The Experts below are selected from a list of 3759 Experts worldwide ranked by ideXlab platform
Etienne Decroly - One of the best experts on this subject based on the ideXlab platform.
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structure and sequence requirements for RNA Capping at the venezuelan equine encephalitis virus RNA 5 end
Journal of Virology, 2021Co-Authors: Oney Ortega Granda, Bruno Canard, Bruno Coutard, Etienne Decroly, Nadia Rabah, Coralie Valle, Ashleigh ShannonAbstract:Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5' end. The Capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The Capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that Capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the Capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral Capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.
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Mutations on VEEV nsP1 relate RNA Capping efficiency to ribavirin susceptibility
Antiviral Research, 2020Co-Authors: Nadia Rabah, Bruno Canard, Etienne Decroly, Gilles Querat, Oney Ortega Granda, Bruno CoutardAbstract:Alphaviruses are arthropod-borne viruses of public health concern. To date no efficient vaccine nor antivirals are available for safe human use. During viral replication the nonstructural protein 1 (nsP1) catalyzes Capping of genomic and subgenomic RNAs. The Capping reaction is unique to the Alphavirus genus. The whole three-step process follows a particular order: (i) transfer of a methyl group from S-adenosyl methionine (SAM) onto a GTP forming m7GTP; (ii) guanylylation of the enzyme to form a m7GMP-nsP1adduct; (iii) transfer of m7GMP onto 5′-diphosphate RNA to yield capped RNA. Specificities of these reactions designate nsP1 as a promising target for antiviral drug development. In the current study we performed a mutational analysis on two nsP1 positions associated with Sindbis virus (SINV) ribavirin resistance in the Venezuelan equine encephalitis virus (VEEV) context through reverse genetics correlated to enzyme assays using purified recombinant VEEV nsP1 proteins. The results demonstrate that the targeted positions are strongly associated to the regulation of the Capping reaction by increasing the affinity between GTP and nsP1. Data also show that in VEEV the S21A substitution, naturally occurring in Chikungunya virus (CHIKV), is a hallmark of ribavirin susceptibility. These findings uncover the specific mechanistic contributions of these residues to nsp1-mediated methyl-transfer and guanylylation reactions.
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the methyltransferase domain of the sudan ebolavirus l protein specifically targets inteRNAl adenosines of RNA substrates in addition to the cap structure
Nucleic Acids Research, 2018Co-Authors: Baptiste Martin, Bruno Canard, Bruno Coutard, Theo Guez, Guido C Paesen, Francoise Debart, Jeanjacques Vasseur, J M Grimes, Etienne DecrolyAbstract:Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA Capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2’O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2’O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2’O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting inteRNAl adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2’O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA inteRNAl methylations in the viral replication.
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discovery of novel dengue virus ns5 methyltransferase non nucleoside inhibitors by fragment based drug design
European Journal of Medicinal Chemistry, 2017Co-Authors: Bruno Coutard, Etienne Decroly, Gilles Querat, Fatiha Benmansour, Iuni Trist, Andrea Brancale, Karine BarralAbstract:Abstract With the aim to help drug discovery against dengue virus (DENV), a fragment-based drug design approach was applied to identify ligands targeting a main component of DENV replication complex: the NS5 AdoMet-dependent mRNA methyltransferase (MTase) domain, playing an essential role in the RNA Capping process. Herein, we describe the identification of new inhibitors developed using fragment-based, structure-guided linking and optimization techniques. Thermal-shift assay followed by a fragment-based X-ray crystallographic screening lead to the identification of three fragment hits binding DENV MTase. We considered linking two of them, which bind to proximal sites of the AdoMet binding pocket, in order to improve their potency. X-ray crystallographic structures and computational docking were used to guide the fragment linking, ultimately leading to novel series of non-nucleoside inhibitors of flavivirus MTase, respectively N-phenyl-[(phenylcarbamoyl)amino]benzene-1-sulfonamide and phenyl [(phenylcarbamoyl)amino]benzene-1-sulfonate derivatives, that show a 10–100-fold stronger inhibition of 2′-O-MTase activity compared to the initial fragments.
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the viral Capping enzyme nsp1 a novel target for the inhibition of chikungunya virus infection
Scientific Reports, 2016Co-Authors: Leen Delang, Etienne Decroly, Ali Tas, Gilles Querat, Irina C Albulescu, T De Burghgraeve, N Segura A Guerrero, Alba Gigante, Geraldine PiorkowskiAbstract:The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a novel class of small molecules ([1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA Capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus Capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.
Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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RNA Capping progress and prospects
RNA, 2015Co-Authors: Stewart ShumanAbstract:The 5′ m7GpppN cap, co-discovered by Shatkin, Furuichi, and Moss in 1975, is the signature feature of eukaryal cellular and viral messenger RNA that confers mRNA stability and efficient translation. Cap formation entails three sequential enzymatic modifications targeted to nascent pre-mRNAs synthesized by cellular or viral RNA polymerases. First, the 5′ triphosphate end of the pre-mRNA is hydrolyzed to a diphosphate by RNA 5′ triphosphatase (RTPase). Second, the diphosphate RNA is capped with GMP by RNA guanylyltransferase (GTase) via a two-step mechanism: (i) reaction of GTase with GTP to form a covalent enzyme-(lysyl-Nζ)-GMP intermediate and PPi and (ii) transfer of GMP from GTase to the ppRNA end to form GpppRNA. Third, the GpppRNA cap is converted to m7GpppRNA by AdoMet:RNA(guanine-N7)-methyltransferase (MTase). This pathway was elucidated between 1975 and 1984 via the analysis of the purified vaccinia virus Capping enzyme, a heterodimer of 97 kDa and 33 kDa subunits that catalyzes all three steps in cap formation. The same biochemical pathway (though not the same organization of the Capping apparatus) is conserved in all eukaryal taxa.
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The polynucleotide ligase and RNA Capping enzyme superfamily of covalent nucleotidyltransferases.
Current Opinion in Structural Biology, 2004Co-Authors: Stewart Shuman, Christopher D. LimaAbstract:ATP- and NAD+-dependent DNA ligases, ATP-dependent RNA ligases and GTP-dependent mRNA Capping enzymes comprise a superfamily of proteins that catalyze nucleotidyl transfer to polynucleotide 5′ ends via covalent enzyme-(lysyl-N)–NMP intermediates. The superfamily is defined by five peptide motifs that line the nucleotide-binding pocket and contribute amino acid sidechains essential for catalysis. Early crystal structures revealed a shared core tertiary structure for DNA ligases and Capping enzymes, which are composed minimally of a nucleotidyltransferase domain fused to a distal OB-fold domain. Recent structures of viral and bacterial DNA ligases, and a fungal mRNA Capping enzyme underscore how the substrate-binding and chemical steps of the ligation and Capping pathways are coordinated with large rearrangements of the component protein domains and with remodeling of the atomic contacts between the enzyme and the nucleotide at the active site. The first crystal structure of an RNA ligase suggests that contemporary DNA ligases, RNA ligases and RNA Capping enzymes evolved by fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.
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Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Stewart ShumanAbstract:RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases. Here, we identified another RNA ligase from the bacterial domain—a second RNA ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA Capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.
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what messenger RNA Capping tells us about eukaryotic evolution
Nature Reviews Molecular Cell Biology, 2002Co-Authors: Stewart ShumanAbstract:The 5′ cap is a unique feature of eukaryotic cellular and viral messenger RNA that is absent from the bacterial and archaeal domains of life. The cap is formed by three enzymatic reactions at the 5′ terminus of nascent mRNAs. Although the Capping pathway is conserved in all eukaryotes, the structure and genetic organization of the component enzymes vary between species. These differences provide insights into the evolution of eukaryotes and eukaryotic viruses.
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characterization of an atp dependent dna ligase encoded by chlorella virus pbcv 1
Journal of Virology, 1997Co-Authors: J. L. Van Etten, Stewart ShumanAbstract:We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA Capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.
Bruno Coutard - One of the best experts on this subject based on the ideXlab platform.
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structure and sequence requirements for RNA Capping at the venezuelan equine encephalitis virus RNA 5 end
Journal of Virology, 2021Co-Authors: Oney Ortega Granda, Bruno Canard, Bruno Coutard, Etienne Decroly, Nadia Rabah, Coralie Valle, Ashleigh ShannonAbstract:Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5' end. The Capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The Capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that Capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the Capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral Capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.
