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. 1998 May;72(5):3691–3697. doi: 10.1128/jvi.72.5.3691-3697.1998

Hepatitis C Virus Core Protein Binds to the Cytoplasmic Domain of Tumor Necrosis Factor (TNF) Receptor 1 and Enhances TNF-Induced Apoptosis

Nongliao Zhu 1, Ali Khoshnan 2, Robert Schneider 2, Masayuki Matsumoto 2, Gunther Dennert 1, Carl Ware 3, Michael M C Lai 1,2,*
PMCID: PMC109590  PMID: 9557650

Abstract

The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin β receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathione S-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the “death domain” of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-κB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-κB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.

Hepatitis C virus (HCV) is a major cause of non-A, non-B acute and chronic hepatitis, which frequently leads to liver cirrhosis and hepatocellular carcinoma (11). The molecular mechanism of HCV pathogenesis remains largely unknown. HCV is classified as a member of the Flaviviridae family. Its 9.5-kb positive-sense, single-stranded RNA genome encodes a polyprotein, which is processed into three structural proteins at the amino-terminal end and six nonstructural proteins at the carboxyl-terminal end (27, 30). The viral core protein consists of 191 amino acids (aa) and has an apparent molecular mass of 21 kDa. It is a major component of viral nucleocapsids and also functions as a transcriptional regulator of various viral and cellular promoters (20, 33), potentially deranging normal cellular functions. In addition, the core protein may cooperate with the ras oncogene and transform primary rat embryo fibroblasts into a tumorigenic phenotype (32). It activates the c-myc promoter and Rous sarcoma virus long terminal repeat but suppresses the promoters for c-fos, retinoblastoma susceptibility gene, beta interferon gene, β-actin gene, and the human immunodeficiency virus type 1 long terminal repeat (20, 33).

We have previously shown that the HCV core protein interacts with the cytoplasmic domain of lymphotoxin β receptor (LTβR), a member of the tumor necrosis factor receptor (TNFR) family (26). LTβR is involved in the regulation of lymph node development (34) and possibly other additional immune functions (13). Thus, the binding of the HCV core protein to LTβR could potentially affect immune functions of the host. This finding prompted us to examine whether the HCV core protein can also bind TNFR 1, which is the prototype TNFR family member. The signal transduction pathways of TNFR 1 as the primary receptor mediating TNF induction have been extensively studied. Cross-linking of TNFR 1 often induces apoptosis or causes inflammatory responses (2). The cytoplasmic tail of TNFR 1 contains a stretch of sequence, termed the death domain, which is required for the major outcomes of TNF induction, i.e., cell death signaling and NF-κB activation (17). However, TNFR itself does not contain an intrinsic catalytic activity; thus, it most likely exerts its signaling through cellular transducers. It has been reported that the death domain serves as binding sites for some of these transducers (8). If the core protein binds to the cytoplasmic region of TNFR 1, especially the death domain, it may conceivably disrupt or enhance the association of cellular transducers, thereby affecting their signaling.

Here we demonstrate that the core protein indeed binds to the death domain of TNFR 1. As a consequence of this binding, cells are sensitized to TNF-induced apoptosis. In some cell lines, the core protein also suppresses the activation of RelA/NF-κB by TNF. The involvement of the core protein in TNF signaling has direct clinical relevance, as TNF is one of the major mediators of cell death in chronic liver diseases (15, 19). These results suggest that the core protein can exert a cytotoxic effect through the TNF signaling pathway and may account for HCV-induced hepatitis.

MATERIALS AND METHODS

Cell lines.

BC10ME is a mouse fibrosarcoma cell line derived from the B/C-N cell line (10). HeLa (human epitheloid carcinoma), HepG2 (human hepatocellular carcinoma), and Cos7 (monkey kidney) cell lines were purchased from the American Type Culture Collection, Rockville, Md. All cells were grown in Dulbecco’s modified Eagle medium (DMEM; Irvine Scientific) supplemented with 10% fetal bovine serum (Gemini Bio-products, Inc.).

