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. 2016 Nov 28;6:37829. doi: 10.1038/srep37829

Figure 3. SMS deficiency inhibits JEV attachment.

Figure 3

(a) To assess JEV attachment in tMEFs, medium of Vero cells infected with JEV (MOI = 1) for 48 h was used for short term (15 min) infection. Wild type (WT) and SMS knockout (SMS DKO) MEFs were infected with medium containing JEV for 15 min, washed, and harvested. JEV-E and actin were detected by western blot and quantified. The values represent the mean ± SD (n = 3). *P < 0.005. Full-length blots are presented in Supplementary Figure S4. (b) DiL-labelled JEV was added to poly-d-lysin-coated coverslip, stained with anti-JEV-E antibody and Alexa 488-conjugated rat IgG. Scale bars, 10 μm. (c) WT and SMS DKO MEFs were treated with DiL-labeled JEV and CT-b-AF488 for 15 min at 4 °C and 37 °C to detect the attachment and internalization of JEV. After fixation, nuclei were stained with Hoechst 33342. Scale bars, 10 μm. (d) To assess the uptake of JEV, virus entry assay was performed. Cells were infected with medium containing JEV for 1 h on ice, washed, and incubated for 15 min at 37 °C. After treatment with trypsin to remove cell surface virus, cells were harvested for real-time PCR. JEV RNA levels in the cells were normalized with β-actin mRNA. The values represent the mean ± SD (n = 3). *P < 0.005.