The Experts below are selected from a list of 162 Experts worldwide ranked by ideXlab platform
Jeffrey S. Kieft - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of a polerovirus exoribonuclease resistant rna shows how diverse sequences are integrated into a conserved fold
RNA, 2020Co-Authors: Anna-lena Steckelberg, David A. Costantino, Quentin Vicens, Jay C Nix, Jeffrey S. KieftAbstract:Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5' to 3' exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a Dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from Dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences.
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a folded viral noncoding rna blocks host cell exoribonucleases through a conformationally dynamic rna structure
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A. Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5′ → 3′ cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in Dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a Dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5′ end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the Dianthovirus xrRNAs undergo conformational changes and can use “codegradational remodeling,” exploiting the exoribonucleases’ degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
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a folded viral noncoding rna blocks host cell exoribonucleases through programmed remodeling of rna structure
bioRxiv, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A. Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5'-->3' cellular exoribonucleases (xrRNAs) to produce biologically active viral non-coding RNAs were discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, it was unknown if this RNA structure-dependent mechanism exists elsewhere and if so, whether a singular RNA fold is required. Here, we demonstrate the existence of authentic RNA structure-dependent xrRNAs in Dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These novel xrRNAs have no sequence similarity to known xrRNAs, thus we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a Dianthovirus xrRNAs by x-ray crystallography, we reveal a complex fold that is very different from the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature that is created by a different set of intramolecular contacts; this may be a defining structural feature of xrRNAs. Remarkably, the Dianthovirus xrRNA can use 'co-degradational remodeling,' exploiting the exoribonuclease's degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
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Exoribonuclease-Resistant RNAs Exist within both Coding and Noncoding Subgenomic RNAs
'American Society for Microbiology', 2018Co-Authors: Anna-lena Steckelberg, Quentin Vicens, Jeffrey S. KieftAbstract:During infection, viruses often produce subgenomic RNAs (sgRNAs) that either serve as the template for protein synthesis or act as “riboregulators” that interact with and influence the viral and cellular machinery. Recently, a mechanism for producing sgRNAs was found that depends on the presence of specifically structured RNA elements (xrRNAs). However, the degree to which this mechanism is used, where the elements are found, their structural diversity, and what types of sgRNAs are produced by this pathway were unclear. This article describes the discovery of these structured RNA elements in two large families of plant viruses and shows that they are used to produce both protein-coding sgRNAs and “riboregulatory” RNAs. These discoveries provide evidence that xrRNA-based RNA maturation pathways may be more widespread than previously anticipated and that they are involved in producing a variety of RNAs of diverse functions.Many viruses produce protein-coding and noncoding subgenomic RNAs (sgRNAs) that are critical for infection. A recently discovered pathway for viral sgRNA production uses exoribonuclease-resistant RNAs (xrRNAs), discrete folded RNA elements that block the processive exoribonucleolytic degradation of RNA. xrRNAs are widespread in animal-infecting flaviviruses but had been found only in three members of the plant virus genus Dianthovirus. Also, xrRNAs had been found only in the 3′ untranslated regions (3′UTRs) of viral RNAs, where they produce noncoding sgRNAs. The degree to which xrRNA elements exist in other viruses, the conservation of their ring-like fold, and the ability of xrRNAs to operate in diverse contexts were unknown. Using computational tools and biochemical assays, we discovered xrRNA elements pervading two large families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Comparison of the sequences and functional requirements suggests that all adopt the characteristic ring-like fold. Unexpectedly, many of these newly discovered xrRNAs are located in intergenic regions rather than 3´UTRs, and some are associated with the 5′ ends of subgenomic RNAs that encode viral proteins. This suggests that xrRNAs are involved in the production of both coding and noncoding subgenomic RNAs and can operate as part of broader mechanisms to regulate RNA levels and protein expression. These discoveries expand the potential roles for xrRNAs and suggest that xrRNAs may represent a more general strategy for RNA maturation and maintenance than previously known
Steven A. Lommel - One of the best experts on this subject based on the ideXlab platform.
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a sobemovirus coat protein gene complements long distance movement of a coat protein null Dianthovirus
Virology, 2004Co-Authors: Anton S Callaway, Carol G George, Steven A. LommelAbstract:Red clover necrotic mosaic virus (RCNMV; genus Dianthovirus) and Turnip rosette virus (TRoV; genus Sobemovirus) are taxonomically and ecologically distinct plant viruses. In addition, the two genera differ in the role of coat protein (CP) in cell-to-cell movement. However, both are small icosahedral viruses requiring CP for systemic movement in the host vasculature. Here, we show that the TRoV CP gene is capable of facilitating the vascular movement of a Dianthovirus. Substitution of the RCNMV CP gene with the TRoV CP gene permits movement of the resulting chimeric virus to non-inoculated leaves. RCNMV lacking a CP gene or containing a non-translatable TRoV CP gene do not move systemically. This report introduces the molecular characterization of TRoV and describes the unprecedented complementation of systemic movement function by intergenic complete substitution of a plant virus CP gene.