Eukaryotic expression vectors.

For stable expression of the HCV core protein, we constructed a plasmid containing the human elongation factor 1α (EF) promoter (21). For this purpose, plasmid pEF321neo (kindly provided by Tatsuo Miyamura, National Institute of Health, Tokyo, Japan) was digested with HindIII and NheI. The fragment was cloned into pcDNA3.1 (Invitrogen) at HindIII and NruI sites such that the human cytomegalovirus promoter was replaced with the EF promoter. The resulting vector, designated pcDEF, was digested with BamHI and ligated with a PCR-generated HCV core gene (encoding aa 1 to 191) with attached BamHI sites. The resulting plasmids, pcDEF-TW and pcDEF-RH, contain the HCV core gene of the Taiwan isolate (7) and the southern California isolate (24), respectively, downstream of the EF promoter. These plasmids also contain the neomycin resistance gene.

GST binding assay.

Various portions of the cytoplasmic domain of TNFR 1 were inserted in frame into the pGEX-4T-1 expression vector (Novagen). Glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli BL21(DE) (Novagen) and purified with glutathione-Sepharose 4B beads as specified by the manufacturer (Pharmacia Biotech). 35S-labeled in vitro-translated HCV core protein (3 μl) was incubated with various GST-TNFR 1 fusion proteins at 4°C for 2 h in binding buffer (40 mM HEPES [pH 7.5], 100 mM KCl, 0.1% Nonidet P-40, 20 mM 2-mercaptoethanol). Approximately 2 μg of each fusion protein, as determined by Coomassie blue staining of the partially purified protein, and same amount of 35S-labeled core protein were used in each assay. After four washes with the same buffer, the bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by autoradiography (26).

Generation of permanent cell lines expressing the HCV core protein.

For stable expression of the HCV core protein, plasmids containing HCV core protein-coding sequences under the control of EF promoter were used. Cell lines were transfected with various plasmids by using DOTAP (Boehringer Mannheim) and selected with 0.6 mg of G418 (Life Technologies Inc.) per ml. Empty vectors were also transfected into cells, and stable cell clones were selected to serve as controls. All of the selected permanent cell clones were maintained in the presence of G418 (0.3 mg/ml) throughout the experiment.

Membrane flotation analysis.

The method used was a slight modification of the published procedures (26). Plasmids containing the HCV core and TNFR 1-coding sequences under the control of T7 promoter were transfected into Cos7 cells infected with VT7 (14), a recombinant vaccinia virus that expresses the T7 RNA polymerase. Fourteen hours posttransfection, the cells were suspended in hypotonic lysis buffer and separated into membrane and cytosol fractions by sucrose gradient centrifugation (10, 55, and 72% sucrose step gradients for 14 h at 38,000 rpm in a Beckman SW55Ti rotor). The proteins were detected by immunoblotting using an anticore polyclonal antibody (26) and anti-human soluble TNFR 1 antibody (R&D Systems).

TNF cytotoxicity assay and annexin V apoptosis assay.

A colorimetric assay to quantitate cytotoxic effects of TNF was performed as previously described (12). Approximately 4 × 104 cells were added to each well of a 96-well plate in DMEM without phenol red (Irvine Scientific), supplemented with 10% fetal bovine serum and 1 μg of actinomycin D (Boehringer Mannheim) per ml. For induction of TNF cytotoxicity, recombinant murine TNF (Gibco BRL) was used for BC10ME cell lines, and recombinant human TNF (Gibco BRL) was used for HeLa cell lines. To assay HepG2 cells, anti-human sTNFR 1 detection antibody (R&D Systems) was used to induce oligomerization of TNFR 1. One of the 96 wells contained only medium to serve as a background control for each assay. The cells were incubated for 18 h at 37°C before the medium was replaced by DMEM containing 1 mg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Research Organics, Inc.) per ml. After incubation for 3 h, the medium was removed, 100 μl of methylsulfoxide (EM Science) was added to each well, and the plates were incubated for 20 min at room temperature. The optical density at 560 nm (OD560) was read by a 96-well microplate autoreader (Bio-TEK Instruments). The percentage of cell death was calculated based on the OD560 according to the following formula: percentage of cell death = 100 − (OD560 of TNF-treated sample/OD560 of untreated sample). Assays were performed in triplicate. Standard deviation was calculated from data of independently derived cell clones in separate experiments.

