Cells, viruses, and transfection

The 293A, Vero, HeLa, Huh7, SH-SY5Y, 293T-derived knockout cell lines [82], and HCT116-derived knockout cell lines [68] were cultured in Dulbecco’s modified Eagle medium (DMEM)-high glucose (Nacalai Tesque, Kyoto, Japan) containing 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% (v/v) fetal calf serum (FCS), in a humidified atmosphere containing 5% CO₂ at 37° C. C6/36 cells were cultured in Schneider’s insect medium (Merck KGaA, Darmstadt, Germany) containing 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% (v/v) FCS at 25°C. The JEV AT31 strain (kindly provided by Eiji Konishi, Osaka University, Japan) was propagated in 293A cells. PEI MAX (Polysciences, Warrington, PA, USA) or Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) were used for transfection according to the manufacturer’s protocols.