ANXA2 stabilizes mTOR at the plasma membrane to facilitate autophagic flux for CSFV release

Classical swine fever virus (CSFV), a member of the Flaviviridae family, remains a major pathogen responsible for substantial economic losses in the global swine industry. Autophagy plays a critical role in the life cycle and virulence of CSFV, however, the mechanisms through which the virus regulates autophagy are still not fully understood. In this study, we identified ANXA2, a calcium-dependent phospholipid-binding protein, within autophagy-derived vesicles that facilitate CSFV transmission. We demonstrated that ANXA2 modulates CSFV release in a manner dependent on autophagy. Moreover, multiple lines of evidence, including Western blot, LC3 puncta formation, tandem fluorescence assay, and electron microscopy, consistently showed that ANXA2 promotes CSFV-induced autophagy. Mechanistically, ANXA2 overexpression reduced mTOR phosphorylation, while its knockout increased phosphorylation. Comprehensive binding assays revealed that both ANXA2 and the CSFV envelope protein E2 interact with mTOR with high affinity. Domain mapping further indicated that ANXA2 and E2 bind to distinct regions of mTOR, suggesting a synergistic mechanism for autophagy activation. Confocal microscopy showed that ANXA2 facilitates mTOR accumulation at the plasma membrane during infection. Importantly, relocalizing ANXA2 to mitochondria attenuated CSFV-induced autophagy. Collectively, these results indicate that ANXA2 modulates CSFV-triggered autophagy by controlling mTOR subcellular localization, thereby influencing viral production. This study unveils a novel strategy by which CSFV co-opts the ANXA2-mTOR axis to manipulate autophagic processes, highlighting potential targets for future antiviral interventions.