For apoptosis assay, an annexin V-fluorescein isothiocyanate (FITC)-labeled kit was used as instructed by the manufacturer (R&D Systems). The procedure measures an early event in apoptosis, in which phosphatidylserine from the inner plasma membrane migrates to the outer leaflet of the plasma membrane (25, 40). Briefly, cells were treated with different concentrations of recombinant human TNF (R&D Systems) in the presence of 10 μg of cycloheximide per ml for 2 h. Both attached and nonattached cells were harvested, washed in phosphate-buffered saline (PBS), and suspended in annexin binding buffer at a concentration of 106 cells/ml. Approximately 105 cells were incubated in the presence of optimized amounts of FITC-conjugated annexin V and propidium iodide for 15 min. The percentage of apoptotic cells was evaluated by flow cytometry with a FACStar plus (Becton Dickinson) according to the instructions provided by the supplier.

Flow cytometry analysis.

Approximately 2 × 106 cells were detached from plates with 5 mM EDTA in PBS. After several washes with PBS, the cells were incubated in blocking solution (PBS containing 5% bovine serum albumin) at room temperature for 5 min and then with anti-human sTNFR 1 detection antibody (4 μg/ml) at 4°C for 6 h in the blocking solution. The cells were incubated with 5 μg/ml of FITC-conjugated mouse anti-goat immunoglobulin G (IgG; heavy and light chains; Jackson ImmunoResearch Laboratories, Inc.) in the blocking solution at 4°C for 2 h. The cells were fixed with 1% methanol-free formaldehyde for 20 min on ice and then subjected to flow cytometry analysis using a FACStar plus (Becton Dickinson) with 2.5-W argon-krypton and 488-nm 200-mW water-cooled laser. Cells incubated with secondary antibody only were used as negative controls. Cells not exposed to either antibody served as background controls.

RelA/NF-κB nuclear translocation analysis.

Approximately 106 BC10ME control (BC10/EF) and core-expressing (BC10/CORE) cells were either treated with murine TNF (10 ng/ml) for 20 min at 37°C or left untreated. The cell pellets were resuspended in 100 μl of buffer A (10 mM HEPES-KOH [pH 7.8], 10 mM KCl, 1.5 mM MgCl2, 20% glycerol, 0.5 mM dithiothreitol) and incubated on ice for 15 min; 0.5% of Nonidet P-40 was then added to lyse the cells. After centrifugation at 8,000 rpm for 10 min at 4°C in a microcentrifuge, the supernatant (cytoplasmic extract) was collected, and the pellet was resuspended in buffer C (20 mM HEPES-KOH [pH 7.8], 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol) and incubated at 4°C for 30 min. The supernatant (nuclear extract) obtained from centrifugation (14,000 rpm for 10 min at 4°C in a microcentrifuge) was collected. The protein concentration of each extract was determined by Bio-Rad protein assay. Approximately 60 μg of total protein from each extract was then loaded for SDS-PAGE and immunoblotted for RelA (Santa Cruz Biotechnology, Inc.), actin (Santa Cruz Biotechnology, Inc.), and the core protein.

RESULTS

HCV core protein interacts with the cytoplasmic tail of TNFR 1 in vitro.

