4.7 Article

Molecular characterization of a dual inhibitory and mutagenic activity of 5-fluorouridine triphosphate on viral RNA synthesis. Implications for lethal mutagenesis

Journal

JOURNAL OF MOLECULAR BIOLOGY
Volume 382, Issue 3, Pages 652-666

Publisher

ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.07.033

Keywords

foot-and-mouth disease virus; quasispecies; error catastrophe; nucleoside analogue; antiviral strategy

Funding

  1. Ministerio de Educacion y Ciencia [BFU 2005-00863]
  2. CSIC (Consejo Superior de Investigaciones Cientificas) [2005-20F-0221]
  3. Fundacion Ramon Areces
  4. CIBERehd (Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas)
  5. Instituto de Salud Carlos III
  6. Comunidad Autonoma de Madrid
  7. CIBERehd

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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) ill 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 ill 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 5fluorouridine triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe. (C) 2008 Elsevier Ltd. All rights reserved.

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