Similar to that of other enteroviruses,the replication of enterovirus 71 (EV71) occurs on rearranged membranous structures called replication organelles (ROs).Phosphatidylinositol 4-kinase Ⅲ (PI4KB),which is required by enteroviruses for RO formation,yields phosphatidylinositol-4-phosphate (PI4P) on ROs.PI4P then binds and induces conformational changes in the RNA-dependent RNA polymerase (RdRp) to modulate RdRp activity.Here,we targeted 3D polymerase,the core enzyme of EV71 ROs,and found that the host factor Annexin A2 (ANXA2) can interact with 3D polymerase and promote the replication of EV71.Then,an experiment showed that the annexin domain of ANXA2,which possesses membrane-binding capacity,mediates the interaction of ANXA2 with EV713D polymerase.Further research showed that ANXA2 is localized on ROs and interacts with PI4KB.Overexpression of ANXA2 stimulated the formation of PI4P,and the level of PI4P was decreased in ANXA2-knockout cells.Furthermore,ANXA2,PI4KB,and 3D were shown to be localized to the viral RNA replication site,where they form a higher-order protein complex,and the presence of ANXA2 promoted the PI4KB-3D interaction.Altogether,our data provide new insight into the role of ANXA2 in facilitating formation of the EV71 RNA replication complex.