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.

BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV) can be cured of virus by treatment with the antiviral nucleoside analogue ribavirin. To study whether the process involved an increase in the number of mutations in the mutant spectrum of the viral population, viral genomes were cloned from persistently infected cells treated or untreated with ribavirin. An increase of up to 10-fold in mutation frequencies associated with ribavirin treatment was observed in the viral genomes from the treated cultures as compared with parallel, untreated cultures. To address the possible mechanisms of enhanced mutagenesis, we investigated the mutagenic effects of ribavirin together with guanosine, and mycophenolic acid in the presence or absence of guanosine. Changes in the intracellular nucleotide concentrations were determined for all treatments. The results suggest that the increased mutation frequencies were not dependent on nucleotide pool imbalances or due to selection of preexisting genomes but they were produced by a mutagenic action of ribavirin.

RNA viruses replicate near the error threshold for maintenance of genetic information, and an increase in mutation frequency during replication may drive RNA viruses to extinction in a process termed lethal mutagenesis. This report addresses the efficiency of extinction (versus escape from extinction) of foot-andmouth disease virus (FMDV) by combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H). Selection of G-or H-resistant, extinctionescape mutants occurred with low-fitness virus only in the absence of FU and with high-fitness virus with some mutagen-inhibitor combinations tested. The combination of FU, G, and H prevented selection of extinctionescape mutants in all cases examined, and extinction of high-fitness FMDV could not be achieved by equivalent inhibitory activity exerted by the nonmutagenic agents. The G-resistant phenotype was mapped in nonstructural protein 2C by introducing the relevant mutations in infectious cDNA clones. Decreases in FMDV infectivity were accompanied by modest decreases in the intracellular and extracellular levels of FMDV RNA, maximal intracellular concentrations of FU triphosphate, and a decrease in the intracellular concentrations of UTP. In addition to indicating a key participation of mutagenesis in virus extinction, the results suggest that picornaviruses provide versatile experimental systems to approach the problem of extinction failure associated with inhibitor-escape mutants during treatments based on enhanced mutagenesis.The error rate during template copying by viral RNA-dependent RNA polymerases and reverse transcriptases is close to the maximum tolerable for the maintenance of the genetic information of the virus. This is supported by theoretical predictions (reviewed in references 14 and 15) and experimental results on the adverse effects of enhanced mutagenesis on infectivity during cytolytic or persistent infections of viruses as diverse as picornaviruses (1, 9, 10, 23, 35, 52), retroviruses and retroviral vectors (29,30,37), the rhabdovirus vesicular stomatitis virus (VSV) (23), the flavivirus GB virus B (27), the arenavirus lymphocytic choriomeningitis virus (LCMV) (21, 46), and Hantaan virus (50).Virus extinction by enhanced mutagenesis was accompanied by increases in mutant spectrum complexity, as quantitated by mutation frequency and Shannon entropy (1,21,35,46,52). LCMV showed a very high sensitivity to extinction by 5-fluorouracil (FU) during persistent infections in BHK-21 cells (21, 46), and recently it has been shown that administration of FU to mice prevented the establishment of a persistent LCMV infection (46), providing the first evidence of feasibility of a lethal mutagenesis approach to control viral infections in vivo.Studies with the important animal pathogen foot-and-mouth disease virus (FMDV) have documented that low viral relative fitness and low viral load favored FMDV extinction by enhanced mutagenesis (35,52). The effect of fitness was evidenced using FMDV clo...