The nucleoside analogue ribavirin (R) is mutagenic for foot-and-mouth disease virus (FMDV). Passage of FMDV in the presence of increasing concentrations of R resulted in the selection of FMDV with the amino acid substitution M296I in the viral polymerase (3D). Measurements of progeny production and viral fitness with chimeric viruses in the presence and absence of R documented that the 3D substitution M296I conferred on FMDV a selective replicative advantage in the presence of R but not in the absence of R. In polymerization assays, a purified mutant polymerase with I296 showed a decreased capacity to use ribavirin triphosphate as a substrate in the place of GTP and ATP, compared with the wild-type enzyme. The results suggest that M296I has been selected because it attenuates the mutagenic activity of R with FMDV. Replacement M296I is located within a highly conserved stretch in picornaviral polymerases which includes residues that interact with the template-primer complex and probably also with the incoming nucleotide, according to the three-dimensional structure of FMDV 3D. Given that a 3D substitution, distant from M296I, was associated with resistance to R in poliovirus, the results indicate that picornaviral polymerases include different domains that can alter the interaction of the enzyme with mutagenic nucleoside analogues. Implications for lethal mutagenesis are discussed.

A mutant poliovirus (PV) encoding a change in its polymerase (3Dpol) at a site remote from the catalytic center (G64S) confers reduced sensitivity to ribavirin and forms a restricted quasispecies, because G64S 3Dpol is a high-fidelity enzyme. A foot-and-mouth disease virus (FMDV) mutant that encodes a change in the polymerase catalytic site (M296I) exhibits reduced sensitivity to ribavirin without restricting the viral quasispecies. In order to resolve this apparent paradox, we have established a minimal kinetic mechanism for nucleotide addition by wild-type (WT) FMDV 3Dpol that permits a direct comparison to PV 3Dpol as well as to FMDV 3Dpol derivatives. Rate constants for correct nucleotide addition were on par with those of PV 3Dpol, but apparent binding constants for correct nucleotides were higher than those observed for PV 3Dpol. The A-to-G transition frequency was calculated to be 1/20,000, which is quite similar to that calculated for PV 3Dpol. The analysis of FMDV M296I 3Dpol revealed a decrease in the calculated ribavirin incorporation frequency (1/8,000) relative to that (1/4,000) observed for the WT enzyme. Unexpectedly, the A-to-G transition frequency was higher (1/8,000) than that observed for the WT enzyme. Therefore, FMDV selected a polymerase that increases the frequency of the misincorporation of natural nucleotides while specifically decreasing the frequency of the incorporation of ribavirin nucleotide. These studies provide a mechanistic framework for understanding FMDV 3Dpol structure-function relationships, provide the first direct analysis of the fidelity of FMDV 3Dpol in vitro, identify the ␤9-␣11 loop as a (in)fidelity determinant, and demonstrate that not all ribavirin-resistant mutants will encode high-fidelity polymerases.

Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin (R)selected for viruses with decreased sensitivity to R, which included different mutations in their polymerase (3D): G64S located in the finger subdomain in the case of PV and M296I located within loop ␤9-␣11 at the active site in the case of FMDV. To investigate why disparate substitutions were selected in two closely related 3Ds, we constructed FMDVs with a 3D that included either G62S (the equivalent replacement in FMDV of PV G64S), M296I, or both substitutions. G62S, but not M296I, inflicts upon FMDV a strong selective disadvantage which is partially compensated for by the substitution M296I. The corresponding mutant polymerases, 3D(G62S), 3D(M296I), and 3D(G62S-M296I), were analyzed functionally and structurally. G62S in 3D impairs RNA-binding, polymerization, and R monophosphate incorporation activities. The X-ray structures of the 3D(G62S)-RNA, 3D(M296I)-RNA, and 3D(G62S-M296I)-RNA complexes show that although the two positions are separated by 13.1 Å, the loops where the replacements reside are tightly connected through an extensive network of interactions that reach the polymerase active site. In particular, G62S seems to restrict the flexibility of loop ␤9-␣11 and, as a consequence, the flexibility of the active site and its ability to bind the RNA template. Thus, a localized change in the finger subdomain of 3D may affect the catalytic domain. The results provide a structural interpretation of why different amino acid substitutions were selected to confer R resistance in closely related viruses and reveal a complex network of intra-3D interactions that can affect the recognition of both the RNA template and incoming nucleotide.Ribavirin (1-␤-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) (R) is a clinically important nucleoside analogue that exhibits antiviral activity against a broad spectrum of RNA viruses (17). R displays several antiviral mechanisms of action, including lethal mutagenesis (loss of infectivity associated with an increase in the mutation rate) (7,9,21,23). The 5Ј-triphosphorylated form of R (RTP) can be incorporated by the viral polymerases into the nascent RNA, acting as either an adenylate or a guanylate analogue, inducing base transitions. Ambiguous utilization of RTP by RNA-dependent RNA polymerases during genome replication may lead to virus extinction (1, 6, 7, 33).As extensively documented for nonmutagenic antiviral inhibitors, selection of mutagen-resistant viruses may be a problem for the efficacy of antiviral treatments based on lethal mutagenesis. Serial passages of foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of R resulted in the selection of a mutant virus containing the amino acid substitution M296I in polymerase 3D. Measurements of viral fitness and progeny production suggested that M296I was selected because it decreased the mutagenic activity of R on FMDV (28). The mutant polymerase restricted the incorporation of RTP during RNA synt...