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. 2013 Jan;87(1):148-62.
doi: 10.1128/JVI.01399-12. Epub 2012 Oct 10.

Japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism

Manjula Kalia  1 , Renu Khasa, Manish Sharma, Minu Nain, Sudhanshu Vrati
Affiliations

Japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism

Manjula Kalia et al. J Virol. 2013 Jan.

Abstract

Japanese encephalitis virus (JEV) is a mosquito-borne pathogenic flavivirus responsible for acute viral encephalitis in humans. The cellular entry of JEV is poorly characterized in terms of molecular requirements and pathways. Here we present a systematic study of the internalization mechanism of JEV in fibroblasts and neuroblastoma cells. To verify the roles of distinct pathways of cell entry, we used fluorescently labeled virus particles, a combination of pharmacological inhibitors, RNA interference (RNAi), and dominant-negative (DN) mutants of regulatory proteins involved in endocytosis. Our study demonstrates that JEV infects fibroblasts in a clathrin-dependent manner, but it deploys a clathrin-independent mechanism to infect neuronal cells. The clathrin-independent pathway requires dynamin and plasma membrane cholesterol. Virus binding to neuronal cells leads to rapid actin rearrangements and an intact and dynamic actin cytoskeleton, and the small GTPase RhoA plays an important role in viral entry. Immunofluorescence analysis of viral colocalization with endocytic markers showed that JEV traffics through Rab5-positive early endosomes and that release of the viral nucleocapsid occurs at the level of the early and not the late endosomes.