To determine whether the HCV core protein binds to TNFR 1, we first used a GST fusion protein pull-down assay. In vitro-translated [35S]methionine-labeled HCV core protein (aa 1 to 191) was incubated with GST-TNFR 1 fusion proteins containing different portions of the cytoplasmic domain. The results showed that core protein binds to the cytoplasmic domain of TNFR 1. In contrast, core protein did not bind to the GST-CD40 (cytoplasmic domain), another member of the TNFR family (2) (Fig. 1). To map the core protein-interacting domain in TNFR 1, a series of deletion or point mutants were constructed, expressed as GST fusion proteins (Fig. 1a), and used for interaction with in vitro-translated core protein. The results (Fig. 1b) indicated that a C-terminal region of 62 aa from aa 345 to 407, in the cytoplasmic domain of TNFR 1 contains the critical residues for interaction with core protein. This entire region was required for efficient interaction, since either half of this region retained only about 10% of the binding activities. This region corresponds to the previously identified death domain of TNFR 1, which is required for cell death signaling (38). A point mutation at aa 345 from phenylalanine (F) to alanine (A), which has a dominant negative effect on TNFR 1-mediated cytotoxicity signaling (17), reduced binding approximately fourfold. The extracellular domain of TNFR 1 did not interact with the core protein (data not shown). The N-terminal 115 aa of the core protein, which contains the hydrophilic portion of the protein, is sufficient for interaction with TNFR 1 (data not shown).

FIG. 1.

FIG. 1

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In vitro interaction of the HCV core protein with the cytoplasmic domain of TNFR 1 in a GST-binding assay. (a) Schematic representation of deletion and point mutants of GST-TNFR 1 (cytoplasmic domain) fusion proteins. The darkened region indicates the death domain. The numbers represent the starting and the ending amino acid residues of the TNFR 1 open reading frame in each construct. Binding of each protein to the HCV core protein is summarized at the right. (b) 35S-labeled in vitro-translated HCV core protein (3 μl) was incubated with different GST-TNFR 1 fusion proteins. The bound core protein was separated by SDS-PAGE and detected by autoradiography. The input HCV core protein (1 μl; core probe) used was run in parallel. Shown is a representative result of at least three separate experiments.

Association of the HCV core protein with TNFR 1 in mammalian cells.

The potential core protein-TNFR 1 interactions were then demonstrated in vivo by using membrane flotation analysis (26) as shown in Fig. 2. Based on the observations that the truncated core protein was in the cytosol and nucleus, in contrast to the full-length core protein, which was associated with the endoplasmic reticulum (36), and that the truncated core protein was expressed to a higher level than the full-length core protein (unpublished observation), we used a truncated core protein (aa 1 to 153) to study its interaction with TNFR 1. If the truncated core protein binds to TNFR1, which is an integral membrane protein, the core protein is expected to be translocated from the cytosol to the membrane fractions of the cells. It should be noted that this truncated core protein retains the domain responsible for the binding to TNFR 1, as determined by GST pull-down results. For this purpose, Cos7 cells were either transfected with the HCV core protein (aa 1 to 153) alone or cotransfected with the core protein and TNFR 1. Cell lysates from the transfected cells were separated into membrane and cytosol fractions by sucrose gradient centrifugation. The results showed that when expressed alone, the N-terminal 153 aa of the core protein remained mainly in the cytosol fractions (Fig. 2a). However, when coexpressed with the full-length TNFR 1 (aa 1 to 426), a significant proportion (47%) of the core protein shifted from the cytosol to the membrane fractions (Fig. 2b), colocalizing with TNFR 1. In contrast, when cotransfected with a C-terminus-truncated TNFR 1 (aa 1 to 308), which retained the transmembrane domain but not the death domain, the core protein remained exclusively in the cytosol whereas the truncated TNFR 1 was still in the membrane fraction (Fig. 2c), suggesting a complete disruption of the TNFR 1-core protein interaction. These data showed that core protein can bind to the membrane-associated TNFR 1 in the cells and that this interaction occurs between the death domain of TNFR 1 and the N-terminal 153 aa of the HCV core protein, consistent with the in vitro binding data.

FIG. 2.

