Genotypic and phenotypic characterization of West Nile virus NS5 methyltransferase mutants

•Mutation of the viral NS5 methyltransferase active site attenuates flaviviruses.•Single site mutation of NS5-K61A or E218A strongly attenuates West Nile virus.•Simultaneous double mutation of NS5-K61A/E218A does not attenuate West Nile virus.•When combined, NS5-K61A/E218A mutations are capable of reversion to wild-type. Although West Nile virus (WNV) causes annual cases of neurological disease and deaths in humans, a vaccine has not been licensed for human use. Several WNV genes have been targeted for mutagenesis in attempts to generate live attenuated vaccine candidates, including the non-structural protein NS5. Specifically, mutation of WNV NS5-K61A or NS5-E218A in the catalytic tetrad of the methyltransferase decreases enzyme activity of the NS5 protein and correspondingly attenuates the virus in mice. In this report, NS5-K61A, NS5-E218A, and a double mutant encoding both mutations (NS5-K61A/E218A) were compared both in vitro and in vivo. Each single mutant was strongly attenuated in highly susceptible outbred mice, whereas the double mutant unexpectedly was not attenuated. Sequencing analysis demonstrated that the double mutant was capable of reversion at both residues NS5-61 and NS5-218, whereas the genotype of the single mutants did not show evidence of reversion. Overall, either NS5-K61A or NS5-E218A methyltransferase mutations could be potential mutations to include in a candidate live WNV vaccine; however, multiple mutations in the catalytic tetrad of the methyltransferase are not tolerated.