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Mutations on VEEV nsP1 relate RNA Capping efficiency to ribavirin susceptibility
Antiviral Research, 2020Co-Authors: Nadia Rabah, Bruno Canard, Etienne Decroly, Gilles Querat, Oney Ortega Granda, Bruno CoutardAbstract:Alphaviruses are arthropod-borne viruses of public health concern. To date no efficient vaccine nor antivirals are available for safe human use. During viral replication the nonstructural protein 1 (nsP1) catalyzes Capping of genomic and subgenomic RNAs. The Capping reaction is unique to the Alphavirus genus. The whole three-step process follows a particular order: (i) transfer of a methyl group from S-adenosyl methionine (SAM) onto a GTP forming m7GTP; (ii) guanylylation of the enzyme to form a m7GMP-nsP1adduct; (iii) transfer of m7GMP onto 5′-diphosphate RNA to yield capped RNA. Specificities of these reactions designate nsP1 as a promising target for antiviral drug development. In the current study we performed a mutational analysis on two nsP1 positions associated with Sindbis virus (SINV) ribavirin resistance in the Venezuelan equine encephalitis virus (VEEV) context through reverse genetics correlated to enzyme assays using purified recombinant VEEV nsP1 proteins. The results demonstrate that the targeted positions are strongly associated to the regulation of the Capping reaction by increasing the affinity between GTP and nsP1. Data also show that in VEEV the S21A substitution, naturally occurring in Chikungunya virus (CHIKV), is a hallmark of ribavirin susceptibility. These findings uncover the specific mechanistic contributions of these residues to nsp1-mediated methyl-transfer and guanylylation reactions.
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the methyltransferase domain of the sudan ebolavirus l protein specifically targets inteRNAl adenosines of RNA substrates in addition to the cap structure
Nucleic Acids Research, 2018Co-Authors: Baptiste Martin, Bruno Canard, Bruno Coutard, Theo Guez, Guido C Paesen, Francoise Debart, Jeanjacques Vasseur, J M Grimes, Etienne DecrolyAbstract:Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA Capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2’O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2’O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2’O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting inteRNAl adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2’O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA inteRNAl methylations in the viral replication.
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discovery of novel dengue virus ns5 methyltransferase non nucleoside inhibitors by fragment based drug design
European Journal of Medicinal Chemistry, 2017Co-Authors: Bruno Coutard, Etienne Decroly, Gilles Querat, Fatiha Benmansour, Iuni Trist, Andrea Brancale, Karine BarralAbstract:Abstract With the aim to help drug discovery against dengue virus (DENV), a fragment-based drug design approach was applied to identify ligands targeting a main component of DENV replication complex: the NS5 AdoMet-dependent mRNA methyltransferase (MTase) domain, playing an essential role in the RNA Capping process. Herein, we describe the identification of new inhibitors developed using fragment-based, structure-guided linking and optimization techniques. Thermal-shift assay followed by a fragment-based X-ray crystallographic screening lead to the identification of three fragment hits binding DENV MTase. We considered linking two of them, which bind to proximal sites of the AdoMet binding pocket, in order to improve their potency. X-ray crystallographic structures and computational docking were used to guide the fragment linking, ultimately leading to novel series of non-nucleoside inhibitors of flavivirus MTase, respectively N-phenyl-[(phenylcarbamoyl)amino]benzene-1-sulfonamide and phenyl [(phenylcarbamoyl)amino]benzene-1-sulfonate derivatives, that show a 10–100-fold stronger inhibition of 2′-O-MTase activity compared to the initial fragments.
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crystal structure and functional analysis of the sars coronavirus RNA cap 2 o methyltransferase nsp10 nsp16 complex
PLOS Pathogens, 2011Co-Authors: Etienne Decroly, Mickael Bouvet, Laure Gluais, Andrew Sharff, Isabelle Imbert, Claire Debarnot, Francois Ferron, Bruno Coutard, Nicolas Papageorgiou, Gérard BricogneAbstract:Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA Capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 A resolution, which shows nsp10 bound to nsp16 through a ∼930 A2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA Capping enzymes in (+)RNA viruses.
Bruno Canard - One of the best experts on this subject based on the ideXlab platform.
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structure and sequence requirements for RNA Capping at the venezuelan equine encephalitis virus RNA 5 end
Journal of Virology, 2021Co-Authors: Oney Ortega Granda, Bruno Canard, Bruno Coutard, Etienne Decroly, Nadia Rabah, Coralie Valle, Ashleigh ShannonAbstract:Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5' end. The Capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The Capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that Capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the Capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral Capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.
