Sequential structures provide insights into the fidelity of RNA replication

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 ESRF