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. 1999 Feb;73(2):1672-81.
doi: 10.1128/JVI.73.2.1672-1681.1999.

Hepatitis C virus core protein enhances NF-kappaB signal pathway triggering by lymphotoxin-beta receptor ligand and tumor necrosis factor alpha

L R You  1 , C M Chen, Y H Lee
Affiliations

Hepatitis C virus core protein enhances NF-kappaB signal pathway triggering by lymphotoxin-beta receptor ligand and tumor necrosis factor alpha

L R You et al. J Virol. 1999 Feb.

Abstract

Our previous study indicated that the core protein of hepatitis C virus (HCV) can associate with tumor necrosis factor receptor (TNFR)-related lymphotoxin-beta receptor (LT-betaR) and that this protein-protein interaction plays a modulatory effect on the cytolytic activity of recombinant form LT-betaR ligand (LT-alpha1beta2) but not tumor necrosis factor alpha (TNF-alpha) in certain cell types. Since both TNF-alpha/TNFR and LT-alpha1beta2/LT-betaR are also engaged in transcriptional activator NF-kappaB activation or c-Jun N-terminal kinase (JNK) activation, the biological effects of the HCV core protein on these regards were elucidated in this study. As demonstrated by the electrophoretic mobility shift assay, the expression of HCV core protein prolonged or enhanced the TNF-alpha or LT-alpha1beta2-induced NF-kappaB DNA-binding activity in HuH-7 and HeLa cells. The presence of HCV core protein in HeLa or HuH-7 cells with or without cytokine treatment also enhanced the NF-kappaB-dependent reporter plasmid activity, and this effect was more strongly seen with HuH-7 cells than with HeLa cells. Western blot analysis suggested that this modulation of the NF-kappaB activity by the HCV core protein was in part due to elevated or prolonged nuclear retention of p50 or p65 species of NF-kappaB in core protein-producing cells with or without cytokine treatment. Furthermore, the HCV core protein enhanced or prolonged the IkappaB-beta degradation triggering by TNF-alpha or LT-alpha1beta2 both in HeLa and HuH-7 cells. In contrast to that of IkappaB-beta, the increased degradation of IkappaB-alpha occurred only in LT-alpha1beta2-treated core-producing HeLa cells and not in TNF-alpha-treated cells. Therefore, the HCV core protein plays a modulatory effect on NF-kappaB activation triggering by both cytokines, though the mechanism of NF-kappaB activation, in particular the regulation of IkappaB degradation, is rather cell line and cytokine specific. Studies also suggested that the HCV core protein had no effect on TNF-alpha-stimulated JNK activity in both HeLa and HuH-7 cells. These findings, together with our previous study, strongly suggest that among three signaling pathways triggered by the TNF-alpha-related cytokines, the HCV core protein potentiates NF-kappaB activation in most cell types, which in turn may contribute to the chronically activated, persistent state of HCV-infected cells.

