Fig 1.

JEV internalization is dynamin dependent. (A) Neuro2a cells grown on coverslips were either untreated or treated with 80 μM dynasore for 1 h, following which they were infected with JEV (MOI, 0.4) in the presence of the inhibitor. At 24 hpi cells were fixed and stained with anti-JEV E primary and Alexa 488 anti-mouse IgG secondary antibodies. Cells were mounted in DAPI containing antifade reagent and imaged at ×20 on a confocal microscope. Cell nuclei can be seen in blue (DAPI) and JEV E in green. Bar, 20 μm. (B) Neuro2a cells were infected with JEV (MOI, 10) in the presence of 80 μM dynasore for 1 h, following which the endocytosed viral load was estimated by qRT-PCR of JEV positive-strand RNA. (C) Quantitation of JEV infection (MOI, 0.4) in Neuro2a, SH-SY5Y, and Vero cell lines in the presence of 80 μM dynasore was done by microscopy and image analysis as described in Materials and Methods. The results are normalized to solvent-treated control cells. (D) After 24 hpi (MOI, 0.4), the virus titer (mean ± SD) in culture supernatants was calculated by plaque assay. (E) Neuro2a cells transfected with GFP or GFP-dyn2-K44A were infected with JEV (MOI, 1; ∼30% infection in GFP-transfected cells). Cells were fixed at 24 hpi, stained with anti-JEV E primary and Alexa 568 anti-mouse IgG secondary antibody, and imaged at ×60 on a confocal microscope. Bar, 10 μm. (F) JEV infection in Neuro2a, SH-SY5Y, and Vero cells expressing GFP and GFPdyn2K44A was quantitated as described above. Infection was normalized to GFP-transfected cells. For panels B, C, and F, Student's t test was used to generate P values. **, P < 0.01; *, P < 0.05.