title: Molecular characterization of a dual inhibitory and mutagenic activity of 5-fluorouridine triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.
abstract: 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.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049, Madrid, Spain.
authors.forename: Rubén
authors.initials: R
authors.lastname: Agudo
authors.forename: Armando
authors.initials: A
authors.lastname: Arias
authors.forename: Nonia
authors.initials: N
authors.lastname: Pariente
authors.forename: Celia
authors.initials: C
authors.lastname: Perales
authors.forename: Cristina
authors.initials: C
authors.lastname: Escarmís
authors.forename: Alberto
authors.initials: A
authors.lastname: Jorge
authors.forename: Anabel
authors.initials: A
authors.lastname: Marina
authors.forename: Esteban
authors.initials: E
authors.lastname: Domingo
mesh_terms: D000818:Animals; D000963:Antimetabolites / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D005472:Fluorouracil / Q000378:metabolism; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D006801:Humans; D016296:Mutagenesis; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000378:metabolism; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology
chemical_list: D000963:Antimetabolites; D012367:RNA, Viral; D014764:Viral Proteins; C025635:5-fluorouridine 5'-triphosphate; D014542:Uridine Monophosphate; D012324:RNA-Dependent RNA Polymerase; D005472:Fluorouracil; D014544:Uridine Triphosphate