FIG. 2

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In vivo interaction between the HCV core protein and the cytoplasmic tail of TNFR 1 shown by membrane flotation analysis. Cos7 cells transfected with various plasmids were separated into membrane and cytosol fractions as described in Materials and Methods. The core protein and TNFR 1 were detected by immunoblotting with the appropriate antibodies. (a) The core protein expressed alone; (b) coexpression of the core protein with the full-length TNFR 1 (aa 1 to 426); (c) coexpression of the core protein and a C-terminus-truncated TNFR 1 (aa 1 to 308).

HCV core protein sensitizes three cell lines to TNF-induced cell death.

Since the death domain of TNFR 1 is involved in cell death signaling and NF-κB activation (38), the interaction between HCV core protein and the death domain of TNFR 1 could potentially affect TNF cytotoxicity and/or NF-κB activation. We first examined the effects of the core protein on TNF-induced cell death in several cell lines. Permanent cell clones that stably express the HCV core protein were established in BC10ME cells, HepG2 cells, and HeLa cells. Different cell clones expressed comparable amounts of core protein, as demonstrated by immunoblotting using a polyclonal antibody against the core protein (Fig. 3a to c). Control cell clones transformed with the empty expression vector were also obtained. These cells were tested for TNF-induced cytotoxicity by MTT assay (10). The results showed that BC10ME cell lines expressing core protein (BC10/CORE) were significantly more sensitive to TNF-induced cell death than control cell lines (BC10/EF) at low TNF concentrations, with P values less than 0.001 (Fig. 3a). This difference was not observed at high TNF concentrations (>1 ng/ml), when the majority of both core-expressing cells and control cells were killed. Since these data were averaged from eight independently derived cell clones expressing core protein and their counterparts transformed with the empty vector, this difference could not have been due to individual clonal variations. Similar results were obtained for HepG2 cell clones. In this case, the cells were treated with anti-TNFR 1 antibody, which induced cross-linking of TNFR 1 and triggered TNFR 1 signal transduction, since HepG2 cell lines are not very sensitive to TNF. The results showed that the core protein-expressing HepG2 cell lines (HepG2/CORE) were approximately twice as sensitive to cell death induced by cross-linking with anti-TNFR 1 antibody as the control cell lines (HepG2/EF) (Fig. 3b). Since the antibody specific for the human sTNFR 1 was used to induce HepG2 cell death, the enhanced cell death in core-expressing HepG2 cells was most likely mediated by TNFR 1 but not other members of the TNFR family. A similar enhanced sensitivity to TNF was observed in HeLa cells expressing core protein (Fig. 3c). In this case, the transformed cell clones were pooled, instead of being maintained as individual clones, for assays. The sensitization of HeLa cells to TNF-induced cell death by the core protein was also demonstrated by the annexin V assay (25), which detects early membrane changes associated with apoptosis (Fig. 3d). The results showed that HeLa cells expressing the core protein were threefold more sensitive to TNF-induced apoptosis than control cells. Since the pooled HeLa cell clones were used in these studies, the observed difference could not have been due to clonal variations. Based on these above results for different cell lines, we conclude that there is an approximately twofold increase in TNF-induced cell killing in the presence of core protein. These results suggest that the binding of core protein to the cytoplasmic domain of TNFR may alter the sensitivity of TNFR response to TNF.

FIG. 3.

FIG. 3

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Effects of HCV core protein expression on the sensitivity of various cell lines to TNF-induced cell death. Various stably transformed cell lines were treated with different concentrations of TNF or anti-TNFR 1 antibody as indicated for each experiment. Cells were assayed by the MTT assay (a to c) and by the annexin V assay (d). (a) BC10/CORE and control BC10/EF cells (n = 8 independent clones examined) incubated with murine TNF (mTNF). The inset shows the immunoblotting of the core protein in two representative BC10/EF and BC10/CORE clones. (b) HepG2/EF and HepG2/CORE cells (n = 4) incubated with anti-human TNFR 1 antibody. The inset shows the immunoblotting of the core protein in two representative HepG2/EF and HepG2/CORE clones. (c) HeLa/EF and HeLa/CORE cells incubated with human TNF (hTNF). The inset shows the immunoblotting of the core protein in HeLa/EF and HeLa/CORE cell pools. (d) HeLa/EF and HeLa/CORE cell clones incubated with human TNF in the annexin V apoptosis assay.

