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.