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Esteban Domingo - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterization of a dual inhibitory and mutagenic activity of 5-Fluorouridine Triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.
Journal of molecular biology, 2008Co-Authors: Rubén Agudo, Armando Arias, Nonia Pariente, Celia Perales, Cristina Escarmís, Alberto Jorge, Anabel Marina, Esteban DomingoAbstract:The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine Triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-Fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-Fluorouridine Triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.
Rubén Agudo - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterization of a dual inhibitory and mutagenic activity of 5-Fluorouridine Triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.
Journal of molecular biology, 2008Co-Authors: Rubén Agudo, Armando Arias, Nonia Pariente, Celia Perales, Cristina Escarmís, Alberto Jorge, Anabel Marina, Esteban DomingoAbstract:The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine Triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-Fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-Fluorouridine Triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.
Domingo Esteban - One of the best experts on this subject based on the ideXlab platform.
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Molecular Characterization of a Dual Inhibitory and Mutagenic Activity of 5-Fluorouridine Triphosphate on Viral RNA Synthesis. Implications for Lethal Mutagenesis
'Elsevier BV', 2021Co-Authors: Agudo Rubén, Arias Armando, Pariente Nonia, Perales Celia, Escarmís Cristina, Jorge Alberto, Marina Anabel, Domingo EstebanAbstract:The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine Triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-Fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-Fluorouridine Triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe. © 2008 Elsevier Ltd. All rights reserved.This work was supported by grant BFU 2005-00863 from Ministerio de Educación y Ciencia, Proyecto Intramural de Frontera 2005-20F-0221 from CSIC (Consejo Superior de Investigaciones Científicas), and Fundación Ramón Areces. CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. R.A. is the recipient of a predoctoral fellowship from Comunidad Autónoma de Madrid, A.A. is the recipient of an I3P contract from CSIC, and C.P. is the recipient of a contract from CIBERehd
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Design, synthesis and X-Ray of Foot-and-mouth disease virus (Fluoro)uridylylated peptides linked to the genome (VPgpU and VPgpFU)
2016Co-Authors: Sonia De ,castro, Domingo Esteban, Ferrer-orta Cristina, Verdaguer Núria, Fernández-cureses Gloria, Camarasa Rius, María JoséAbstract:Póster presentado en el 3rd Antiviral Congress, celebrado del 12 al 14 de octubre de 2014 en Amsterdam (Holanda)Foot-and-mount disease virus (FMDV), belonging to the picornavirus family, is responsible for the highly contagious Foot-and-mounth disease (FMD) of cloven-hoofed bovids. The viral RNA synthesis, is initiated by a viral protein genome-linked (VPg) of 20-24 aminoacids. Unliquely of other picornavirus, FMDV-RNA encodes three distinguishable copies of this protein (VPg1, VPg2, VPg3). Each of them attached to genomic RNA, and therefore believed to be functionally equivalent. During replication initiation, the first step is the binding of a UMP to the hydroxyl group of Tyr3 in the VPg protein. The virally encoded RNA-dependent RNA polymerase (3D) requires the uridylylated VPg form as the primer for both positive- and negative-strand synthesis. 5-Fluorouridine Triphosphate (FUTP) is a potent competitive inhibitor of VPg-1 uridylylation. By peptide analysis, a VPg fragment containing FUMP covalently attached to Tyr has been identified. However, the molecular basis of this phenomenon is still unknown. To investigate the role of FUMP, the synthesis and X-ray studies of simplified models of VPg-1 containing U or FU, in peptides of diferent lenghts linked through the hydroxyl group of Tyr3 will be presented. Interestingly, X-ray structure of 3D-pol with a 15-mer peptide FMDV/VPg-FU showed a significant distortion of b9-b11 loop of the polymerasePeer Reviewe
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Sequential structures provide insights into the fidelity of RNA replication
'Proceedings of the National Academy of Sciences', 2015Co-Authors: Ferrer-orta Cristina, Arias Armando, Escarmís Cristina, Domingo Esteban, Pérez-luque Rosa, Verdaguer NúriaAbstract:RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin Triphosphate and 5-Fluorouridine Triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3′-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases. © 2007 by The National Academy of Sciences of the USA.Work in Barcelona was supported by Grant BFU2005-02376/BMC from the Ministerio de Educación y Ciencia (M.E.C.) (to N.V.) and work in Madrid by Grant BFU2005-00863/BMC from the M.E.C. (to E.D.) and by the Fundación R. Areces. Work in Barcelona and Madrid was further supported by Proyecto Intramural de Frontera (CSIC) (to A.A. and C.F.-O.) and an I3P contract from CSIC (to A.A.). X-ray data were collected at the EMBL protein crystallography beam lines ID14.2 and ID14.4 at European Synchrotron Radiation Facility (ESRF) (Grenoble) within a block allocation group (BAG Barcelona). Financial support was provided by the ESRFPeer Reviewe
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Sequential structures provide insights into the fidelity of RNA replication
National Academy of Sciences, 2026Co-Authors: Ferrer-orta Cristina, Arias Armando, Escarmís Cristina, Domingo Esteban, Pérez-luque Rosa, Verdaguer NúriaAbstract:RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin Triphosphate and 5-Fluorouridine Triphosphate with different RNAs as template–primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3′-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases
Verdaguer Núria - One of the best experts on this subject based on the ideXlab platform.
