TEXT

Top Abstract Text References

Hepatitis C virus (HCV) is a positive-strand RNA virus that has been identified as the major causative agent of posttransfection non-A, non-B hepatitis (25, 51). Its persistent infection may result in chronic active hepatitis, cirrhosis, and hepatocellular carcinoma (19, 83). Intriguingly, HCV persists despite the presence of virus-specific cytotoxic T-lymphocytes (11, 20, 50, 80). The reason for the failure of host immune response to resolve HCV infection is not known. This could be due in part to the effect of viral gene products on the host immune system, as had been noted for several viruses (36). Of at least 10 viral proteins encoded by the HCV genome (10, 37, 59, 100), its nucleocapsid core protein may have such a feature.

Several studies suggested that the core protein of HCV possesses several distinguishing properties. It is phosphorylated (87) and has both cytoplasmic and nuclear localization (61, 86, 88). Additionally, the core protein has regulatory roles in viral and cellular genes and also has effects on cell growth and proliferation (21, 65, 73-77, 87, 88, 113). Recently, studies from several laboratories, including ours, have identified several cellular factors that can associate with the HCV core protein. For example, the core protein forms the complex with apolipoprotein AII of the lipid droplet, which in turn may contribute to the liver steatosis in HCV-infected chimpanzee or humans (11). The interaction between the HCV core protein and the tumor necrosis factor receptor (TNFR)-related lymphotoxin- receptor (LT-R) (3, 15, 28) was also demonstrated by two different groups (21, 65). This interaction modulates one of the biological activities, i.e., cytolytic activity, of LT-R triggering by its recombinant ligand (LT-12) (17, 18, 108) in HeLa cells but not in HuH-7 cells (21). Moreover, the HCV core protein also interacts with TNFR 1 (TNFRI) (113), although its effect on TNF-induced cytolytic activity still remains controversial (21, 76, 113). Like the TNF ligand receptor family (reviewed in references 1, 39, 97, and 103), the LT-R is also engaged in activation of the transcriptional factor NF-B and c-Jun in some cell types (22, 62, 71). Conceivably, the interaction of HCV core protein and LT-R or TNFRI may potentiate their NF-B or c-Jun N-terminal kinase (JNK) signaling pathways.

The NF-B signaling pathway is a key component of the cellular response to a variety of extracellular stimuli, including TNF-, interleukin-1 (IL-1) and phorbol ester (reviewed in references 4, 8, 98, and 105). This transcriptional factor, known to regulate a large number of genes involved in inflammatory response, cell proliferation, and apoptosis, is composed of homo- and heterodimers of Rel family proteins (reviewed in references 4, 5, and 10). These family proteins include at least the following five distinct members: C-Rel, p50, p52, p65 (RelA), and RelB; of these, the p50/p65 heterodimer is the most abundant and ubiquitous (reviewed in references 4 and 10). In the uninduced cells, NF-B is sequestered in the cytoplasm by binding to a labile IB family protein with a regulatory and inhibitory function, of which IB- and IB- appear to be the key members (106). Upon induction by several agents, including virus, inflammatory cytokines, and stresses, the intracellular signaling pathways that generally converge on IB rapid phosphorylation and/or modification and subsequent degradation in the proteasome are activated (reviewed in references 4 and 8), thus allowing NF-B complexes to enter the nucleus and activate the target genes. After degradation, the IB- is rapidly replenished by NF-B-mediated transcription of IB- gene (57, 93), which then constitutes the autoregulatory loop of NF-B-IB activation. Of note, unlike IB-, which elicits only transient NF-B activation, the IB- degradation causes a sustained activation of NF-B due to a large lag period of IB- resynthesis (99). Recent studies have identified two cytokine-inducible IB kinases (IKK), termed IKK and IKK, which appear to form heterodimers in the large multiple complex (700 kDa) and catalyze IB site-specific phosphorylation (reviewed in references 64, 90, and 105). In spite of this tight regulatory loop for NF-B activation, this transcriptional factor is activated by different viral proteins with oncogenic potential, such as human T-cell leukemia virus type 1 Tax (35, 43, 54, 92), Epstein-Barr virus latent membrane protein 1 (LMP-1) (40), the X protein of hepatitis B virus (24, 91).

The second branch of the stress response is the JNK pathway, which targets to the activation of transcriptional factor AP-1, ATF-2, and E1K-1 (29, 38, 49, 52, 53, 109). The signal transduction cascade of JNK activation is well defined and involves small GTP-binding proteins (Cdc42 and Rac), p21-activated protein kinase, and mitogen-activated protein kinase kinase kinase members (MEKK1 and MKK4) (27, 30, 56, 68, 84, 111). Many stimuli that induce NF-B, such as TNF-, UV irradiation, and lipopolysaccharide, also activate the JNK cascade (29, 79), thereby raising the possibility that the two pathways share common signal transduction components. Supporting this notion is the fact that TRAF2 and MEKK1 are two critical components of both the JNK and NF-B stress response pathways (44, 58, 67, 79, 89), although contradictory findings were also reported (60). Despite these discrepancies, these two signal pathways diverge at a discrete level. For example, while JNK and its target c-Jun are critical mediators of apoptosis induced by TNF- or ceramide (104, 110), the NF-B in general has an anti-apoptotic effect (12, 60, 101, 107).

