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. 2017 Feb 28;91(6):e02274-16.
doi: 10.1128/JVI.02274-16. Print 2017 Mar 15.

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

Minu Nain  1   2 , Sriparna Mukherjee  3 , Sonali Porey Karmakar  1 , Adrienne W Paton  4 , James C Paton  4 , M Z Abdin  2 , Anirban Basu  3 , Manjula Kalia  5 , Sudhanshu Vrati  5   6
Affiliations

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

Minu Nain et al. J Virol. .

Abstract

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.

Keywords: Japanese encephalitis virus; flavivirus; host-cell interactions; receptors; viral replication; virus entry.

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Figures

FIG 1
FIG 1
JEV ED3 binds to Neuro2a cells and inhibits JEV infection. (A) His-tagged recombinant JEV ED3 and JEV NS3 were expressed in E. coli and purified on an Ni-NTA column. The purified proteins were electrophoresed on an SDS-PAGE gel, followed by Coomassie staining or Western blotting with JEV E, JEV NS3, and His tag antibodies. (B) Alexa 568-coupled JEV ED3 was added to Neuro2a cells on ice for 1 h. The cells were washed, fixed, and imaged on a confocal microscope. Cells were similarly treated with Alexa 568-labeled JEV NS3 protein as a negative control. (Left) JEV ED3 or NS3 binding on cells. (Middle) DIC image of the field. (Right) Merge of the two images. Bar, 10 μm. (C) Neuro2a cells were incubated with JEV ED3 or NS3 proteins at the indicated concentrations on ice for 1 h, followed by infection with JEV at an MOI of 0.1 or 1. At 24 h p.i., JEV RNA levels were determined by qRT-PCR (left), and the infectious-virus titer (right) in the culture soup was determined by plaque assay. (Left) Relative JEV RNA for each condition normalized to mock treatment. (Right) Absolute values of JEV titers. Viral RNA level or titers in protein-treated cells were compared with those in the mock-treated cells. **, P < 0.01. Each experiment was done with biological duplicates, and similar trends were observed in four independent experiments. The error bars indicate SD.
FIG 2
FIG 2
JEV ED3 interacts with GRP78. (A) JEV-ED3-interacting proteins from plasma membrane fractions of Neuro2a cells were separated on a 2D gel and silver stained. Four unique spots (circled) were excised and subjected to mass spectroscopy analysis. The table on the right shows mass spectrometry scores and identities for the unique spots seen on the 2D gel. (B) Neuro2a cell lysate was incubated with JEV ED3, followed by immunoprecipitation (IP) with either mouse IgG or mouse JEV E antibody (ab81193). The input sample (IS) and the immunoprecipitated samples were electrophoresed and Western blotted with JEV E and GRP78 antibodies. (C) The cDNAs for JEV ED3 and mouse GRP78 were cloned in pACT and pBIND vectors, respectively, of the mammalian two-hybrid system. HEK 293T cells were mock transfected or transfected with positive-control (Ctl) vectors: ED3-pACT plus pBIND, pACT plus GRP78-pBIND, and ED3-pACT plus GRP78-pBIND. The cells were lysed 24 h posttransfection, and luciferase activity was determined. The luciferase activity in cells transfected with positive-control plasmids was taken as 1 for normalization. The luciferase activity in transfected cells was compared to that in mock-transfected cells; **, P < 0.01. Each experiment was performed with biological duplicates, and the data presented are from three independent experiments. The error bars indicate SD.
FIG 3
FIG 3
GRP78 localizes on the plasma membrane in mammalian cells. (A) (Middle) Primary mouse cortical neurons and Neuro2a and Huh-7 cells were fixed, permeabilized, and stained with GRP78 and Alexa 488 anti-rabbit antibodies. (Left) As a negative control for specificity of antibody staining, cells were incubated with Alexa 488 anti-rabbit antibody alone. (Right) Cells were incubated with rabbit GRP78 antibody on ice for 1 h, followed by incubation with Alexa 488 anti-rabbit antibody. The cells were fixed and imaged on a confocal microscope. The nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Total cell lysates and the purified plasma membrane fractions of Neuro2a and Huh-7 cells were Western blotted with GRP78 antibody. Western blotting with GAPDH and calreticulin antibodies was done to rule out contamination with ER membranes and to validate the protein fractionation.
FIG 4
FIG 4