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(F)uridylylated peptides linked to VPg1 of foot-and-mouth disease virus (FMDV): Design, synthesis and X-Ray crystallography of the complexes with FMDV RNA-Dependent RNA Polymerase
'MDPI AG', 2019Co-Authors: Sonia De ,castro, Ferrer-orta Cristina, Verdaguer Núria, Fernández-cureses Gloria, Mills A., Gago Federico, Camarasa Rius, María JoséAbstract:Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5′-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3D. 5-Fluorouridine Triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3D. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3D in the presence of VPg1-FUMP showed an 8 Å movement of the β9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3D with VPg1-UMP. The conformational rearrangement of this loop preceding the 3D B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.Financial support was also provided by the ESRF. Work in Madrid was supported by the Spanish Government (MINECO/FEDER project SAF2015-64629-C2) and Comunidad de Madrid (BIPEDD-2_CM ref. S-2010/BMD-2457). Work in Barcelona was supported by Grants BIO2017-83906-P (Ministry of Science Innovation and Universities (MCIU), Spain–European Fund for Economic and Regional Development (FEDER)) and Maria de Maeztu Unit of Excellence MDM-2014-0435Peer Reviewe
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Design, synthesis and X-Ray of Foot-and-mouth disease virus (Fluoro)uridylylated peptides linked to the genome (VPgpU and VPgpFU)
2016Co-Authors: Sonia De ,castro, Domingo Esteban, Ferrer-orta Cristina, Verdaguer Núria, Fernández-cureses Gloria, Camarasa Rius, María JoséAbstract:Póster presentado en el 3rd Antiviral Congress, celebrado del 12 al 14 de octubre de 2014 en Amsterdam (Holanda)Foot-and-mount disease virus (FMDV), belonging to the picornavirus family, is responsible for the highly contagious Foot-and-mounth disease (FMD) of cloven-hoofed bovids. The viral RNA synthesis, is initiated by a viral protein genome-linked (VPg) of 20-24 aminoacids. Unliquely of other picornavirus, FMDV-RNA encodes three distinguishable copies of this protein (VPg1, VPg2, VPg3). Each of them attached to genomic RNA, and therefore believed to be functionally equivalent. During replication initiation, the first step is the binding of a UMP to the hydroxyl group of Tyr3 in the VPg protein. The virally encoded RNA-dependent RNA polymerase (3D) requires the uridylylated VPg form as the primer for both positive- and negative-strand synthesis. 5-Fluorouridine Triphosphate (FUTP) is a potent competitive inhibitor of VPg-1 uridylylation. By peptide analysis, a VPg fragment containing FUMP covalently attached to Tyr has been identified. However, the molecular basis of this phenomenon is still unknown. To investigate the role of FUMP, the synthesis and X-ray studies of simplified models of VPg-1 containing U or FU, in peptides of diferent lenghts linked through the hydroxyl group of Tyr3 will be presented. Interestingly, X-ray structure of 3D-pol with a 15-mer peptide FMDV/VPg-FU showed a significant distortion of b9-b11 loop of the polymerasePeer Reviewe
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Sequential structures provide insights into the fidelity of RNA replication
'Proceedings of the National Academy of Sciences', 2015Co-Authors: Ferrer-orta Cristina, Arias Armando, Escarmís Cristina, Domingo Esteban, Pérez-luque Rosa, Verdaguer NúriaAbstract:RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin Triphosphate and 5-Fluorouridine Triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3′-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases. © 2007 by The National Academy of Sciences of the USA.Work in Barcelona was supported by Grant BFU2005-02376/BMC from the Ministerio de Educación y Ciencia (M.E.C.) (to N.V.) and work in Madrid by Grant BFU2005-00863/BMC from the M.E.C. (to E.D.) and by the Fundación R. Areces. Work in Barcelona and Madrid was further supported by Proyecto Intramural de Frontera (CSIC) (to A.A. and C.F.-O.) and an I3P contract from CSIC (to A.A.). X-ray data were collected at the EMBL protein crystallography beam lines ID14.2 and ID14.4 at European Synchrotron Radiation Facility (ESRF) (Grenoble) within a block allocation group (BAG Barcelona). Financial support was provided by the ESRFPeer Reviewe
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Sequential structures provide insights into the fidelity of RNA replication
National Academy of Sciences, 2026Co-Authors: Ferrer-orta Cristina, Arias Armando, Escarmís Cristina, Domingo Esteban, Pérez-luque Rosa, Verdaguer NúriaAbstract:RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin Triphosphate and 5-Fluorouridine Triphosphate with different RNAs as template–primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3′-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases
Celia Perales - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterization of a dual inhibitory and mutagenic activity of 5-Fluorouridine Triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.
Journal of molecular biology, 2008Co-Authors: Rubén Agudo, Armando Arias, Nonia Pariente, Celia Perales, Cristina Escarmís, Alberto Jorge, Anabel Marina, Esteban DomingoAbstract:The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine Triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-Fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-Fluorouridine Triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.