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. 2013 Dec;13(23-24):3442-56.
doi: 10.1002/pmic.201300001. Epub 2013 Dec 2.

Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Tsuey-Ching Yang  1 , Shih-Wein Li, Chien-Chen Lai, Kai-Zen Lu, Man-Tzu Chiu, Tsung-Han Hsieh, Lei Wan, Cheng-Wen Lin
Affiliations

Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Tsuey-Ching Yang et al. Proteomics. 2013 Dec.

Abstract

Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFNβ-induced responses, for example, expression of IFN-stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress-induced-phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca(2+) levels while inhibiting nuclear translocalization of STAT1 and NFAT-1 in response to IFNβ, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca(2+) /calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5-mediated inhibition of IFNβ-induced responses, for example, IFN-sensitive response element driven luciferase, STAT1-dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5-expressing cells in response to IFNβ. Results prove Ca(2+) , calreticulin, and calcineurin involvement in STAT1-mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.

Keywords: Calreticulin; Interferon; Japanese encephalitis virus; Microbiology; Non-structural protein 5; STAT1.

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Figures

Figure 1
Figure 1
Expression of JEV NS5 protein in TE671 human medulloblastoma cells. Cells transfected with pCR3.1‐Flag (control vector; left) or pCR3.1‐JEV NS5‐Flag (right) were selected by 2‐wk incubation with G418. Expression of Flag‐epitope tagged protein was examined by fluorescence microscopy (A). Also, Western blot to detect expressed NS5 was conducted via anti‐Flag antibody (B). Lysates from cells transfected with pCR3.1‐Flag (lane 1) or pCR3.1‐JEV NS5‐Flag (lane 2) were analyzed by 10% SDS‐PAGE prior to blotting, resulting blots probed with anti‐Flag tag antibody.
Figure 2
Figure 2
Inhibitory effects of JEV NS5 protein on IFNβ‐induced responses. (A) To analyze ISRE promoter activity, vector control and NS5‐expressing cells were transiently cotransfected with reporter plasmid containing firefly luciferase under control of ISRE and internal control reporter pRluc‐C1. After 4‐h IFNβ treatment, firefly luciferase and renilla luciferase were measured; firefly luciferase activity normalized to renilla luciferase activity is reported. (B) To analyze ISRE‐driven gene expression, vector control and NS5‐expressing cells were treated with or without 1000 U/mL IFNβ for 8 h; relative levels of PKR and OAS mRNAs were gauged by quantitative real‐time PCR, relative fold levels of PKR or OAS mRNA presented as ratio of PKR or OAS mRNA/GAPDH mRNA. (C) To analyze STAT1 phosphorylation, Western blot of lysates from cells treated with IFNβ for 0, 30, 60, or 120 min was performed by anti‐phospho‐STAT1 (Tyr701) and anti‐β actin antibody as internal control.
Figure 3
Figure 3
Western blot and MS/MS analysis of protein profiling in NS5‐expressing and vector control cells in response to IFNβ. (A) Enlarged images of cyclophilin A and calreticulin protein spots in 2D gels from vector control and NS5‐expressing cells in response to IFNβ treatment. (B, C) Nanoelectrospray mass spectrum of two charged ion peaks for thioredoxin (spot ID 10) was shown. (D) To analyze cyclophilin A and calreticulin protein levels, vector control and NS5‐expressing cells in the presence or absence of IFNβ were harvested for Western blot with anti‐cyclophilin A, anti‐calreticulin, and anti‐β‐actin antibodies. (E) To quantify relative calreticulin mRNA levels, total RNA extracts from both cells were analyzed by quantitative PCR and normalized by GAPDH mRNA, presented as relative ratio.
Figure 4
Figure 4
Functional characterization of calreticulin with siRNA‐mediated gene silencing. (A) To detect intracellular Ca2+, cells transfected with control or calreticulin siRNA were harvested 4 h posttreatment with or without IFNβ, stained with FLUO3/AM and analyzed by flow cytometry. To analyze subcellular localization of STAT1 (B, C) and NFAT‐1 (D, E), vector control (B, D) and NS5‐expressing cells (C, E) transfected with control or calreticulin siRNA were tested by immunfluorescent staining with anti‐STAT1 or anti‐NFAT‐1 antibodies.
Figure 5
Figure 5
Effects of cyclosporin A (CsA) on NS5‐modulated suppression of IFNβ‐induced response. (A) For cytopathic effect assays, cells were treated singly or with both CsA and IFNβ for 48 h, imaging analyzed by light microscopy. (B) To analyze ISRE‐driven promoter activity, vector control and NS5‐expressing cells were transiently cotransfected with dual‐luciferase reporters, treated singly or with both CsA and IFNβ for 4 h. Firefly and renilla luciferase were measured, with relative firefly luciferase activity normalized to renilla luciferase activity. Relative mRNA levels of PKR (C) and ISG15 (D) were measured by quantitative PCR and normalized by GAPDH mRNA, presented as relative ratio.
Figure 6
Figure 6
Effect of CsA on IFNβ‐induced phosphorylation and nuclear translocation of STAT1 in NS5‐expressing cells. (A) To analyze tyrosine phosphoryl‐ated STAT1, vector control and NS5‐expressing cells were treated singly or with both IFNβ and cyclosporine A for 0, 30, 60, or 120 min. Lysates were subjected to Western blot, probed with anti‐phospho‐STAT1 (Tyr701). For subcellular location of STAT1, vector control (B) and NS5‐expressing (C) cells were treated singly or with both CsA and IFNβ for 24 h, then washed, fixed, and reacted with anti‐STAT1 and FITC‐conjugated anti‐mouse IgG antibodies. Finally, cells were stained with DAPI for 10 min, imaging analyzed by immunofluorescent microscopy.
Figure 7
Figure 7
Functional analysis of calcineurin with siRNA‐mediated gene silencing. To analyze subcellular localization of STAT1, vector control (A) and NS5‐expresing cells (B) transfected with control or calreticulin siRNA were tested using immunfluorescent staining with anti‐STAT1 antibodies. Relative mRNA levels of PKR (C) and IL‐4 (D) were measured by quantitative PCR and normalized by GAPDH mRNA, presented as relative ratio.
Figure 8
Figure 8
Effect of cyclosporine A on JEV replication in human medulloblastoma cells. (A) To analyze calreticulin expression, TE671 cells were infected with JEV at MOIs of 0.1, 0.5, 1, and 2, then harvested 2 days post infection. Lysates subjected to Western blot were probed with anticalreticulin. (B) TE671 cells were infected with JEV at the MOI of 0.5 and treated with IFNβ alone or both IFNβ and cyclosporine A for 48‐h and cultured supernatants harvested to determine virus yields. BHK‐21 cell monolayers were incubated with serial dilution of cultured supernatant at 37°C for 1 h, then overlaid with MEM medium containing 1.1% methylcellulose. After 3 days of incubation, BHK‐21 cell monolayers were stained with naphthol blue‐black dye and viral plaques counted. (C) For analysis effect of CsA on apoptosis, JEV‐infected, TE671 cells were infected with JEV at MOI of 0.5 and treated with IFNβ alone or with IFNβ plus CsA for 2 days, then harvested, stained with both Annexin V‐FITC and PI, and analyzed for percent of cells in apoptosis, using flow cytometry.
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