Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH 2 -terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.Foot-and-mouth disease virus (FMDV) 1 is the prototype member of the Aphthovirus genus of the Picornaviridae family, and the etiological agent of the economically most important disease of farm animals (1, 2). The cost of the FMD outbreak that took place in 2001 in the United Kingdom has been evaluated in 6 billion pounds sterling (3), and it has been estimated that a large FMD outbreak in northern Europe or the United States would entail losses exceeding 100 billion dollars (4). Probably its most devastating effects are felt in underdeveloped countries because of the severe trade restrictions imposed by the disease (5). Difficulties for the control of FMD stem from the wide host range of FMDV, variability in pathogenic manifestations, antigenic diversity, high infectivity and transmissibility, capacity to establish persistent, asymptomatic infection affecting farm ruminants and also wildlife species, and the limited efficacy of current vaccines (1, 2). Some of these difficulties have their origin in a basic feature of FMDV biology, namely the error-prone nature of FMDV RNA replication, determined by the limited template copying fidelity of the RNAdependent RNA polymerase (RDRP) 3D (6). To understand FMDV RNA synthesis at the molecular level, to interpret the copying-fidelity properties of the replication machinery, and to design antiviral compounds against FMD as a potential new approach to control the disease, a knowledge of the structure of the polymerase 3D is essential.Here, we report the structure of the RDRP of FMDV in a free form at 1.9-Å resolution and in complex with a template-primer RNA decanucleotide at 3.0-Å resolution. These structures represent the first complete three-dimensional structure of a picornavirus polymerase and provide new insights into the structural basis for the FMDV 3D function and the structure-based design of antiviral compounds against an important group of animal pathogens. EXPERIMENTAL PROCEDURESCloning of FMDV C-S8c1 3D Polymerase in Plasmid pET-28a-The genomic region coding for the 3D polymerase of FMDV was amplified by PCR from the infectious plasmid pMT28, 2 containing a complete copy of the genome of our standard FMDV C-S8c1, and cloned in...

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Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix a8 of the fingers domain and helix a13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction.

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.

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.5-fluorouracil ͉ foot-and-mouth disease virus ͉ replication fidelity ͉ ribavirin ͉ RNA elongation