Fig 3.

Clathrin is not required for JEV entry. (A) B104 cells were pretreated with chlorpromazine and infected with JEV. After 1 h of internalization, extracellular virus was inactivated with proteinase K and the cell pellets were plated onto B104 cells to determine internalized virus by an infectious center assay. Bar graphs represent the percentage of internalized virus with respect to a control without drug treatment. Cell viability upon drug treatments was unaffected, as represented by the line graphs. Results are presented as the means ± SD of three independent experiments. (B) B104 cells were pretreated with chlorpromazine and inoculated with JEVpv or VSVpv. GFP-positive cells were determined by flow cytometry. Results are expressed as infectious units (IU)/milliliter and are presented as the means ± SD of three independent experiments. (C) B104 cells were left untreated (control) or were treated with 20 μM chlorpromazine for 1 h at 37°C and then incubated with 10 μg/ml AF 488-labeled transferrin for 30 min at 37°C. Nuclei were stained with DAPI. Bars, 10 μm. (D and E) B104 cells were transfected with a GFP-only construct (GFP), the GFP-tagged wild-type construct (EPS 15 WT), or a DN EPS 15-expressing construct (EPS 15 DN) and then infected with JEV. (D) The number of JEV E-expressing cells was determined by immunofluorescence and normalized to the value for the GFP-only control. Results are presented as the means ± SD of three independent experiments. (E) B104 cells transfected with the GFP, EPS 15 WT (Dyn WT), or EPS 15 DN (Dyn DN) construct were incubated with 10 μg/ml AF 555-labeled transferrin for 30 min at 37°C. The cells then were fixed and stained with DAPI. Bars, 10 μm. (F, G, H, and I) B104 cells were transfected with the construct containing siRNA targeting clathrin heavy-chain (siCHC) or nontargeting siRNA (siCtrl) and infected with JEV (F) or JEVpv or VSVpv (G). (F) The internalized viruses (bar graphs) and cell viability (line graphs) of transfected cells were determined as in panel A, and the results were normalized to the value for the siCtrl. Results are presented as the means ± SD of three independent experiments. (G) GFP-positive cells were determined as in panel B, and the results are presented as the means ± SD of three independent experiments. (H) B104 cells transfected with siCtrl or siCHC were incubated with 10 μg/ml AF 488-labeled transferrin for 30 min at 37°C. Cells then were fixed and stained with DAPI. Bars, 10 μm. (I) To verify clathrin knockdown, protein samples from cells expressing each siRNA construct were analyzed by immunoblotting for clathrin. *, P < 0.05.