Substitutions M478K and A482G in the polymerase of yellow fever virus confer resistance to sofosbuvir and uprifosbuvir in vitro

The continuous re-emergence of yellow fever virus (YFV) poses a serious public health threat, with no approved specific Antiviral drugs for treatment. Sofosbuvir and uprifosbuvir, developed for treatment of chronic hepatitis C, are nucleotide analogs with anti-YFV activity in vitro, and sofosbuvir has been used off-label in YFV-infected individuals. To study the barrier to resistance of these drugs, escape experiments were performed with the YFV 17D vaccine strain in human hepatoma Huh7.5 cells. Compared to the original virus, sofosbuvir and uprifosbuvir escape variants exhibited 8.6- to 10.7-fold increased 50% effective concentration (EC₅₀) values. Escape viruses developed putative resistance-associated-substitutions (Ras) M478K and A482G in the NS5 RNA-dependent RNA polymerase domain. Reverse-engineered YFV 17D mutants demonstrated that A482G was the primary determinant of drug resistance, resulting in 5.3- to 5.7-fold increased EC₅₀; however, the substitutions acted synergistically when combined, leading to 12.3- and 11.1-fold increased EC₅₀. A482G conferred slight cross-resistance to bemnifosbuvir, while no cross-resistance was observed for remdesivir, galidesivir, or NITD008. Escape and mutant viruses had slight delays in growth kinetics in Huh7.5 cells and were viable in C6/36 and Aag2-AF5 mosquito cells. Structural analysis of a predicted AlphaFold3 YFV NS5-RNA structure suggests that M478K and A482G induce conformational changes in RdRp motif F, which may reduce sofosbuvir and uprifosbuvir affinities. Sofosbuvir Ras described for Other orthoflaviviruses did not substantially affect drug susceptibility in reverse-engineered YFV 17D. This study demonstrates that YFV can develop resistance to sofosbuvir and uprifosbuvir with only two NS5 substitutions that have slight impact on viral fitness.