Foot-and-mouth disease virus low-fidelity polymerase mutants are attenuated

Previous studies have shown that RNA viruses can be attenuated by either increased or decreased viral polymerase replication fidelity. Although foot-and-mouth disease virus (FMDV) high-fidelity RNA-dependent RNA polymerase (RdRp) variants with an attenuated phenotype have been isolated using mutagens, no FMDV mutant with a low-fidelity polymerase has been documented to date. Here, we describe the generation of several FMDV RdRp mutants using site-directed mutagenesis via a reverse genetic system. Mutation frequency assays confirmed that five rescued FMDV RdRp mutant populations had lower replication fidelity than the wild-type virus population, which allowed us to assess the effects of the change in replication fidelity on the virus phenotype. These low-fidelity FMDV RdRp mutants showed increased sensitivity to ribavirin or 5-fluorouracil (5-FU) treatment without a loss of growth capacity in cell cultures. In addition, decreased fitness and attenuated virulence were observed for the RdRp mutants with lower fidelity. Importantly, based on a quantitative analysis for fidelity and virulence, we concluded that lower replication fidelity is associated with a more attenuated virus phenotype. These results further contribute to our understanding of the replication fidelity of polymerases of RNA viruses and its relationship to virulence attenuation.