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Sequence element required for efficient -1 ribosomal frameshifting in red clover necrotic mosaic Dianthovirus.
Virology, 1998Co-Authors: Kook-hyung Kim, Steven A. LommelAbstract:Abstract The RNA−1 of the bipartite red clover necrotic mosaic Dianthovirus (RCNMV) genome encodes the 88-kDa polymerase. The polymerase is translated from both 5′ proximal and internal open reading frames by a −1 ribosomal frameshifting event. A shifty heptanucleotide conforming to the simultaneous slippage model is identified, and a downstream stem-loop structure and atypical pseudoknot are predicted. A β-glucuronidase reporter assay identified a 118-nucleotide element containing both the shifty heptanucleotide and the predicted secondary structures that were required for efficient −1 ribosomal frameshift expression in vivo . A series of site-directed and compensatory mutations affecting the base-paired regions of the predicted secondary structure were introduced into a RCNMV RNA−1 cDNA clone from which infectious transcripts were derived. Mutations that destroyed the predicted pseudoknot had no effect on frameshifting efficiency in vitro or infectivity of the virus, whereas mutations destabilizing the stem-loop structure abolished both ribosomal frameshifting in vitro and biological activity. These results demonstrate the essential role of a predicted secondary structure that does not involve a pseudoknot in the expression of the RCNMV polymerase by ribosomal frameshifting.
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Tobamovirus and Dianthovirus movement proteins are functionally homologous.
Virology, 1995Co-Authors: Donna Giesman-cookmeyer, Steven A. Lommel, S. Silver, Andrew A. Vaewhongs, C M DeomAbstract:The movement proteins (MPs) of tobacco mosaic tobamovirus (TMV) and red clover necrotic mosaic Dianthovirus (RCNMV) enlarge plasmodesmata size exclusion limits, transport RNA from cell to cell, and bind nucleic acids in vitro. Despite these functional similarities, they have no sequence homology. However, they do appear to have similar secondary structures. We have used transgenic plants expressing either the TMV MP or the RCNMV MP, and a chimeric TMV that encodes the RCNMV MP as its only functional MP gene, to demonstrate that the MPs of TMV and RCNMV are functionally homologous. Further, both TMV and RCNMV can act as helper viruses to allow the cell-to-cell movement of the heterologous movement-defective viruses. These data support the conclusion that, despite other differences, such as particle morphology, host range, and sequence, TMV and RCNMV share a common mechanism for cell-to-cell movement.
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nucleotide sequence of carnation ringspot Dianthovirus rna 1
Journal of General Virology, 1994Co-Authors: Eugene V Ryabov, Steven A. Lommel, E V Generozov, T L Kendall, S K ZavrievAbstract:The nucleotide sequence of carnation ringspot virus (CRSV) RNA-1, the type member of the Dianthovirus genus, has been determined. The 3756 nucleotide genomic RNA-1 contains three large open reading frames (ORFs), capable of encoding 27K, 54K and 38K polypeptides. In addition, a small ORF encoding a 10K polypeptide at the 3′ terminus of the RNA has been identified. The gene organization of CRSV RNA-1 is similar to those of red clover necrotic mosaic (RCNMV) and sweet clover necrotic mosaic (SCNMV) diantho-viruses with the exception that CRSV RNA-1 contains the additional 3′-terminal ORF. The 27K and 54K proteins possess significant sequence similarity to corresponding polypeptides of the other Dianthoviruses. The 54K protein also contains the conserved RNA-dependent RNA polymerase motif. The identification of a shifty heptanucleotide preceding the p27 ORF termination codon and a predicted secondary structure following the terminator suggest that a translational frameshifting event allows translation to continue past the p27 ORF into the p54 ORF, which is in the -1 frame, generating an 88K fusion protein. Amino acid sequence alignment of the 38K protein with the corresponding RCNMV and SCNMV polypeptides indicate that this is the viral capsid protein.
