Abstract
<jats:p>Seasonal influenza causes up to 650,000 deaths annually and remains a persistent pandemic threat due to zoonotic strains crossing the species barrier. Current antivirals, which target neuraminidase or the viral polymerase, have limited efficacy and rapidly select for resistant variants, highlighting the urgent need for new therapeutic strategies. The heterotrimeric influenza polymerase (FluPol), comprising PB2, PB1, and PA subunits, is essential for viral replication and harbors virus-specific protein-protein interfaces that are potential drug targets. We focused on the highly conserved PA-PB1 interface, where the N-terminal peptide of PB1 binds the PA C-terminal domain with high af-finity. Disrupting this interaction abrogates polymerase function, halting viral replication. Using phage display, we identified PB1-derived peptides with enhanced affinity for PA and characterized their binding via biophysical methods and X-ray crystallography. Lead peptides efficiently disrupt-ed the PB1-PA interaction and inhibited polymerase activity in cell-based assays. To address pep-tide delivery challenges, we expressed these inhibitors intracellularly from synthetic mRNA formu-lated in lipid nanoparticles, achieving robust inhibition of viral replication in cultured cells. This work establishes intracellularly expressed peptide inhibitors as a viable antiviral strategy and pro-vides a generalizable framework for targeting essential protein-protein interactions of influenza and other RNA viruses.</jats:p>