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

Abstract

During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life 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 entry of JEV into BHK-21 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.

Keywords: Japanese encephalitis virus; Rab protein; endocytosis.

FIG 1

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.

FIG 2

JEV entry depends on dynamin. (A) Dynasore inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h, the medium was replaced, and then the cells were inoculated with JEV (MOI of 5) at 4°C 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 37°C 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 as determined by cell viability assay as described in Materials and Methods. (C and D) Transferrin uptake was blocked by dynasore. Cells were treated with 100 μM dynasore or dimethyl sulfoxide (DMSO) for 1 h at 37°C, incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C, and transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (E and F) The inhibitory effect of the dynamin DN construct on transferrin uptake was determined by confocal microscopy. Cells transfected with the GFP-tagged dynamin WT or DN construct were incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 37°C. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (G) Transfected BHK-21 cells were infected with JEV (MOI of 0.1); at 24 hpi, the cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (H) At least 300 cells transfected with the indicated plasmids were screened for JEV infection, and values are expressed as percentages of the number of infected cells observed in the control experiment. Results are presented as the means ± SD of data from three independent experiments. *, P < 0.05; **, P < 0.01. a.u., arbitrary units.

FIG 3

Effects of EPS15 Δ95/295 overexpression and chlorpromazine on JEV infection. (A) Chlorpromazine (CPZ) inhibited JEV entry but not binding. Cells were pretreated with subtoxic doses at 37°C for 1 h, the medium was replaced, and then cells were inoculated with JEV (MOI of 5) at 4°C 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) Chlorpromazine (CPZ) inhibited JEV infection. Cells were pretreated with increasing concentrations of CPZ for 1 h at 37°C, the medium was replaced, and then cells were infected with JEV (MOI of 0.05). At 24 hpi, cells were lysed to quantitate viral RNA copy number by RT-qPCR. The horizontal line shows results of subtoxic doses of CPZ drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. (C and D) Transferrin uptake was blocked by CPZ. Cells were treated with CPZ or DMSO for 1 h at 37°C, incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C, and transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensities from two independent experiments. (E and F) The inhibitory effect of the EPS15 DN construct on transferrin uptake was determined by confocal microscopy. Cells transfected with the EPS15 WT or DN plasmid construct were incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C and transferred to 37°C for 30 min. Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensity from two independent experiments. (G) Cells transfected with the EPS15 WT or DN plasmid construct were infected with JEV (MOI of 0.1). At 24 hpi, the cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (H) At least 300 cells transfected with the indicated plasmids were counted and scored as positive or negative for JEV infection. Values are expressed as percentages of the number of infected cells observed in the control experiment. Results are presented as the means ± SD of data from three independent experiments. *, P < 0.05; **, P < 0.01.

FIG 4

Clathrin is required for JEV entry. (A and B) Transferrin uptake was blocked in siCHC-transfected cells. Cells were transfected with siCHC or a control siRNA (siCtrl), incubated with 10 μg/ml Alexa Fluor 568-labeled transferrin for 30 min at 4°C, and then transferred to 37°C for 30 min. Nuclei were stained with 4′,6′-diamidino-2-phenylindole. (B) Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. Tfn uptake is represented as means and standard errors of the means of integrated fluorescence intensities from two independent experiments. (C) At 48 h posttransfection, cells were infected with JEV (MOI of 0.1) for 24 h. Cells were fixed and processed for confocal microscopy with anti-JEV E and anti-clathrin antibodies as indicated. Nuclei were stained with 4′,6′-diamidino-2-phenylindole. (D) At least 300 transfected cells were scored as positive or negative for JEV infection; values are expressed as percentages of the number of infected cells observed in the control experiment. (E) Effect of CHC knockdown on JEV infectivity was determined by Western blotting. siCHC- or siCtrl-transfected cells were infected with JEV (MOI of 0.05). At 24 hpi, expressed CHC and JEV NS5 proteins were probed with anti-JEV NS5 and anti-clathrin antibodies as indicated. (F) CHC knockdown inhibited JEV infection. siCHC- or siCtrl-transfected cells were infected with JEV (MOI of 0.05 of 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. (G) Colocalization of clathrin with JEV during the early stage of infection. Cells grown on glass coverslips in six-well plates were infected with JEV (MOI of 10) at 4°C for 1 h and then shifted to 37°C for 1, 3, or 5 min. Cells were then fixed with 4% PFA, stained with mouse anti-JEV E antibody (4B4) and rabbit anti-clathrin, and examined by confocal microscopy. Bar, 10 μm. *, P < 0.05; **, P < 0.01.

FIG 5

Caveolae are not required for JEV entry. The effect of dominant negative caveolin on CTB uptake or JEV infection was determined by confocal microscopy. (A and B) BHK-21 cells transfected with plasmid constructs expressing GFP-tagged WT and DN caveolin were incubated with 10 μg/ml Alexa Fluor 568-labeled CTB for 30 min at 4°C and then transferred to 37°C for 30 min. Cells were fixed and stained with 4′,6′-diamidino-2-phenylindole. (B) Total fluorescence intensity per cell was calculated using Nikon NIS-Elements AR, version 4.5, analysis software. CTB uptake is represented as means and standard errors of the means of integrated fluorescence intensities from two independent experiments. (C) Transfected cells were infected with JEV (MOI of 0.1). At 24 hpi, cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (D) At least 300 transfected cells were scored as positive or negative for JEV infection; values are expressed as percentages of the number of infected cells observed in the control experiment. (E and F) Effect of caveolin depletion on JEV propagation. Cells transfected with the indicated siRNAs were infected with JEV (MOI of 0.05) and incubated for 24 h to allow virus propagation. (E) The silencing efficiency of siCav and the level of JEV NS5 were analyzed using anti-caveolin-1 antibody or anti-JEV NS5 monoclonal antibody. (F) Infected cells were lysed to quantitate viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. (G) BHK-21 cells grown on glass coverslips in six-well plates were infected with JEV (MOI of 10) at 4°C for 1 h and then shifted to 37°C. At different time intervals monolayers were fixed with 4% PFA and stained with mouse anti-JEV E (4B4) and anti-caveolin-1 antibodies and examined by confocal microscopy. Bar, 10 μm. **, P < 0.01.

