Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. cycle of flaviviruses. IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that Clozic entry of JEV into BHK-21 Clozic cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow. within the family 0.05; **, 0.01. JEV entry depends on dynamin. Previous studies have shown that dynamin is involved in flavivirus (33, 34) and JEV endocytosis (12, 13). Here, we used dynasore, a Clozic cell-permeable, noncompetitive dynamin GTPase activity inhibitor, to analyze the role of dynamin during JEV infection of BHK-21 cells. We observed that dynasore inhibits JEV entry into cells but does not inhibit JEV binding. The results showed that after infection at 37C for 1 h, 100 M dynasore caused a 78.3% reduction of viral RNA copy numbers and that the size of the reduction was dose dependent (Fig. 2A). At 24 hpi, viral RNA copies were significantly reduced in a dose-dependent manner in cells pretreated with dynasore; at 100 M dynasore, we observed a greater than 95% reduction in JEV infection, but no cytotoxicity was observed in the cells treated with up to 100 M dynasore (Fig. 2B). A significant reduction in the signal intensity of fluorescently labeled transferrin (Tfn) was observed in 100 M dynasore-treated cells Rabbit Polyclonal to Desmin compared to that in untreated cells, indicating a block in transferrin uptake (Fig. 2C and ?andD).D). To further examine the role of dynamin in clathrin-mediated endocytosis, Tfn uptake was monitored by confocal microscopy in cells transfected with constructs of wild-type (WT) and DN (K44A) Clozic dynamin II (Fig. 2E and ?andF).F). We next examined JEV infection in cells overexpressing DN dynamin II using confocal microscopy. JEV infection was reduced in cells transfected with the DN construct compared to levels in cells transfected with the WT construct (Fig. 2G). Overexpression of DN dynamin II resulted in approximately 87.8% inhibition of the number of JEV-infected cells compared to the number of cells overexpressing the WT construct (Fig. 2H). These results demonstrated that JEV entry requires dynamin. Open in a separate window FIG 2 JEV entry depends on dynamin. (A) Dynasore inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at Clozic 37C for 1 h, the medium was replaced, and then the cells were inoculated with JEV (MOI of 5) at 4C for 1 h. At 0 h (binding) or 1 h (entry) postinfection, cells were lysed to determine viral RNA copy number by RT-qPCR. (B) Dynasore inhibited JEV infection. Cells were pretreated with increasing concentrations of dynasore at 37C for 1 h, the medium was replaced, and then the cells were inoculated with JEV (MOI of 0.05). At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. The horizontal line shows results of subtoxic doses of dynasore on BHK-21 cells.