induced in the intercellular fluid of leaves of plants grown in the presence of strain CHA0 as in the intercellular fluid of leaves of plants immunized by a previous TNV inoculation on a lower leaf [...]
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induction of systemic resistance of Tobacco to Tobacco Necrosis Virus by the root colonizing pseudomonas fluorescens strain cha0 influence of the gaca gene and of pyoverdine production
Phytopathology, 1994Co-Authors: M Maurhofer, Jean-pierre Métraux, Carsten Hase, P Meuwly, Genevieve DefagoAbstract:Pseudomonas fluorescens strain CHA0, which suppresses various plant diseases caused by soilborne pathogens, also can restrict leaf disease. Plants of Nicotiana glutinosa and of two cultivars of N. tabacum were grown in autoclaved natural soil previously inoculated with strain CHA0. After 6 wk, all the plants tested showed resistance in leaves to infection with Tobacco Necrosis Virus (TNV) to the same extent as plants previously immunized with TNV (induced resistance control). Polyacrylamide gel electrophoresis and enzyme assays showed that the same amount of PR proteins (Pr-1 group proteins, beta-1,3-glucanases, and endochitinases) was induced in the intercellular fluid of leaves of plants grown in the presence of strain CHA0 as in the intercellular fluid of leaves of plants immunized by a previous TNV inoculation on a lower leaf. Strain CHA0 was reisolated from the roots but could not be detected in stems or leaves. Strain CHA96, a gacA (global activator)-negative mutant of strain CHA0 defective in the production of antibiotics and in the suppression of black root rot of Tobacco, had the same capacity to induce PR proteins and resistance against TNV as did the wild-type strain. CHA400, a pyoverdine-negative mutant of strain CHA0 with the same capacity to suppress black root rot of Tobacco and take-all of wheat as the wild-type strain, was able to induce PR proteins but only partial resistance against TNV. P3, another P. fluorescens wild-type strain, does not suppress diseases caused by soilborne pathogens and induced neither resistance nor PR proteins in Tobacco leaves. Root colonization of Tobacco plants with strain CHA0 and its derivatives as well as leaf infection with TNV caused an increase in salicylic acid in leaves. These results show that colonization of Tobacco roots by strain CHA0 reduces TNV leaf Necrosis and induces physiological changes in the plant to the same extent as does induction of systemic resistance by leaf inoculation with TNV
Jean-pierre Métraux - One of the best experts on this subject based on the ideXlab platform.
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transport of salicylic acid in Tobacco Necrosis Virus infected cucumber plants
Plant Physiology, 1996Co-Authors: Wolfgang Molders, A J Buchala, Jean-pierre MétrauxAbstract:The transport of salicylic acid (SA) was studied in cucumber (Cucumis sativus L.) using 14C-labeled benzoic acid that was injected in the cotyledons at the time of inoculation. Primary inoculation with Tobacco Necrosis Virus (TNV) on the cotyledons led to an induction of systemic resistance of the first primary leaf above the cotyledon against Colletotrichum lagenarium as early as 3 d after inoculation. [14C]SA was detected in the phloem or in the first leaf 2 d after TNV inoculation, whereas [14C]benzoic acid was not detected in the phloem during the first 3 d after TNV inoculation of the cotyledons, indicating phloem transport of [14C]SA from cotyledon. In leaf 1, the specific activity of [14C]SA decreased between 1.7 and 8.6 times compared with the cotyledons, indicating that, in addition to transport, leaf 1 also produced more SA. The amount of SA transported after TNV infection of the cotyledon was 9 to 160 times higher than in uninfected control plants. Thus, SA can be transported to leaf 1 before the development of systemic acquired resistance, and SA accumulation in leaf 1 results both from transport from the cotyledon and from synthesis in leaf 1.
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induction of systemic resistance of Tobacco to Tobacco Necrosis Virus by the root colonizing pseudomonas fluorescens strain cha0 influence of the gaca gene and of pyoverdine production
Phytopathology, 1994Co-Authors: M Maurhofer, Jean-pierre Métraux, Carsten Hase, P Meuwly, Genevieve DefagoAbstract:Pseudomonas fluorescens strain CHA0, which suppresses various plant diseases caused by soilborne pathogens, also can restrict leaf disease. Plants of Nicotiana glutinosa and of two cultivars of N. tabacum were grown in autoclaved natural soil previously inoculated with strain CHA0. After 6 wk, all the plants tested showed resistance in leaves to infection with Tobacco Necrosis Virus (TNY) to the same extent as plants previously immunized with TNY (induced resistance control). Polyacrylamide gel electrophoresis and enzyme assays showed that the same amount of PR proteins (PR-1 group proteins, β-1,3-glucanases, and endochitinases) was induced in the intercellular fluid of leaves of plants grown in the presence of strain CHA0 as in the intercellular fluid of leaves of plants immunized by a previous TNV inoculation on a lower leaf [...]
