Journal: Journal of Virology

Article Title: Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells

doi: 10.1128/JVI.01113-17

Figure Lengend Snippet: JEV entry and infection require acidic endosomal pH. (A, C, and E) Chloroquine, NH4Cl, and bafilomycin A1 (Baf A1) inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h and inoculated with JEV (MOI of 5) at 4°C for 1 h. At 0 h (binding) or 1 h (entry) postinfection at 37°C, cells were lysed to determine viral RNA copy number by RT-qPCR. (B, D, and F) Chloroquine, NH4Cl, and bafilomycin A1 inhibit JEV infection. Cells were pretreated with subtoxic doses at 37°C for 1 h and then inoculated with JEV (MOI of 0.05) at 37°C for 1 h. At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. Horizontal lines show results of subtoxic doses of these three drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. (G) Effect of V-ATPase knockdown on JEV infectivity was determined by Western blotting. siV-ATPase- or siCtrl-transfected cells were infected with JEV (MOI of 0.05). At 24 hpi the expression of V-ATPase or JEV NS5 was probed with anti-V-ATPase or anti-JEV NS5 antibody as indicated. (H) V-ATPase knockdown inhibited JEV infection. siV-ATPase- or siCtrl-transfected cells were infected with JEV (MOI of 0.05 or 0.01). At 24 hpi, infected cells were lysed to determine viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: For RNA knockdown, BHK-21 cells were seeded in a six-well plate at 2.5 × 10 5 cells/well and then transfected with 100 nM siRNA using Lipofectamine 3000 according to the manufacturer's instructions. siRNA duplexes used in the study are as follows: clathrin heavy chain (sc-35067), caveolin-1 (sc-29241), V-ATPase B1 (sc-36787), siRab5 (sc-36344), siRab7 (sc-29460), siRab9 (sc-44065), the negative-control siRNA (sc-37007) (all, Santa Cruz Biotechnology), and siRab11 (317644A10; Invitrogen).

Techniques: Infection, Binding Assay, Quantitative RT-PCR, Viability Assay, Western Blot, Transfection, Expressing