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Mutations on VEEV nsP1 relate RNA Capping efficiency to ribavirin susceptibility
Antiviral Research, 2020Co-Authors: Nadia Rabah, Bruno Canard, Etienne Decroly, Gilles Querat, Oney Ortega Granda, Bruno CoutardAbstract:Alphaviruses are arthropod-borne viruses of public health concern. To date no efficient vaccine nor antivirals are available for safe human use. During viral replication the nonstructural protein 1 (nsP1) catalyzes Capping of genomic and subgenomic RNAs. The Capping reaction is unique to the Alphavirus genus. The whole three-step process follows a particular order: (i) transfer of a methyl group from S-adenosyl methionine (SAM) onto a GTP forming m7GTP; (ii) guanylylation of the enzyme to form a m7GMP-nsP1adduct; (iii) transfer of m7GMP onto 5′-diphosphate RNA to yield capped RNA. Specificities of these reactions designate nsP1 as a promising target for antiviral drug development. In the current study we performed a mutational analysis on two nsP1 positions associated with Sindbis virus (SINV) ribavirin resistance in the Venezuelan equine encephalitis virus (VEEV) context through reverse genetics correlated to enzyme assays using purified recombinant VEEV nsP1 proteins. The results demonstrate that the targeted positions are strongly associated to the regulation of the Capping reaction by increasing the affinity between GTP and nsP1. Data also show that in VEEV the S21A substitution, naturally occurring in Chikungunya virus (CHIKV), is a hallmark of ribavirin susceptibility. These findings uncover the specific mechanistic contributions of these residues to nsp1-mediated methyl-transfer and guanylylation reactions.
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the methyltransferase domain of the sudan ebolavirus l protein specifically targets inteRNAl adenosines of RNA substrates in addition to the cap structure
Nucleic Acids Research, 2018Co-Authors: Baptiste Martin, Bruno Canard, Bruno Coutard, Theo Guez, Guido C Paesen, Francoise Debart, Jeanjacques Vasseur, J M Grimes, Etienne DecrolyAbstract:Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA Capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2’O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2’O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2’O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting inteRNAl adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2’O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA inteRNAl methylations in the viral replication.
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insights into RNA synthesis Capping and proofreading mechanisms of sars coronavirus
Virus Research, 2014Co-Authors: Marion Sevajol, Etienne Decroly, Bruno Canard, Lorenzo Subissi, Isabelle ImbertAbstract:The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30 kb. In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3′–5′ exoribonuclease activities involved in RNA Capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world.
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the viral RNA Capping machinery as a target for antiviral drugs
Antiviral Research, 2012Co-Authors: Francois Ferron, Barbara Selisko, Etienne Decroly, Bruno CanardAbstract:Most viruses modify their genomic and mRNA 5'-ends with the addition of an RNA cap, allowing efficient mRNA translation, limiting degradation by cellular 5'-3' exonucleases, and avoiding its recognition as foreign RNA by the host cell. Viral RNA caps can be synthesized or acquired through the use of a Capping machinery which exhibits a significant diversity in organization, structure and mechanism relative to that of their cellular host. Therefore, viral RNA Capping has emerged as an interesting field for antiviral drug design. Here, we review the different pathways and mechanisms used to produce viral mRNA 5'-caps, and present current structures, mechanisms, and inhibitors known to act on viral RNA Capping.
Barbara Selisko - One of the best experts on this subject based on the ideXlab platform.
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the viral RNA Capping machinery as a target for antiviral drugs
Antiviral Research, 2012Co-Authors: Francois Ferron, Barbara Selisko, Etienne Decroly, Bruno CanardAbstract:Most viruses modify their genomic and mRNA 5'-ends with the addition of an RNA cap, allowing efficient mRNA translation, limiting degradation by cellular 5'-3' exonucleases, and avoiding its recognition as foreign RNA by the host cell. Viral RNA caps can be synthesized or acquired through the use of a Capping machinery which exhibits a significant diversity in organization, structure and mechanism relative to that of their cellular host. Therefore, viral RNA Capping has emerged as an interesting field for antiviral drug design. Here, we review the different pathways and mechanisms used to produce viral mRNA 5'-caps, and present current structures, mechanisms, and inhibitors known to act on viral RNA Capping.