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Figures

Fig 1
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.
Fig 2
Fig 2
DiD-labeled JEV is internalized independently of clathrin-mediated endocytosis cargo transferrin. (A) DiD-labeled JEV particles were spotted onto a poly-d-lysine-coated coverslip, immunostained with JEV E antibody, and imaged on a 60× objective. Images are presented as gray scales for individual colors and were pseudocolored as indicated prior to being merged. Insets show magnified views of the region corresponding to the asterisk. Bar, 10 μm. (B) DiD-labeled JEV and unlabeled JEV were processed identically, and infectious viral titers were calculated by plaque assays. (C) DiD-labeled JEV (pseudocolored in green) was allowed to bind to Neuro2a cells on ice for 1 h, washed with chilled PBS, and then incubated at 37°C for different times in the presence of Alexa 568/Alexa 647-Tf (pseudocolored in red). Cells were imaged at ×60. The upper right panel represents a differential interference contrast (DIC) image (superimposed with fluorescence from the DiD channel, pseudocolored in green) of the fluorescent image in the upper left panel. Note that DiD-labeled JEV endosomes remain segregated from Tf endosomes until 15 min (arrowheads), and some overlap is detected only by 30 min (arrows). Bar, 5 μm.
Fig 3
Fig 3
JEV entry and infection in neuronal cells are not inhibited by chlorpromazine treatment. (A) Neuro2a, SH-SY5Y, and Vero cells were pretreated for 30 min with chlorpromazine at the indicated concentrations and given a pulse of Alexa 488-Tf for 10 min. Cells were fixed, and transferrin uptake was quantified by flow cytometry. The averages ± SDs of measured geometric means of internalized Tf in control and chlorpromazine-treated cells are shown. (B) Neuro2a, SH-SY5Y, and Vero cells were pretreated for 30 min with 50 μM chlorpromazine and infected with JEV (MOI, 0.4) in the presence of inhibitor. Infection was scored as described in Materials and Methods. Student's t test was used to generate P values. *, P < 0.05. (C) Neuro2a cells were infected with JEV (MOI, 10) in the presence of 50 μM chlorpromazine for 1 h, and the amount of virus endocytosed was estimated by qRT-PCR of JEV positive-strand RNA. (D) Virus titers (mean ± SD) in culture supernatants at 24 hpi (MOI, 0.4) were calculated by plaque assay.
Fig 4
Fig 4
JEV infection in neuronal cells is clathrin independent. (A and B) Neuro2a cells were transfected with plasmids for GFP-Eps15-DIIIδ2 (A) and GFP-Eps15-DIII (B) and 24 h later were given a pulse of Alexa 568-Tf for 10 min. Images are presented as gray scales for individual colors and were pseudocolored as indicated prior to being merged. Note that the majority of cells transfected with Eps15-DIII (arrows) show a block in Tf uptake, whereas cells transfected with control Eps15-DIIIδ2 (arrows) show Tf uptake. Bar, 20 μm. (C) Quantitation of Tf internalization in Neuro2a cells transfected with GFP-Eps15 constructs DIIIδ2 and DIII was done as described in Materials and Methods. (D) Neuro2a, SH-SY5Y, and Vero cells transfected with GFP-Eps15-DIIIδ2 and GFP-Eps15 DIII were infected with JEV (MOI, 1). At 24 hpi, cells were fixed and stained for JEV E antigen. Infection was scored by counting the number of GFP-transfected cells stained versus unstained for JEV E and normalized to GFP-expressing infected cells. (E) Virus titers (mean ± SD) in culture supernatants of transfected cells at 24 hpi (MOI, 1) were calculated by plaque assay. (C and D) Student's t test was used to generate P values; **, P < 0.01; *, P < 0.05.
Fig 5
Fig 5
Depletion of clathrin light-chain and clathrin heavy-chain proteins has no effect on JEV infection of neuronal cells. (A) Neuro2a cells were transfected with either GFP-Retro Q shRNA plasmid vector alone (left panel) or GFP-CLC shRNA (right panel) and 72 h later were given a 10-min pulse of Alexa 647-Tf (pseudocolored in red). Note that cells expressing vector alone show efficient Tf uptake, while cells expressing CLC shRNA show a block in Tf endocytosis. Bar, 5 μm. (B) Quantitation of Tf uptake was done as described in Materials and Methods. (C) Western blots showing depletion of CLC in Neuro2a and Vero cells transfected with CLCshRNA plasmid for 72 h and loading control actin. (D) Neuro2a and Vero cells were transfected with shRNA plasmids directed against clathrin light chain (CLC) or control mock plasmid. After 72 h, cells were infected with JEV (MOI, 1). Cells were scored at 24 hpi by immunofluorescence staining of JEV E antigen. Infection was quantified as described above. (E) JEV titers in Neuro2a and Vero cells transfected with control and CLCshRNA plasmids, estimated at 24 hpi (MOI, 1). (F) Western blot showing depletion of CHC in Neuro2a cells transfected with shRNA plasmids for 72 h and loading control actin. (G) JEV infection in Neuro2a cells in the background of CHC depletion was scored as described above. (H) Virus titers (mean ± SD) in culture supernatants of Neuro2a cells depleted of CHC, infected at an MOI of 1, were calculated at 24 hpi by plaque assays. Student's t test was used to generate P values. **, P < 0.01; *, P < 0.05.
Fig 6
Fig 6
Cholesterol is required for JEV infection. (A) Neuro2a and Vero cells were treated with the indicated concentrations of MβCD or filipin for 1 h and infected with JEV (MOI, 0.4) in the presence of inhibitor. At 24 hpi, cells were fixed and infection was quantified as described in Materials and Methods. (B) Neuro2a cells were treated with the indicated concentrations of MβCD or filipin for 1 h and infected with JEV (MOI, 10) in the presence of inhibitor. The endocytosed viral load was estimated by qRT-PCR of JEV positive-strand RNA. Student's t test was used to generate P values. **, P < 0.01; *, P < 0.05.
Fig 7
Fig 7
Role of actin and myosin motors in JEV entry in neuronal cells. (A) JEV (MOI, 10) was added to Neuro2a cells and left for the indicated times at 37°C, following which cells were washed with chilled PBS, fixed, and stained with Alexa 546-phalloidin. Images were taken at ×60. Bar, 10 μm. (B) Neuro2a and Vero cells were treated with inhibitors cytochalasin D, latrunculin, jasplakinolide, and blebbistatin at the indicated concentrations. Cells were infected with JEV (MOI, 0.4) in the presence of inhibitor, and infection was scored as described in Materials and Methods. (C) Neuro2a cells with treated with inhibitors at the indicated concentrations and infected with JEV at an MOI of 10 in the presence of the inhibitor. Virus entry was quantified by qRT-PCR. Student's t test was used to generate P values. **, P < 0.01; *, P < 0.05.
Fig 8
Fig 8
The GTPase Rho A is required for JEV infection of neuronal cells. (A to C) Neuro2a cells were transfected with myc-tagged constructs of wt RhoA, RhoA L63, RhoA N19, Rac1 L61, Rac1 N17, wt Cdc42, Cdc42 L61, and Cdc42 N17. The GTPase activity of the overexpressed constructs was quantified using GTPase-specific ELISA as described in Materials and Methods. (D) Neuro2a cells transfected with the wt, DA, and DN constructs of Rho, Rac, and Cdc42 were infected with JEV (MOI, 1). At 24 hpi, double immunofluorescence staining was done for myc and JEV E. Cells staining positive for both myc and JEV E were scored and normalized to wt RhoA-expressing infected cells. (E) Time course of Rho activation in response to JEV binding using Rho GTPase-specific ELISA as described in Materials and Methods. (F) Neuro2a cells were serum starved for 2 h before treatment with CT04 inhibitor for another 2 h. Cells were infected with JEV (MOI, 0.4) in the presence of inhibitor. Infection was quantified as described in Materials and Methods. (G) Virus endocytosis (MOI, 10) was quantified in control and CT04-treated cells by qRT-PCR. Student's t test was used to generate P values, **, P < 0.01; *, P < 0.05.
Fig 9
Fig 9
JEV infection requires trafficking through Rab5-positive compartments but is independent of Rab7. (A) DiD-labeled JEV (pseudocolored in green) was added to Neuro2a cells transfected with dsRed-Rab5 or GFP-Rab7 (pseudocolored in red) on ice for 1 h. Cells were warmed to 37°C for the indicated times, washed with low-pH buffer, fixed, and imaged. DiD-labeled JEV colocalization with Rab5 is seen only by 30 min postinternalization (lower left panel, arrowheads). Bar, 10 μm. (B) Neuro2a and Vero cells were pretreated with 100 nM bafilomycin before infection with JEV. Infection was quantified as described in Materials and Methods. (C) Neuro2a and Vero cells transfected with GFP, GFP-wt Rab5, GFP-Rab5dn, GFP-wt Rab7, or GFP-Rab7dn were infected with JEV and processed at 24 hpi as described above. Infection was normalized to cells transfected with GFP alone. Student's t test was used to generate P values. **, P < 0.01.
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