FIG 6

Role of macropinocytosis on JEV binding, entry, and infection. Cells were pretreated with subtoxic doses of EIPA or wortmannin, as indicated, for 1 h at 37°C. Drugs were present in the medium during the adsorption period. BHK-21 cells were infected with JEV (MOI of 5) (A and D) or DF-1 cells were infected with NDV (MOI = 5) (C and F) at 4°C for 1 h and then at 37°C for 0 h (binding) or 1 h (entry); the infected cells were lysed to determine viral RNA copy number by RT-qPCR. (B and E) cells were infected with JEV (MOI of 0.05) at 37°C; at 24 hpi, viral RNA copy number was determined by RT-qPCR. The horizontal lines show results of subtoxic doses of EIPA and wortmannin on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. Results are presented as the means ± SD of three independent experiments. **, P < 0.01.

FIG 7

Effects of MβCD on JEV infection. (A) Cell viability upon MβCD treatment was assessed as described previously. (B) Cells were treated with 5 mM MβCD for 1 h and then infected with JEV (MOI of 0.5) in the presence of the inhibitor. At 24 hpi, cells were fixed and stained with an anti-JEV E antibody. Bar, 10 μm. (C)For cholesterol depletion of cell membrane, cells were pretreated with the indicated concentrations of MβCD for 1 h at 37°C, washed twice with medium, infected with JEV (MOI of 0.05) at 37°C for 1 h, washed twice with medium, and then incubated with fresh medium at 37°C for 8 or 24 h. (D) MβCD was mixed with JEV (MOI of 0.05) and incubated at 37°C for 1 h. BHK-21 cells were infected with the treated JEV at 37°C for 1 h. The treated virus was subjected to ultracentrifugation through a 20% sucrose cushion at 70,000 × g for 2 h to remove MβCD, resuspended in medium containing 10% FCS, and filtered before being used to infect cells at 37°C for 8 or 24 h. (E) Cells were infected with JEV (MOI of 0.05) for 8 and 24 h and then treated with the indicated concentrations of MβCD. The medium was replaced, and cells were then incubated for 12 h at 37°C. All infected cells were lysed to quantitate viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of data from three independent experiments. **, P < 0.01.

FIG 8

Effects of microtubules and actin cytoskeleton on JEV infection. (A and C) Cells were pretreated with subtoxic doses of nocodazole or jasplakinolide at 37°C for 1 h, the medium was replaced, and then cells were 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 and D) Cells were pretreated with subtoxic doses of nocodazole or jasplakinolide 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. The horizontal lines show results of subtoxic doses of the two drugs on BHK-21 cells as determined by cell viability assay as described in Materials and Methods. Results are presented as the means ± SD of data from three independent experiments. **, P < 0.01.

FIG 9

Effects of Rab on JEV infection. (A) The effect of dominant negative Rab proteins on JEV infection was determined by confocal microscopy. Cells transfected with plasmids expressing GFP-tagged Rab5, Rab7, Rab9, and Rab11 WT and DN constructs were infected with JEV (MOI of 0.1). At 24 hpi, the cells were fixed with 4% PFA, reacted with anti-JEV E antibody, and visualized by confocal microscopy. Bar, 10 μm. (B) At least 300 transfected cells were scored as positive or negative for JEV infection; values are expressed as percentages of the number of infected cells observed in the control experiment. Results are presented as the means ± SD of data from three independent experiments. **, P < 0.01. (C) JEV NS5 protein and the knockdown efficiency of Rab5, Rab7, Rab9, and Rab11 were determined using Western blotting. Cells were transfected with either an siRNA control (siCtrl) or siRNAs targeting Rab5, Rab7, Rab9, and Rab11 (siRab5, siRab7, siRab9, and siRab11) for 48 h. Cell lysates were subject to SDS-PAGE and Western blotting using the indicated antibodies. (D) Rab5 and Rab11 depletion reduced JEV propagation. Cells transfected with the indicated siRNA were infected with JEV (MOI of 0.05) and incubated at 37°C for 24 h to allow viral propagation. The infected cells were lysed to determine viral RNA copy number by RT-qPCR. Results are presented as the means ± SD of three independent experiments. **, P < 0.01.

FIG 10

Colocalization of Rab with JEV during the early stage of infection. BHK-21 cells grown on glass coverslips in six-well plates were infected with JEV (MOI of 10) at 4°C for 1 h and then shifted to 37°C. At different time intervals, monolayers were fixed with 4% PFA and stained with mouse anti-JEV E (4B4), rabbit anti-Rab5, rabbit anti-Rab7, rabbit anti-Rab9, or rabbit anti-Rab11 antibody and examined by confocal microscopy. Bar, 10 μm.