Genotypic and phenotypic characterization of West Nile virus NS5 methyltransferase mutants

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 out-bred 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. (C) 2019 Elsevier Ltd. All rights reserved.