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nucleotide sequence of carnation ringspot Dianthovirus rna 2
Journal of General Virology, 1992Co-Authors: T L Kendall, Steven A. LommelAbstract:RNA-2 of carnation ringspot virus (CRSV), the type member of the Dianthovirus group, has been cDNA cloned and sequenced. CRSV RNA-2 is 1394 nucleotides in length and contains a single open reading frame encoding a 304 amino acid polypeptide of 33.8K. Amino acid sequence alignment of this polypeptide with the cell-to-cell movement proteins encoded by RNA-2 of red clover necrotic mosaic virus (RCNMV) Australian (Aus) and Czechoslovakian (TpM-34) isolates indicates 59.6% and 55.7% sequence identity, respectively. The N-terminal 230 amino acids are more highly conserved, with 64.3% and 62.6% sequence identity, respectively. The cell-to-cell movement proteins of the two RCNMV isolates are themselves 82.5% and 91.7% identical when the amino-terminal 230 amino acids are compared. Structural prediction comparison of the RCNMV-Aus, RCNMV-TpM-34 and tobacco mosaic virus cell-to-cell movement proteins to the putative CRSV RNA-2-encoded movement protein suggests that even though no primary amino acid sequence similarity exists, the movement protein polypeptides are possibly similar in structure and function.
Anna-lena Steckelberg - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of a polerovirus exoribonuclease resistant rna shows how diverse sequences are integrated into a conserved fold
RNA, 2020Co-Authors: Anna-lena Steckelberg, David A. Costantino, Quentin Vicens, Jay C Nix, Jeffrey S. KieftAbstract:Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5' to 3' exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a Dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from Dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences.
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a folded viral noncoding rna blocks host cell exoribonucleases through a conformationally dynamic rna structure
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A. Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5′ → 3′ cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in Dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a Dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5′ end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the Dianthovirus xrRNAs undergo conformational changes and can use “codegradational remodeling,” exploiting the exoribonucleases’ degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
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a folded viral noncoding rna blocks host cell exoribonucleases through programmed remodeling of rna structure
bioRxiv, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A. Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5'-->3' cellular exoribonucleases (xrRNAs) to produce biologically active viral non-coding RNAs were discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, it was unknown if this RNA structure-dependent mechanism exists elsewhere and if so, whether a singular RNA fold is required. Here, we demonstrate the existence of authentic RNA structure-dependent xrRNAs in Dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These novel xrRNAs have no sequence similarity to known xrRNAs, thus we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a Dianthovirus xrRNAs by x-ray crystallography, we reveal a complex fold that is very different from the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature that is created by a different set of intramolecular contacts; this may be a defining structural feature of xrRNAs. Remarkably, the Dianthovirus xrRNA can use 'co-degradational remodeling,' exploiting the exoribonuclease's degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
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Exoribonuclease-Resistant RNAs Exist within both Coding and Noncoding Subgenomic RNAs
'American Society for Microbiology', 2018Co-Authors: Anna-lena Steckelberg, Quentin Vicens, Jeffrey S. KieftAbstract:During infection, viruses often produce subgenomic RNAs (sgRNAs) that either serve as the template for protein synthesis or act as “riboregulators” that interact with and influence the viral and cellular machinery. Recently, a mechanism for producing sgRNAs was found that depends on the presence of specifically structured RNA elements (xrRNAs). However, the degree to which this mechanism is used, where the elements are found, their structural diversity, and what types of sgRNAs are produced by this pathway were unclear. This article describes the discovery of these structured RNA elements in two large families of plant viruses and shows that they are used to produce both protein-coding sgRNAs and “riboregulatory” RNAs. These discoveries provide evidence that xrRNA-based RNA maturation pathways may be more widespread than previously anticipated and that they are involved in producing a variety of RNAs of diverse functions.Many viruses produce protein-coding and noncoding subgenomic RNAs (sgRNAs) that are critical for infection. A recently discovered pathway for viral sgRNA production uses exoribonuclease-resistant RNAs (xrRNAs), discrete folded RNA elements that block the processive exoribonucleolytic degradation of RNA. xrRNAs are widespread in animal-infecting flaviviruses but had been found only in three members of the plant virus genus Dianthovirus. Also, xrRNAs had been found only in the 3′ untranslated regions (3′UTRs) of viral RNAs, where they produce noncoding sgRNAs. The degree to which xrRNA elements exist in other viruses, the conservation of their ring-like fold, and the ability of xrRNAs to operate in diverse contexts were unknown. Using computational tools and biochemical assays, we discovered xrRNA elements pervading two large families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Comparison of the sequences and functional requirements suggests that all adopt the characteristic ring-like fold. Unexpectedly, many of these newly discovered xrRNAs are located in intergenic regions rather than 3´UTRs, and some are associated with the 5′ ends of subgenomic RNAs that encode viral proteins. This suggests that xrRNAs are involved in the production of both coding and noncoding subgenomic RNAs and can operate as part of broader mechanisms to regulate RNA levels and protein expression. These discoveries expand the potential roles for xrRNAs and suggest that xrRNAs may represent a more general strategy for RNA maturation and maintenance than previously known
K W Buck - One of the best experts on this subject based on the ideXlab platform.
