Genome-wide CRISPR screen identifies RNF24 as a critical host factor for foot-and-mouth disease virus entry.

Background: Foot-and-mouth disease virus (FMDV) causes substantial economic losses in global livestock production; however, the key host factors supporting its early infection process remain poorly characterized. Methods: In this study, we performed an unbiased genome-wide CRISPR/Cas9 knockout screening using porcine cells to screen and identify host factors involved in FMDV infection. Results: We identified that the E3 ubiquitin ligase RNF24 supports efficient FMDV entry. RNF24 depletion inhibits viral entry and replication, whereas its overexpression enhances viral infectivity. Mechanistically, RNF24 preferentially promotes K27-linked non-degradative polyubiquitination of leupaxin (LPXN) at lysine 162, driving LPXN's trafficking to the plasma membrane. At the membrane, LPXN assembles a ternary integrin-LPXN-VP1 complex that strengthens virus-receptor interactions and promotes viral adsorption and entry. Disruption of this ubiquitination event via the LPXN K162R mutation impairs complex formation and compromises viral entry. Conclusion: Together, our study reveals a ubiquitin-dependent RNF24-LPXN regulatory axis that supports FMDV entry, highlights the role of non-degradative ubiquitination in viral pathogenesis, and proposes this interface as a potential target for antiviral intervention.