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flaviviral methyltransferase RNA interaction structural basis for enzyme inhibition
Antiviral Research, 2009Co-Authors: Mario Milani, Mickael Bouvet, Barbara Selisko, Eloise Mastrangelo, Michela Bollati, Bruno Canard, Etienne Decroly, Martino BolognesiAbstract:Abstract Flaviviruses are the causative agents of severe diseases such as Dengue or Yellow fever. The replicative machinery used by the virus is based on few enzymes including a methyltransferase, located in the N-terminal domain of the NS5 protein. Flaviviral methyltransferases are involved in the last two steps of the mRNA Capping process, transferring a methyl group from S-adenosyl- l -methionine onto the N7 position of the cap guanine (guanine-N7 methyltransferase) and the ribose 2′O position of the first nucleotide following the cap guanine (nucleoside-2′O methyltransferase). The RNA Capping process is crucial for mRNA stability, protein synthesis and virus replication. Such an essential function makes methyltransferases attractive targets for the design of antiviral drugs. In this context, starting from the crystal structure of Wesselsbron flavivirus methyltransferase, we elaborated a mechanistic model describing protein/RNA interaction during N7 methyl transfer. Next we used an in silico docking procedure to identify commercially available compounds that would display high affinity for the methyltransferase active site. The best candidates selected were tested in vitro to assay their effective inhibition on 2′O and N7 methyltransferase activities on Wesselsbron and Dengue virus (Dv) methyltransferases. The results of such combined computational and experimental screening approach led to the identification of a high-potency inhibitor.
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recognition of RNA cap in the wesselsbron virus ns5 methyltransferase domain implications for RNA Capping mechanisms in flavivirus
Journal of Molecular Biology, 2009Co-Authors: Michela Bollati, Mario Milani, Barbara Selisko, Simona Nonnis, Gabriella Tedeschi, Stefano Ricagno, Eloise Mastrangelo, Etienne Decroly, Bruno CoutardAbstract:The mRNA-Capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′–5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; Capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support Capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, N7MeGpppG, and N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities.
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Recognition of the RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-Capping mechanisms in Flavivirus
'Elsevier BV', 2009Co-Authors: Michela Bollati, Mario Milani, Barbara Selisko, Simona Nonnis, Gabriella Tedeschi, Stefano Ricagno, Eloise Mastrangelo, Etienne Decroly, Bruno CoutardAbstract:The mRNA-Capping process starts with the conversion of a 5V-triphosphate end into a 5V-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5V–5Vphosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-L-methionine to N7-guanine (cap 0) and to the ribose 2VOH group (cap 1) of the first RNA nucleotide; Capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2VO-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support Capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-Lmethionine cofactor, S-adenosyl-L-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, N7MeGpppG, and N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate,Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of theWesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7- methyltransferase and 2VO-methyltransferase activities
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Flaviviral methyltransferase/RNA interaction: structural basis for enzyme inhibition
'Elsevier BV', 2009Co-Authors: Mario Milani, Mickael Bouvet, Barbara Selisko, Eloise Mastrangelo, Michela Bollati, Bruno Canard, Etienne Decroly, Martino BolognesiAbstract:Flaviviruses are the causative agents of severe diseases such as Dengue or Yellow fever. The replicative machinery used by the virus is based on few enzymes including a methyltransferase, located in the N-terminal domain of the NS5 protein. Flaviviral methyltransferases are involved in the last two steps of the mRNA Capping process, transferring a methyl group from S-adenosyl-L-methionine onto the N7 position of the cap guanine (guanine-N7 methyltransferase) and the ribose 2'O position of the first nucleotide following the cap guanine (nucleoside-2'O methyltransferase). The RNA Capping process is crucial for mRNA stability, protein synthesis and virus replication. Such an essential function makes methyltransferases attractive targets for the design of antiviral drugs. In this context, starting from the crystal structure of Wesselsbron flavivirus methyltransferase, we elaborated a mechanistic model describing protein/RNA interaction during N7 methyl transfer. Next we used an in silico docking procedure to identify commercially available compounds that would display high affinity for the methyltransferase active site. The best candidates selected were tested in vitro to assay their effective inhibition on 2'O and N7 methyltransferase activities on Wesselsbron and Dengue virus (Dv) methyltransferases. The results of such combined computational and experimental screening approach led to the identification of a high-potency inhibitor
