Annexin A2 orchestrates CSFV entry route selection via the annexin A2/S100A10 heterotetrameric complex.

Classical swine fever virus (CSFV) is a highly contagious pathogen that causes substantial economic losses in swine production, yet the mechanisms governing its cellular entry remain poorly defined. Here, through a combination of pharmacological inhibition, genetic knockout/overexpression, and co-immunoprecipitation, we identify annexin A2 (ANXA2) as a pivotal host determinant of CSFV entry into PK-15 cells. Confocal microscopy revealed that CSFV infection rapidly induced the translocation of ANXA2 to the cell surface, and functional assays confirmed that its plasma membrane localization was essential for efficient infection. Mechanistically, the viral envelope protein E2 specifically interacts with the heterotetrameric A2t complex (ANXA2/S100A10), establishing this complex as a functional cellular receptor for CSFV. ANXA2 also maintains clathrin at the plasma membrane and facilitates clathrin-dependent viral entry, as evidenced by inhibitor and siRNA knockdown studies. Intriguingly, ANXA2 depletion redirects viral entry to a caveolin-1-dependent pathway. We further demonstrate that ANXA2 competitively binds the C-terminal domain of caveolin-1, a site that overlaps with the binding region for the viral Erns protein, thereby suppressing the caveolin-1 route in wild-type cells. In summary, this study identifies the A2t complex as a CSFV receptor and reveals a crucial regulatory role for ANXA2 in viral entry pathway selection. These findings provide new insights into CSFV infection and a rationale for host-directed antiviral strategies.