Annexin A2 facilitates porcine circovirus type 2 infection by mediating viral attachment to host cells and interacting with the capsid protein

Porcine circovirus type 2 (PCV2) causes substantial economic losses globally. Although glycosaminoglycans (GAGs) have been identified as the general cell receptors responsible for PCV2 binding and subsequent cell infection, other critical protein(s) may also be involved. Our objective was to explore key host proteins that mediate PCV2 infection. The host protein annexin A2 (ANXA2) was observed to interact with PCV2 virus-like particles (VLPs) through immunoprecipitation (IP) and liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) experiments. Furthermore, knockdown of ANXA2 or the inhibitor A2ti-1 significantly reduced PCV2 replication in host cells. Notably, the addition of ANXA2-specific antibodies to cell culture or the preincubation of PCV2 with recombinant ANXA2 significantly reduced viral infection, primarily by weakening viral attachment to cells. Confocal microscopy further confirmed the colocalization of PCV2 Cap with endogenous ANXA2 in infected cells, and in vitro GST pull-down assays also demonstrated a direct interaction between these two proteins, confirming the role of ANXA2 as an attachment factor for PCV2 cellular entry. In addition, molecular docking revealed that an interface and multiple residues between PCV2 Cap and ANXA2 were involved in the interaction, which was also verified by truncation assays. In conclusion, ANXA2 on the cell surface may function as an attachment site, promoting viral binding and increasing PCV2 infectivity through interactions with Cap. These findings provide novel insights into the molecular mechanisms of PCV2 infection and new molecular targets for the development of vaccines and therapeutic strategies.