Mechanism of Inhibition of the Active Triphosphate Form of 2′-α-Fluoro,2′-β-bromouridine against Yellow Fever Virus RNA-Dependent RNA Polymerase

Yellow fever virus (YFV) is a deadly mosquito-borne flavivirus that infects approximately 200,000 individuals each year. YFV outbreak and infection are persistent throughout South America and Africa, demonstrating significant epidemic potential. Although an approved and effective vaccine exists, the zoonotic nature of YFV undermines any potential eradication efforts, highlighting the need for effective, direct-acting antivirals. Essential for viral replication, RNA-dependent RNA polymerase (RdRp) is a proven therapeutic target. A prominent example includes sofosbuvir, a 2'-α-fluoro,2'-β-methyluridine prodrug approved for the treatment of hepatitis C virus (HCV), that has demonstrated efficacy against YFV <i>in vitro</i>. A structurally similar 2'-α-fluoro, 2'-β-bromouridine prodrug has exhibited potent anti-YFV activity both <i>in vitro</i> and <i>in vivo</i>. Here, we expressed the full-length nonstructural protein 5 from YFV in insect cells to investigate the active triphosphate form of these prodrugs. Enzyme kinetics indicate that both nucleotide analogs are incorporated less efficiently than UTP. Once incorporated, the analogs inhibit RNA synthesis through immediate chain termination. Omitting the 2'-β-modification alleviates the inhibition of RNA synthesis, highlighting its role in eliciting an antiviral effect. S282T is a well-characterized mutation in motif B of HCV RdRp that confers resistance to sofosbuvir. We discovered that the analogous substitution in YFV (S603T) improved discrimination against the 2'-α-fluoro,2'-β-modified uridine analogs. Collectively, our findings explain their observed anti-YFV activity and identify a conserved mechanism of resistance. Based on its <i>in vivo</i> efficacy and mechanism of action, the 2'-fluoro,2'-bromouridine prodrug shows potential for future therapeutic strategies against YFV.