Identification of the bona fide active center of influenza A virus polymerase acidic protein as the antiviral target

The influenza virus PA protein, a core subunit of RNA polymerase, is critical for viral replication and a key antiviral target. Contrary to the prevailing view that its endonuclease active site resides in the N-terminal domain (PAn, residues 1-319), we identified a C-terminal truncation (PAc, 320-716) of H1N1 PA that retained high endonuclease activity, while PAn showed minimal activity. Crystal structural analysis revealed that PAc binds substrate RNA via the YDS motif (residues 393-395), with mutations abolishing RNA association. Surprisingly, baloxavir acid (BXA), a known PA inhibitor, specifically targeted PAc rather than PAn, supported by molecular docking showing higher binding affinity to PAc. Mutations at PAc-BXA interaction sites identified F707A as a potential drug resistance mutation. Virtual screening targeting PAc identified methotrexate as potent inhibitors (EC ₅₀ < 100 nM), which effectively suppressed viral replication in vitro and alleviated symptoms in influenza-infected mice. Our findings redefine PA’s catalytic architecture and establish PAc as a novel platform for antiviral discovery, offering strategies to combat drug resistance through structure-guided inhibitor design.