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Figures

FIG. 1
FIG. 1
EMSA of LT-α1β2-stimulated NF-κB activation in various HCV core protein-producing cell lines. (A) NF-κB DNA-binding assays with nuclear extracts from untreated (lanes 2 and 7) or LT-α1β2-treated HeLa and HeLa/C190 cells (lanes 3 to 6 and 8 to 11) were performed. The nuclear extracts were prepared as described by Mackay et al. (62) with some modification. Briefly, 2 × 106 cells after being pretreated with recombinant ligand LT-α1β2 (500 ng/ml) (17) (kindly provided by J. L. Browning [Biogen]) for the proper time (30 min to 2 h) were harvested, washed, and suspended in a hypotonic buffer (buffer A) (20 mM HEPES [pH 7.4], 1 mM MgCl2, 10 mM KCl, 0.3% Nonidet P-40, 0.5 mM dithiothreitol [DTT], 0.1 mM EDTA) at 4°C for 30 min. Cell nuclei were collected by centrifugation, and the nuclear proteins were extracted with high-salt buffer (buffer B) (20 mM HEPES [pH 7.4], 20% glycerol, 0.42 M NaCl, 1 mM MgCl2, 10 mM KCl, and 0.5 mM DTT) for 1 h on ice. The supernatants recovered from centrifugation were stored at −70°C and used for an EMSA. For the EMSA, 5 μg of nuclear extracts was incubated with 50 fmol of 32P-end-labeled 45-mer synthetic double-stranded NF-κB oligonucleotide in a binding buffer (10 mM HEPES [pH 7.8], 5 mM MgCl2, 50 mM KCl, 0.5 mM DTT, 10% glycerol) containing 1 μg of poly(dI-dC) and 30 μg of bovine serum albumin. After incubation at room temperature for 45 min, the DNA-protein complex formed was separated from free oligonucleotide on a 4% native polyacrylamide gel using buffer containing 0.25× TBE (22.5 mM Tris-borate, 0.5 mM EDTA [pH 8.0]). After electrophoresis, the gel was dried and visualized with a PhosphorImager. Competition experiments were carried out by including unlabeled oligonucleotides containing either mutated (MT) (40-fold excess) (lanes 6 and 11) or wild-type (WT) (40-fold excess) (lanes 5 and 10) NF-κB binding sites. The main NF-κB-specific band shift induced is indicated. Lane 1, 32P-labeled free oligonucleotide. (B) Binding assays were identical to that described for panel A except that the nuclear extracts were prepared from HuH-7 and HuH-7/C190 cells.
FIG. 2
FIG. 2
EMSA of TNF-α-stimulated NF-κB activation in various HCV core protein-producing cell lines. All experimental conditions were as described in the legend to Fig. 1 except that cells were stimulated with 20 ng of TNF-α/ml for 30, 60, or 120 min, respectively.
FIG. 3
FIG. 3
Analysis of cytokine-stimulated NF-κB-dependent transcriptional activity in HCV core protein-producing cells. (A) HeLa or HeLa/C190 cells seeded at 1.5 × 105 cells/well density were cotransfected with equal amounts (0.4 μg each) of NF-κB-dependent luciferase reporter plasmid and an internal control plasmid carrying the β-galactosidase gene as a reporter with the SuperFect transfection reagent (Qiagen, Hilden, Germany). At 18 h posttransfection, cells were either left untreated (marked with −) or treated with TNF-α (20 ng/ml) or LT-α1β2 (500 ng/ml) for 6 h prior to harvest. After three cycles of freezing and thawing, cells were lysed in 150 μl of lysis buffer (25 mM Tris-HCl [pH 7.8], 70 mM potassium phosphate buffer [pH 7.8], 2.1 mM MgCl2, 0.7 mM DTT, 0.1% Nonidet P-40, and protease inhibitor cocktail [Complete; Boehringer]). Eighty microliters of cell extracts recovered from the centrifugation was then mixed with 250 μl of luciferase assay buffer (43.2 mM glycylglycin [pH 7.8], 22 mM MgSO4, 2.4 mM EDTA, 7.4 mM ATP, 1 mM DTT, and 0.4 mg of bovine serum albumin/ml), and the resulting mixtures were assayed for luciferase activity by using 100 μl of 0.5 mM luciferin (Sigma) as the substrate and measured with AutoLumat LB953 (Berthold, Bad Wildbad, Germany). The β-galactosidase activity in the cell extracts of cotransfected cells was determined essentially as described previously (21). The luciferase activities were normalized on the basis of β-galactosidase expression. The NF-κB-dependent luciferase activity is represented as fold induction relative to that of HeLa cells without treatment. (B) All experimental conditions were similar to those described for panel A except that HuH-7 and HuH-7/C190 cells were used for study. Values shown in all panels are averages (means ± standard deviations) of one representative experiment in which each transfection was performed in triplicate.
FIG. 4
FIG. 4
Western blot analysis of NF-κB/Rel and IκB family proteins in various HCV core protein-producing cell lines. The total cell extracts (60 μg) from various cell lines lysed in 5× sampling buffer (55) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Anti-NF-κB p65, p52, and p50 subunit antibodies (Upstate Biotechnology Inc.) and IκB-α and IκB-β antibody (Santa Cruz) were used at the dilutions suggested by the manufacturer. The antigen-antibody reactions were visualized with horseradish peroxidase-coupled goat anti-rabbit immunoglobulin (Transduction) (1:2,000 dilution) using the enhanced chemiluminescence (ECL) detection system (Amersham). The control cell lysates (lane C) provided by the manufacturers are A341 cells for p65 and p50 and Raji cells for p52.
FIG. 5
FIG. 5
Subcellular distribution of NF-κB family proteins in HCV core protein-producing cells after LT-α1β2 or TNF-α stimulation. Cells were treated with 500 ng of LT-α1β2 ligand/ml (panels A and B) or 20 ng of TNF-α/ml (panels C and D) for 30 or 60 min. The nuclear extracts (40 μg of protein each) prepared from the cytokine-treated or untreated cells were examined for the expression level of NF-κB family proteins (p65, p50, and p52) by immunoblotting using the ECL detection system.
FIG. 6
FIG. 6
Degradation of IκB proteins in various LT-α1β2- or TNF-α-stimulated HCV core protein-producing cells. Cells were stimulated with 500 ng of LT-α1β2 ligand/ml (panels A and B) or 20 ng of TNF-α/ml (panels C and D) and at various time intervals (2 min to 8 h), the total cell extracts were prepared and portions of cell lysates (40 μg of protein each) were examined for the expression level of IκB-α or IκB-β protein by using rabbit polyclonal antibody against human IκB-α or IκB-β (Santa Cruz) and ECL detection system.
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