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Double-stranded RNAs isolated from plant tissue infected with red clover necrotic mosaic virus correspond to genomic and subgenomic
2014Co-Authors: Single-stranded Rnas, T. A. M. Osman, K W BuckAbstract:Red clover necrotic mosaic Dianthovirus (RCNMV) has a genome of two ssRNA species, RNA 1 (4-0 kb) and RNA 2 (1-4 kb). Double-stranded RNA was isolated from Phaseolus vulgaris plants infected separa-tely with five isolates of RCNMV from Czechoslova-kia, Sweden, Scotland and England. In each case three species of dsRNA, designated A, B and C in order of increasing mobility, were solved by gel electrophore-sis in non-denaturing conditions. The mobilities of dsRNA species A and B were similar for all the isolates, but the mobilities of the different species of dsRNA C showed some variation. Southern blotting followed by hybridization with cloned probes showed that dsRNA species A and B are related to RNA 1, whereas dsRNA C is related to RNA 2. In gel electrophoresis
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Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic Dianthovirus.
Journal of General Virology, 1995Co-Authors: Helen Bates, M Farjah, T. A. M. Osman, K W BuckAbstract:A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic Dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl β-d-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the poly-merase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.
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Localization of a single-stranded RNA-binding domain in the movement protein of red clover necrotic mosaic Dianthovirus.
The Journal of general virology, 1993Co-Authors: T A Osman, P Thömmes, K W BuckAbstract:Mutant movement proteins of red clover necrotic mosaic Dianthovirus (RCNMV), consisting of in-frame deletions or fusions with a maltose-binding protein, were produced in Escherichia coli using expression vectors. The ability of the mutant proteins to bind to ssRNA was tested by photochemical cross-linking and gel retardation. The results showed that the region between amino acids 181 and 225 of the RCNMV movement protein contains an ssRNA-binding domain.
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Localization of a single-stranded RNA-binding domain in the movement protein of red clover necrotic mosaic Dianthovirus
1993Co-Authors: T. A. M. Osman, P Thömmes, K W BuckAbstract:Mutant movement proteins of red clover necrotic mosaic Dianthovirus (RCNMV), consisting of in-frame deletions or fusions with a maltose-binding protein, were pro-duced in Escherichia coli using expression vectors. The ability of the mutant proteins to bind to ssRNA was tested by photochemical cross-linking and gel retar-dation. The results showed that the region between amino acids 181 and 225 of the RCNMV movement protein contains an ssRNA-binding domain. There is a growing body of evidence that movement of plant viruses from cell to cell occurs via the intercellular channels known as plasmodesmata, and is mediated by virus-encoded movement proteins (for review, see Hull
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cooperative binding of the red clover necrotic mosaic virus movement protein to single stranded nucleic acids
Journal of General Virology, 1992Co-Authors: T. A. M. Osman, R J Hayes, K W BuckAbstract:The movement protein of red clover necrotic mosaic Dianthovirus was produced in Escherichia coli using an expression vector. Gel retardation analysis and u.v. cross-linking studies showed that the movement protein bound cooperatively to ssRNA and ssDNA, but not to dsDNA. Binding competition experiments established that the movement protein bound to ssRNA and ssDNA with similar affinities and that the binding was not sequence-specific in the experimental conditions employed. A truncated movement protein lacking the C-terminal 88 amino acids was also shown to bind to ssRNA.
David A. Costantino - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of a polerovirus exoribonuclease resistant rna shows how diverse sequences are integrated into a conserved fold
RNA, 2020Co-Authors: Anna-lena Steckelberg, David A. Costantino, Quentin Vicens, Jay C Nix, Jeffrey S. KieftAbstract:Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5' to 3' exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a Dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from Dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences.
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a folded viral noncoding rna blocks host cell exoribonucleases through a conformationally dynamic rna structure
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A. Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5′ → 3′ cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in Dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a Dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5′ end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the Dianthovirus xrRNAs undergo conformational changes and can use “codegradational remodeling,” exploiting the exoribonucleases’ degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
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a folded viral noncoding rna blocks host cell exoribonucleases through programmed remodeling of rna structure
bioRxiv, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A. Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5'-->3' cellular exoribonucleases (xrRNAs) to produce biologically active viral non-coding RNAs were discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, it was unknown if this RNA structure-dependent mechanism exists elsewhere and if so, whether a singular RNA fold is required. Here, we demonstrate the existence of authentic RNA structure-dependent xrRNAs in Dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These novel xrRNAs have no sequence similarity to known xrRNAs, thus we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a Dianthovirus xrRNAs by x-ray crystallography, we reveal a complex fold that is very different from the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature that is created by a different set of intramolecular contacts; this may be a defining structural feature of xrRNAs. Remarkably, the Dianthovirus xrRNA can use 'co-degradational remodeling,' exploiting the exoribonuclease's degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.