An endonuclease inhibitor unlocks the PA-X shutoff mechanism in influenza A virus.

Influenza A virus protein PA-X mediates host shutoff, yet its molecular mechanism and the role of its C-terminal region remain unclear. When ectopically expressed via plasmids, PA-X cleaves its own mRNAs, leading to remarkably low intracellular expression, which makes its characterization challenging. In this study, we utilize an endonuclease inhibitor to suppress PA-X shutoff activity, enabling its robust expression and purification. Our biochemical analyses reveal that PA-X directly binds RNA via its unique C-terminal basic-rich region and induces mRNA degradation with minimal sequence specificity. Furthermore, we find that while PA-X targets a broad range of RNAs, viral RNAs are protected from its endonuclease activity through their assembly into vRNP complexes. These findings provide critical insights into the PA-X-mediated shutoff strategy, revealing how PA-X modulates the cellular environment while sparing the viral genome to favor viral replication.