The sensitization of cells to TNF by HCV core protein is not due to up-regulation of TNFR 1.

The foregoing results for three cell lines of different origins strongly suggest that the core protein may play a general role in sensitizing cells to TNF-induced cell death. It is noteworthy that the core protein by itself is not cytotoxic, since none of the core protein-expressing cell clones showed any cytotoxicity under the culture conditions used. Previously, the core protein has been shown to either stimulate or suppress various cellular or viral promoters (32, 37). Therefore, the observed sensitization of cells by core protein to TNF may have been caused by the up-regulation of TNFR 1 expression rather than alteration in TNFR signal transduction. To rule out this possibility, we first compared TNFR 1 mRNA levels between core protein-expressing and nonexpressing HeLa cells. No significant difference in TNFR 1 mRNA levels between the two cell lines was detected (Fig. 4). We also studied the surface expression of TNFR 1 in HepG2 cells by flow cytometry analysis and found similar surface expression of TNFR 1 regardless of whether core protein was expressed (Fig. 5). We concluded that the surface expression level of TNFR 1 was not altered by core protein expression. Therefore, TNF sensitization of the core-expressing cells was not due to enhanced expression of TNFR 1 resulting from transcriptional up-regulation of TNFR 1 by the core protein.

FIG. 4.

FIG. 4

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Comparison of TNFR 1 mRNA levels in HeLa/EF and HeLa/CORE cell lines by Northern blotting. Approximately 20 μg of total RNA from each cell line was loaded in each lane. The same filter was used for hybridization with a TNFR 1 riboprobe and with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe.

FIG. 5.

FIG. 5

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Comparison of cell surface TNFR 1 expression levels in core-expressing and control HepG2 cell lines by flow cytometry analysis. The cell surface TNFR 1 was detected by a goat anti-human sTNFR 1 (α-TNFRI) as the primary antibody and an FITC-conjugated mouse anti-goat IgG (α-IgG-FITC) as the secondary antibody. Cells exposed to the secondary antibody only (left panels) were used as negative controls. Cells are plotted against their FITC intensity, and the gated percentages of TNFR 1-positive cells of each cell line are shown in parentheses.

Core protein expression suppressed the nuclear translocation of RelA in BC10ME cells but not in HeLa or HepG2 cells.

NF-κB activation, as demonstrated by the rapid nuclear translocation of its major component RelA (1), is also a major outcome of TNFR 1 activation. Therefore, we examined whether HCV core protein could affect NF-κB activation by TNF. We compared the amounts of cytoplasmic and nuclear RelA in BC10/CORE and BC10/EF cells in response to TNF by immunoblotting using the RelA-specific antibody. As expected, we observed a significant increase of nuclear RelA in response to TNF treatment in the control BC10/EF cells, indicating the activation of RelA/NF-κB by TNF (Fig. 6). In BC10/CORE cells, however, no significant increase in the nuclear RelA was detected. The amounts of actin in the various lanes were almost the same, indicating that the difference in RelA was not caused by loading difference. Our results indicated that the nuclear translocation of RelA in response to TNF was suppressed in BC10/CORE cells compared to control BC10/EF cells. In addition, the overall level of RelA expression appeared to be slightly lower in the core protein-expressing cells. Similar observations were made in at least three other independently derived BC10ME cell clones (data not shown).

FIG. 6.

FIG. 6

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Effect of HCV core protein expression on RelA activation by TNF. BC10/EF and BC10/CORE cells were treated with murine TNF (10 ng/ml, final concentration) for 20 min at 37°C and lysed with 0.5% Nonidet P-40. The nuclear (NE) and cytoplasmic (CE) fractions were used for immunoblotting analysis using antibodies specific for RelA, actin, or HCV core protein.

