Inhibitors of pyrimidine synthesis synergize with N4-hydroxycytidine to diminish influenza virus replication

Influenza viruses remain a major threat to both human and animal health, with seasonal outbreaks and the risk of pandemics caused by reassortant strains. Antiviral drugs are needed as a complement to vaccines, but resistance often limits their long-term efficacy. N4-hydroxycytidine (NHC), the active form of Molnupiravir, shows potent activity against influenza A viruses (IAVs) in both cell cultures and animal models, with minimal resistance observed. Building on prior work in SARS-CoV-2, we investigated whether inhibiting pyrimidine biosynthesis could enhance NHC's antiviral activity against IAVs. The combination of NHC with inhibitors of dihydroorotate dehydrogenase (DHODH) or cytidine triphosphate synthases (CTPS1/2) showed strong synergy. This was evident through reduced cytopathic effects, decreased viral RNA and protein, and a marked absence of infectious virus particles. This synergy was consistent across multiple IAV subtypes, including H1N1, H1N2, H3N2, and H5N1. This synergistic effect was reversed by exogenously supplemented pyrimidine nucleosides, confirming nucleotide depletion as a key mechanism. However, some avian IAVs were less sensitive to the treatment in mammalian cells. The PB2-K627E mutation, affecting the interaction with host factor ANP32, modulated NHC efficacy, implicating viral adaptation in drug responsiveness. In a ferret model of H5N1 infection, NHC combined with the CTPS inhibitor STP938 reduced clinical symptoms and lung pathology, with NHC mostly driving antiviral activity and STP938 contributing to disease mitigation. These findings indicate that combining NHC with pyrimidine biosynthesis inhibitors enhances antiviral efficacy against IAVs, especially in rapidly replicating viruses, and may broaden the utility of nucleoside analogues in influenza therapy.