Inhibitors of dihydroorotate dehydrogenase synergize with the broad antiviral activity of 4′-fluorouridine

RNA viruses present a constant threat to human health, often with limited options for vaccination or therapy. Notable examples include influenza viruses and coronaviruses, which have pandemic potential. Filo- and henipaviruses cause more limited outbreaks, but with high case fatality rates. All RNA viruses rely on the activity of a virus-encoded RNA-dependent RNA polymerase (RdRp). An antiviral nucleoside analogue, 4'-Fluorouridine (4'-FlU), targets RdRp and diminishes the replication of several RNA viruses, including influenza A virus and SARS-CoV-2, through incorporation into nascent viral RNA and delayed chain termination. However, the effective concentration of 4'-FlU varied among different viruses, raising the need to fortify its efficacy. Here we show that inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme essential for pyrimidine biosynthesis, can synergistically enhance the antiviral effect of 4'-FlU against influenza A viruses, SARS-CoV-2, henipaviruses, and Ebola virus. Even 4'-FlU-resistant mutant influenza A virus was re-sensitized towards 4'-FlU by DHODH inhibition. The addition of uridine rescued influenza A virus replication, strongly suggesting uridine depletion as a mechanism of this synergy. 4'-FlU was also highly effective against SARS-CoV-2 in a hamster model of COVID. We propose that the impairment of endogenous uridine synthesis by DHODH inhibition enhances the incorporation of 4'-FlU into viral RNAs. This strategy may be broadly applicable to enhance the efficacy of pyrimidine nucleoside analogues for antiviral therapy.