Role of GRP78 in binding and entry of JEV in neuronal cells. (A) Neuro2a cells were mock treated or incubated with 100 μg/ml rabbit IgG or GRP78 C- and N-terminus-specific antibodies on ice for 1 h, followed by the addition of DiI-labeled JEV (MOI, 50) for 1 h. The cells were washed, fixed, and imaged on a confocal microscope. Red fluorescence on the cell membrane indicates virus binding. (B) Quantitation of DiI-labeled JEV binding on cells was done as described in Materials and Methods. Shown are the means of integrated intensities of DiI signals for the different treatments. The intensities recorded under different treatments were compared to that seen on the mock-treated cells (**, P < 0.01). Similar results were seen in two independent experiments. Error bars indicate standard error of the mean (SEM). (C) (Left) Neuro2a cells were transfected with NT or GRP78 siRNAs that caused a significant reduction in GRP78 protein levels in cells 48 h later. (Right) The viability of cells at 48 h of siRNA treatment was tested by MTT assay. The treatment did not affect cell viability. (D) Neuro2a cells were incubated on ice with JEV at an MOI of 5 for 1 h. Mock or proteinase K treatment was followed by incubation at 37°C for 2 h, after which relative endocytosed JEV RNA levels were determined by qRT-PCR. The RNA levels in proteinase K-treated cells were compared to that in the mock-treated cells (taken as 1). The proteinase K treatment efficiently removed membrane-bound virus, resulting in a very significant reduction (**, P < 0.01) in the levels of endocytosed viral RNA. The experiment was performed in biological duplicates, and similar results were seen in three independent experiments. (E) NT control siRNA- or GRP78 siRNA-treated Neuro2a cells were infected with JEV (MOI, 5) 48 h after transfection. This was followed by proteinase K treatment, as described in Materials and Methods, to remove any unbound virus. The endocytosed-virus levels at 2 h p.i. were determined by qRT-PCR for JEV genomic RNA. The level of JEV RNA in NT siRNA-treated cells was taken as 1 for determining the relative RNA levels in GRP78 siRNA-treated cells. The level of endocytosed viral RNA in GRP78 siRNA-treated cells was compared to that seen in mock-treated cells (**, P < 0.01). The experiment was performed in biological duplicates, and the results presented are from three independent experiments. The error bars indicate SD.
FIG 5
FIG 5
GRP78 N-terminal antibody inhibits JEV infection in mammalian cells. Primary mouse cortical neurons and Neuro2a and Huh-7 cells were mock treated or incubated with 100 μg/ml rabbit IgG or GRP78 C- and N-terminus-specific rabbit antibodies on ice for 1 h, followed by JEV infection at an MOI of 1. Viral RNA levels (A) and titers (B) were determined at 24 h p.i. The level of JEV RNA in mock-treated cells was taken as 1 for determining the relative RNA levels. The viral RNA levels and titers were compared to those in mock-treated cells (**, P < 0.01). Each experiment contained biological duplicates, and the data presented are from three independent experiments. The error bars indicate SD.
FIG 6
FIG 6
Antibodies against MHC I inhibit JEV infection. Neuro2a cells were mock treated or incubated with 100 μg/ml rabbit IgG or MHC-I antibody alone or in combination with GRP78 C- and N-terminus-specific rabbit antibodies on ice for 1 h, followed by JEV infection at an MOI of 1. Viral RNA levels (A) and titers (B) were determined at 24 h p.i. The level of JEV RNA in mock-treated cells was taken as 1 for determining the relative RNA levels. The JEV RNA levels and viral titers were compared to those in mock-treated cells (**, P < 0.01). Each experiment contained biological duplicates, and the data presented are from three independent experiments. The error bars indicate SD.
FIG 7
FIG 7
GRP78 colocalizes and interacts with JEV E protein in virus-infected cells. (A) Neuro2a and Huh-7 cells were infected with JEV at an MOI of 5. The cells were fixed 24 h p.i., permeabilized, and stained with GRP78 and JEV E primary antibodies, followed by Alexa 488 anti-rabbit and Alexa 568 anti-mouse antibodies. Cells were imaged on a confocal microscope using a 60×, NA 1.42 objective. Bars, 5 μm. Extensive colocalization of JEV E and GRP78 proteins can be clearly seen in the JEV-infected cells. (B) Neuro2a and Huh-7 cells were infected with JEV at an MOI of 5, and cell lysates were prepared 24 h p.i. They were used for immunoprecipitation with mouse IgG or JEV ED3 (ab81193) antibody. The cell lysates (input samples) and the immunoprecipitates were Western blotted with JEV E (ab26950) and GRP78 antibodies. (C) Lysate from JEV-infected (MOI, 5; 24 h p.i.) Neuro2a cells was immunoprecipitated using rabbit IgG or GRP78 antibody, followed by Western blotting with GRP78 and JEV E (ab81193) antibodies.
