Introduction: Swine influenza virus (SIV) is a highly contagious respiratory pathogen in pigs that causes substantial economic losses in the pig industry. Importantly, pigs act as "mixing vessels" for diverse influenza A viruses (IAVs), facilitating the emergence of novel pandemic strains through reassortment, which represents a continuous global public health threat. IAV replication relies heavily on host cellular machinery, underscoring the importance of elucidating virus-host protein interactions for the development of targeted antiviral therapeutics.

Objectives: This study aims to identify host genes required for SIV replication via a genome-wide CRISPR screen and elucidate the mechanism by which STK11 modulates viral replication.

Methods: A pig genome-scale CRISPR knockout (PigGeCKO) screen was performed in newborn pig trachea (NPTr) cells to identify host genes required for SIV replication. Candidate genes were further validated by generating knockout cell lines using CRISPR/Cas9-mediated gene editing, followed by assessing their impact on IAV replication. The specific lifecycle stage regulated by STK11 and its mechanistic role in viral attachment were determined via Western blotting, confocal microscopy, transmission electron microscopy, and stimulated emission depletion (STED) imaging. In vivo validation of STK11 knockdown effects on IAV replication was conducted in BALB/c mice treated with STK11-targeting siRNA, with outcomes evaluated by survival analysis, body weight monitoring, lung viral titers quantification, immunofluorescence, and histopathology.

Results: STK11 promotes replication of different IAV subtypes in vitro, and STK11 knockdown significantly suppresses SIV replication in vivo. Mechanistically, STK11 depletion impairs viral attachment by altering the organization of sialic acid clusters, mediated through reduced intracellular actin stress fibers via inhibition of RhoA signaling pathway.

Conclusion: We identify STK11 as a novel regulator of IAV attachment and elucidate its mechanistic role in facilitating viral entry. These findings highlight the potential of STK11 to serve as an ideal antiviral target against IAV infection.