Annexin A2 facilitates Japanese encephalitis virus replication by regulating endocytosis and intracellular transport in PK15 cells

Introduction Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes severe encephalitis in humans and reproductive disorders in pigs. However, the host factors that regulate JEV infection and replication remain incompletely understood. Method In this study, we used porcine kidney cells (PK15) infected with the attenuated JEV strain SA14-14-2 to identify host proteins interacting with the JEV envelope (E) protein by immunoprecipitation coupled with LC-MS/MS. Results Annexin A2 (ANXA2) was identified as a candidate host factor associated with JEV infection. ANXA2 expression was increased during JEV infection, and co- immunoprecipitation confirmed its association with the JEV E protein. Inhibition of ANXA2 with antibody reduced JEV RNA replication in a dose-dependent manner. To further define its function, ANXA2 knockout (KO) PK15 cells were generated using CRISPR/Cas9. Compared with wild-type cells, ANXA2-KO cells showed significantly reduced viral RNA accumulation, especially during the early stage of JEV infection (2-6hpi), when the viral entry and initiates replication, lower JEV E protein expression, and decreased progeny virus titers. In addition, ANXA2 deficiency altered the expression of clathrin-mediated endocytosis- related genes and proteins, including CLTC, CLTB, AP2M1, and EPS15, and affected Akt phosphorylation during early infection. Fluorescence recovery after photobleaching analysis (FRAP) of DiD-labeled JEV particles indicated impaired intracellular transport in ANXA2-KO cells. Discussion these findings identify ANXA2 as a host factor that promotes JEV infection and replication, at least in part by regulating clathrin-mediated endocytosis and intracellular transport. ANXA2 may represent a potential target for future antiviral strategies against JEV.