Similar experiments were performed on the core-expressing HeLa and HepG2 cells. However, the extents of NF-κB activation in response to TNF as measured by the increasing level of nuclear RelA were similar between core-expressing and nonexpressing cells (data not shown). Therefore, we conclude that core protein expression in HeLa and HepG2 cells did not block the activation of NF-κB. The difference between BC10ME and HeLa or HepG2 cells could have reflected differences in TNFR signal transduction pathways between murine and human cells.

DISCUSSION

In this report, we have presented evidence that the HCV core protein sensitizes cells to TNF-mediated cytolysis. This phenomenon was shown in three different cell lines. Furthermore, we demonstrated that such sensitization is not due to up-regulation of TNFR 1. The finding that core protein binds to the cytoplasmic domain of TNFR may account for the observed enhanced sensitivity to TNF. It has been well established that the cytoplasmic region of TNFR 1 interacts with a number of cellular proteins which are components of the TNFR 1-mediated signaling complex. At least four such proteins have been identified; RIP and TRAF2 are responsible for TNF-induced JNK or NF-κB activation (23), while FADD and TRADD can induce apoptosis (8, 18). These proteins bind either directly or indirectly to the death domain or its vicinity in TNFR 1. The binding of the HCV core protein to the death domain of TNFR 1, therefore, may potentially disrupt or enhance these interactions and result in alterations of TNFR 1 signaling.

Although the signaling pathways associated with the activation of TNFR 1 that lead to RelA/NF-κB activation and cell killing have been shown to represent two independent pathways (18), recent evidence indicates that RelA/NF-κB activation can block TNF-induced apoptosis in some cell types (3, 43, 44), suggesting the intertwining of the two pathways. An antiapoptotic function of NF-κB has also been demonstrated in RelA/NF-κB knockout mice, which die from extensive apoptosis in the liver during embryogenesis (4). Thus, the suppression of TNF-induced RelA/NF-κB activation by core protein may account for the sensitization of BC10ME cells to TNF-induced cytotoxicity. However, RelA/NF-κB activation was not suppressed by the core protein in HeLa or HepG2 cells. Such variations between these cell lines may reflect the difference in TNF signaling between mouse and human TNFR 1. The finding that both HeLa and HepG2 cells are sensitized by core protein to TNF-induced killing thus indicates that the core protein may affect other NF-κB-independent pathways of TNFR 1 signaling. It is interesting that the core protein-binding site (aa 345 to 407) in TNFR 1 is adjacent to, but distinct from, the major TRADD-binding site (aa 308 to 340) (17). However, the core protein-binding region includes certain sequences within the death domain that can affect the TRADD binding. For example, the aa 345 (F) of TNFR 1 is important for both core protein and TRADD binding as well as for TNFR 1-mediated cytotoxicity (17, 38). It is therefore likely that core protein can also affect other TRADD-mediated signal transduction.

There is also an indication that Fas antigen, another member of the TNFR family, plays an important role in inflammation in the HCV-infected liver (16), particularly in the active inflammation of chronic hepatitis C. Our preliminary results showed that HepG2 cells expressing core protein are also more sensitive to anti-Fas antibody-induced cell killing (data not shown), which is consistent with a recent report (35). This does not come as a surprise, for Fas and TNFR 1 have many redundant or even synergistic functions (9). It is noteworthy that both receptors have a death domain in their intracellular cytoplasmic regions, which is responsible for the induction of apoptosis. We have previously shown that the HCV core protein also binds the cytoplasmic portion of LTβReceptor (26), yet another member of the TNFR family. A recent study showed that HeLa cells expressing the HCV core protein were more sensitive than the control cells to cell killing induced by combination of LTα1β2 and gamma interferon (6). However, the sensitivity of these cells to TNF-induced cell killing was not altered, in contrast to the finding in this study. Furthermore, the sensitivity of Huh7 cells or HepG2 cells to either LTα1β2 or TNF was not affected by the expression of the HCV core protein (6). Thus, these effects were clearly cell line specific. The discrepancies between that report and our study may reflect the differences in the cell lines used and the methods by which the core protein-expressing cell lines were established. In any case, these studies together showed that the expression of HCV core protein may sensitize cells to cell killing mediated by Fas, TNFR 1, or LTβR under various in vitro conditions. The precise effects of the core protein in different cell types in vivo, however, probably will be modulated by many other physiological and pathological factors. The fact that core protein can interact with more than one member of the TNFR family suggests that the core protein may play a profound role in the pathogenesis of HCV. In addition to apoptosis, TNFR 1 can mediate other responses, such as cell proliferation and inflammation. Whether the core protein can modulate these other responses as well is an interesting question.

