Abstract
In various positive-sense single-stranded RNA viruses, a low-fidelity viral RNA-dependent RNA polymerase (RdRp) confers attenuated phenotypes by increasing the mutation frequency. We report a negative-sense single-stranded RNA virus RdRp mutant strain with a mutator phenotype. Based on structural data of RdRp, rational targeting of key residues, and screening of fidelity variants, we isolated a novel low-fidelity mutator strain of influenza virus that harbors a Tyr82-to-Cys (Y82C) single-amino-acid substitution in the PB1 polymerase subunit. The purified PB1-Y82C polymerase indeed showed an increased frequency of misincorporation compared with the wild-type PB1 in an in vitro biochemical assay. To further investigate the effects of position 82 on PB1 polymerase fidelity, we substituted various amino acids at this position. As a result, we isolated various novel mutators other than PB1-Y82C with higher mutation frequencies. The structural model of influenza virus polymerase complex suggested that the Tyr82 residue, which is located at the nucleoside triphosphate entrance tunnel, may influence a fidelity checkpoint. Interestingly, although the PB1-Y82C variant replicated with wild-type PB1-like kinetics in tissue culture, the 50% lethal dose of the PB1-Y82C mutant was 10 times lower than that of wild-type PB1 in embryonated chicken eggs. In conclusion, our data indicate that the Tyr82 residue of PB1 has a crucial role in regulating polymerase fidelity of influenza virus and is closely related to attenuated pathogenic phenotypes in vivoIMPORTANCE Influenza A virus rapidly acquires antigenic changes and antiviral drug resistance, which limit the effectiveness of vaccines and drug treatments, primarily owing to its high rate of evolution. Virus populations formed by quasispecies can contain resistance mutations even before a selective pressure is applied. To study the effects of the viral mutation spectrum and quasispecies, high- and low-fidelity variants have been isolated for several RNA viruses. Here, we report the discovery of a low-fidelity RdRp variant of influenza A virus that contains a substitution at Tyr82 in PB1. Viruses containing the PB1-Y82C substitution showed growth kinetics and viral RNA synthesis levels similar to those of the wild-type virus in cell culture; however, they had significantly attenuated phenotypes in a chicken egg infection experiment. These data demonstrated that decreased RdRp fidelity attenuates influenza A virus in vivo, which is a desirable feature for the development of safer live attenuated vaccine candidates.