FIG 8
FIG 8
Toxicity of subtilase cytotoxin and its biological activity. (A) Neuro2a and Huh-7 cells were treated with SubAA272B or SubAB toxin at the indicated concentrations for 24 h, followed by MTT assay. Shown is cell viability under different treatments, which was not affected by toxin treatment. (B) Purified JEV virions were mock incubated or incubated with SubAA272B or SubAB toxin at the indicated concentrations for 1 h, followed by plaque assays to determine virus viability and infectivity. JEV viability and infectivity were not affected by the toxin treatment. The data shown are representative of the results of three independent experiments, each with biological duplicates. (C) Neuro2a and Huh-7 cells were incubated with SubA272B or SubAB toxin for 2, 4, 6, and 12 h. Cell lysates were run on an SDS-PAGE gel and Western blotted with GRP78, GRP78-C (C-terminal), and GAPDH (loading control) antibodies. GRP78 cleavage is shown by a decrease in the intensity of the 72-kDa band of the full-length GRP78 and appearance of the C-terminal fragment of 28 kDa that can be detected with the C-terminus-specific antibody. The error bars indicate SD.
FIG 9
FIG 9
Role of GRP78 in JEV replication. Primary mouse cortical neurons and Neuro2a and Huh-7 cells were infected with JEV at an MOI of 1, followed by mock treatment or addition of SubAB or SubAA272B toxin at 1 (A), 6 (B), or 12 (C) h p.i. (Left) JEV RNA levels were determined 24 h p.i. by qRT-PCR. The JEV RNA level in mock-treated cells was taken as 1 for determining the relative RNA levels. (Right) Virus titers were determined by plaque assays at 24 h p.i. The JEV RNA levels and viral titers were compared with that in mock-treated cells (*, P < 0.05; **, P < 0.01; ND, not determined). Each experiment contained biological duplicates, and the data presented are from three independent experiments. The error bars indicate SD.
FIG 10
FIG 10
Role of GRP78 in JEV replication. (A) Neuro2a cells were either mock infected (top) or infected with JEV at an MOI of 50 (bottom). At 6 h p.i., the cells were fixed and stained with JEV E antibody and imaged. (Left) DIC images. (Middle) JEV-E staining. (Right) Merge of the two. Bar, 10 μm. (B) Primary mouse cortical neurons and Neuro2a and Huh-7 cells were infected with JEV at an MOI of 50, followed by mock treatment or addition of SubAB or SubAA272B toxin at 1 h p.i. JEV RNA levels were determined at 6 h p.i. by qRT-PCR. The JEV RNA levels were compared with that in the mock-treated cells (**, P < 0.01). Each experiment contained biological duplicates, and the data presented are from three independent experiments. The error bars indicate SD.
FIG 11
FIG 11
SubAB toxin prevents the formation of JEV replication complexes. (A) Neuro2a cells were infected with JEV at an MOI of 1, and 6 h p.i., were mock treated (top) or treated with SubAA272B mutant (middle) or SubAB (bottom) toxin. The cells were fixed at 24 h p.i. and stained with dsRNA (red) and JEV NS3 (green) antibodies. Cells were imaged on a confocal microscope. Bar, 5 μm. (B) The lysates from cells treated as for panel A (JEV at an MOI of 5) were Western blotted to study the levels of JEV E, NS1, and NS3 proteins. GAPDH was used as a loading control.
FIG 12
FIG 12
GRP78 is an important host factor for JEV replication postentry. (A) Neuro2a cells were transfected with 300 ng JEV RNA, and at 6 h posttransfection, the cells were fixed and stained with JEV E antibody. (Left) DIC image. (Middle) JEV E staining. (Right) Merge of the two. Bar, 10 μm. (B to D) Neuro2a cells were transfected with NT or GRP78 siRNAs and 48 h later were transfected with 300 ng purified JEV RNA. At 24 h post-JEV RNA transfection, cells were processed for estimation of relative JEV RNA levels (B) and Western blotted for the indicated antibodies (D), and virus titers in the culture supernatant were determined by plaque assays (C). Each experiment contained biological duplicates, and the data presented in panels B and C are from three independent experiments. (D) The ratios of band intensities for different JEV antibodies and GAPDH are shown below the Western blots. The levels of viral RNA and titers in GRP78 siRNA-treated cells were compared to that in the NT siRNA-treated cells (**, P < 0.01). The error bars indicate SD.
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