Our results suggest that TNF may be an important mediator of HCV pathogenesis. TNF has been shown to elicit an unusually wide range of biological responses, including inflammation, tumor necrosis, cell proliferation, differentiation, and apoptosis, primarily through TNFR 1. Clinical studies have shown that the serum TNF levels in chronic HCV patients were in the range between 3 and 20 pg/ml, which was significantly higher than normal value of 3 pg/ml or lower (28, 41). These values were within the range of TNF concentrations which showed significant difference in cell death between the core-expressing and control cells in our study. Thus, the observed effects of the core protein on the sensitivity of cells to TNF is likely physiologically meaningful. However, the core protein itself does not cause apoptosis of the cells, as permanent cell lines expressing the core protein did not show apparent abnormal growth properties. This finding suggests that the core protein may account for hepatic cell death in HCV infection by an indirect immunologically mediated mechanism. It is interesting that in chronic HCV infection, TNF secretion is elevated (22, 28, 41). The increased sensitivity of the core protein-expressing cells to TNF, coupled with the enhanced secretion of TNF in HCV patients, will make infected hepatic cells particularly vulnerable.

What selective advantage does a viral protein confer to the virus if it enhances the apoptosis of the infected cells as in the case of HCV core protein? One possible scenario is that apoptotic cells can provide an efficient means for the spread of virus after the virus has completed its replication. Viruses within apoptotic bodies can be disseminated with minimum induction of inflammatory and immune responses and without exposure to neutralizing antibodies (39). Indeed, many viral gene products have the ability to induce apoptosis (42, 45).

Several other viruses encode proteins that modulate cellular responses to TNF. For example, adenovirus E1A protein sensitizes cells to TNF-induced cytolysis (46), similar to the effects of the core protein observed here. In contrast, E1B and several E3 proteins antagonize this effect of E1A (39). Vaccinia viruses also encode proteins, e.g., CrmA, that interfere with the activity of TNF (29). Epstein-Barr virus transforming protein LMP-1 binds to TRAF3 (31), a signaling molecule associated with TNFR family members (2). These examples support the notion that TNF has general importance in virus infection.

It is interesting that immunosuppressed liver transplant patients with HCV infection usually had very high HCV load (and presumably high level of the core protein) early after transplantation, yet no liver damage was observed (5). This finding is consistent with the notion as mentioned above that the HCV core protein by itself does not have direct cytopathic effects. It is also conceivable that as for adenovirus, other HCV proteins may have properties to antagonize the effect of the core protein. The delicate balance between these proteins, together with the modulation of the immune system, would determine the pathogenic status of hepatitis C.

The results presented here indicate an intriguing role for the viral core protein in induction of cell death during HCV infection through TNF signaling pathways. The finding also has clinical relevance, as TNF has been established as one of the major mediators of cell death in chronic liver diseases (15, 19), and TNF has been reported to be elevated in chronic HCV infection (22). Thus, TNF may play a significant role in causing hepatocellular damage. The finding that core protein potentiates this effect suggests a potential target for therapy against HCV-induced hepatitis.

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