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induction of systemic resistance of Tobacco to Tobacco Necrosis Virus by the root colonizing pseudomonas fluorescens strain cha0 influence of the gaca gene and of pyoverdine production
Phytopathology, 1994Co-Authors: M Maurhofer, Jean-pierre Métraux, Carsten Hase, P Meuwly, Genevieve DefagoAbstract:Pseudomonas fluorescens strain CHA0, which suppresses various plant diseases caused by soilborne pathogens, also can restrict leaf disease. Plants of Nicotiana glutinosa and of two cultivars of N. tabacum were grown in autoclaved natural soil previously inoculated with strain CHA0. After 6 wk, all the plants tested showed resistance in leaves to infection with Tobacco Necrosis Virus (TNV) to the same extent as plants previously immunized with TNV (induced resistance control). Polyacrylamide gel electrophoresis and enzyme assays showed that the same amount of PR proteins (Pr-1 group proteins, beta-1,3-glucanases, and endochitinases) was induced in the intercellular fluid of leaves of plants grown in the presence of strain CHA0 as in the intercellular fluid of leaves of plants immunized by a previous TNV inoculation on a lower leaf. Strain CHA0 was reisolated from the roots but could not be detected in stems or leaves. Strain CHA96, a gacA (global activator)-negative mutant of strain CHA0 defective in the production of antibiotics and in the suppression of black root rot of Tobacco, had the same capacity to induce PR proteins and resistance against TNV as did the wild-type strain. CHA400, a pyoverdine-negative mutant of strain CHA0 with the same capacity to suppress black root rot of Tobacco and take-all of wheat as the wild-type strain, was able to induce PR proteins but only partial resistance against TNV. P3, another P. fluorescens wild-type strain, does not suppress diseases caused by soilborne pathogens and induced neither resistance nor PR proteins in Tobacco leaves. Root colonization of Tobacco plants with strain CHA0 and its derivatives as well as leaf infection with TNV caused an increase in salicylic acid in leaves. These results show that colonization of Tobacco roots by strain CHA0 reduces TNV leaf Necrosis and induces physiological changes in the plant to the same extent as does induction of systemic resistance by leaf inoculation with TNV
Frank Meulewaeter - One of the best experts on this subject based on the ideXlab platform.
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the 5 and 3 extremities of the satellite Tobacco Necrosis Virus translational enhancer domain contribute differentially to stimulation of translation
RNA, 2002Co-Authors: Rosalinde Van Lipzig, Marc Cornelissen, C W A Pleij, Alexander P Gultyaev, Marc Van Montagu, Frank MeulewaeterAbstract:The translational enhancer domain (TED) of satellite Tobacco Necrosis Virus (STNV) RNA stimulates translation of uncapped RNAs autonomously. Here we set out to identify the 5' and 3' extremities of TED and features of these sequences with respect to translation. We found that both in wheat germ extract and in Tobacco protoplasts, the 5' border is confined to 3 nt. Mutational analysis revealed that the autonomous function of TED is sensitive to 5' flanking sequences. At the 3' end of TED, 23 nt have a cumulative, quantitative effect on translation in wheat germ extract, whereas in Tobacco protoplasts, the most 3' 14 nt of these 23 nt do not enhance translation. The 5' and 3' sequence requirements triggered the development of a new secondary structure model. In this model, TED folds into a phylogenetically conserved stem-loop structure in which the essential 5' nucleotides base-pair with the 3' nucleotides that stimulate translation both in vitro and in vivo. Importantly, the 14 3' nucleotides in TED that stimulate translation in the wheat germ extract only do not require the predicted base-pairing in order to function. The discrepancy between in vitro and in vivo sequence requirements thus correlates with potential base-pairing requirements, opening the possibility that TED contains two functional domains.
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features of the autonomous function of the translational enhancer domain of satellite Tobacco Necrosis Virus
RNA, 1998Co-Authors: Frank Meulewaeter, M Van Montagu, Marc CornelissenAbstract:The RNA of satellite Tobacco Necrosis Virus (STNV) is a monocistronic messenger that lacks both a cap and a poly(A) tail. Translation of STNV RNA in vitro is promoted by a 120-nt translational enhancer domain (TED) in the 3'-untranslated region. TED also stimulates translation of heterologous mRNAs. In this study, we show that TED stimulates translation of a cat mRNA by increasing translation efficiency to the level of capped mRNA. This stimulatory activity is not impaired by translation through TED. TED stimulates translation efficiency from different positions within the mRNA, varying from the 5' end to 940 nt downstream of the coding region. Duplication of TED has an additive effect on translation stimulation only when located at both ends of the mRNA. On dicistronic RNAs, TED stimulates translation of both cistrons to the same extent. These data suggest that TED acts primarily by recruiting the translational machinery to the RNA.
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5 and 3 sequences of satellite Tobacco Necrosis Virus rna promoting translation in Tobacco
Plant Journal, 1998Co-Authors: Frank Meulewaeter, X Danthinne, Marc CornelissenAbstract:Summary The RNA of satellite Tobacco Necrosis Virus (STNV) is a monocistronic messenger that lacks both a 5′ cap and a 3′ poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5′-terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5′ region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5′ 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV-cat RNAs in Tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED-dependent translation in Tobacco was stimulated further by the STNV 5′ 38 nt.
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Expression of Tobacco Necrosis Virus open reading frames 1 and 2 is sufficient for the replication of satellite Tobacco Necrosis Virus
Virology, 1995Co-Authors: Marco Andriessen, Frank Meulewaeter, Marc CornelissenAbstract:Tobacco Necrosis Virus (TNV) is a small icosahedral plant Virus which is often associated with satellite Viruses. The genomic RNA of TNV contains six open reading frames (ORFs), of which ORFs 1 and 2 are thought to encode the viral polymerase. We demonstrate that Tobacco protoplasts transfected with a vector containing TNV ORFs 1 and 2 under the control of the cauliflower mosaic Virus 35S promoter, as well as protoplasts derived from transgenic Nicotiana tabacum containing the same gene(s), support replication of satellite Tobacco Necrosis Virus RNA.
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the 3 untranslated region of satellite Tobacco Necrosis Virus rna stimulates translation in vitro
Molecular and Cellular Biology, 1993Co-Authors: X Danthinne, Frank Meulewaeter, Jef Seurinck, M Van Montagu, Marc CornelissenAbstract:Abstract The RNA of satellite Tobacco Necrosis Virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornaVirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.