In this study, we examined the effects of HCV core protein on the NF-B and JNK signaling pathways induced by LT-12 or TNF- cytokine. Our results as shown here indicated that HCV core protein significantly potentiates NF-B activation pathway triggering by LT-12 or TNF- in HuH-7 cells and to a lesser extent in HeLa cells. These modulation effects are mediated by the differential sensitivity of IB- and IB- degradation in the HCV core protein-producing cells, which in turn increases the nuclear retention of NF-B subunits and potentiates both basal and cytokine-treated NF-B activities. However, no significant modulation effect on JNK activation was detected in those two HCV core protein-producing cells when stimulated by LT-12 or TNF-, suggesting that these two stress responses are differentially regulated by the HCV core protein.

HCV core protein enhances the NF-B DNA-binding activity triggered by LT-₁₂ or TNF- in HeLa and HuH-7 cell lines. To elucidate whether the HCV core protein can modulate the NF-B activation stimulated by LT-₁₂ or TNF-, the levels of induction of NF-B DNA-binding activity in the nuclear extracts of both HCV core protein-expressing HeLa and HuH-7 cells (HeLa/C190 and HuH-7/C190) relative to that of their parental cells were compared at different times (30 min to 2 h) after the cytokine treatment. The NF-B DNA-binding activity was examined by the electrophoretic mobility shift assay (EMSA) with a ³²P-labeled 45-mer oligonucleotide probe (5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3') from the human immunodeficiency virus type 1 long terminal repeat containing two B-binding sites. A mutated oligonucleotide with a single mutated B site (5'-AGTTGAGGCGACTTTCCCAGGC-3') (22 mer; Santa Cruz) was also used to examine the binding specificity of NF-B by EMSA. Results suggested that addition of the LT-₁₂ ligand (500 ng/ml for 30 min to 1 h) greatly enhanced the NF-B DNA-binding activity in HCV core protein-producing HuH-7/C190 and HeLa/C190 cells compared to that in their parental cells without the core protein (Fig. 1). The induction of NF-B DNA-binding activity in LT-₁₂-treated HeLa/C190 cells peaked at 30 min and slightly declined at 60 min (Fig. 1A), while in LT-₁₂-treated HuH-7/C190 cells the induced NF-B activity was still sustained at 60 min (Fig. 1B). A similar finding was obtained with TNF--treated HeLa/C190 cells. Treatment of TNF- (20 ng/ml) in this cell line induced NF-B activity significantly at 30 min, reached plateau at 60 min, and even remained elevated at 2 h. This NF-B induction profile of HeLa/C190 cells is distinct from that observed for HeLa cells, as the NF-B activity in the HeLa cells within the same period was only slightly induced by TNF- (Fig. 2A). A distinct NF-B activation kinetics was also observed for the TNF--treated HuH-7 and HuH-7/C190 cells (Fig. 2B). Although the NF-B DNA-binding activities of these two cell lines were similar at the initial phase (30 min to 1 h) of induction, the activity of HuH-7/C190 at late phase (2 h) of treatment remained stronger than that of HuH-7 cells, suggesting a prolonged NF-B activation in core-producing cells (Fig. 2B). Interestingly, a slight enhancement of NF-B DNA-binding activity was also observed for both core-producing cells without the cytokine treatment (compare lanes 2 and 7 in Fig. 1A and B, and lanes 2 and 8 in Fig. 2A and B), implicating a constitutive activation of NF-B in core-producing cells. It should be noted that the cytokine-induced NF-B DNA-binding activity observed in these EMSAs is specific, since it was ablated by an excess of unlabeled wild-type competitor but not by the mutated one (see lanes 5, 6, 10, and 11 in Fig. 1 and lanes 6, 7, 12, and 13 in Fig. 2). Additionally, the invariant fast-migrating band was a nonspecific complex observed in NF-B EMSA studies.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   EMSA of LT-12-stimulated NF-B activation in various HCV core protein-producing cell lines. (A) NF-B DNA-binding assays with nuclear extracts from untreated (lanes 2 and 7) or LT-12-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-12 (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-B 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-B binding sites. The main NF-B-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.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   EMSA of TNF--stimulated NF-B 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-/ml for 30, 60, or 120 min, respectively.

HCV core protein differentially enhances the NF-B-dependent transcriptional activity triggered by LT-₁₂ or TNF- in HeLa and HuH-7 cells. We next assessed whether this enhancement or persistent induction of NF-B DNA-binding activity in the HCV core protein-producing cells also could reflect on the NF-B-dependent transcriptional activity. To this end, we examined the activity of luciferase reporter plasmid (NF-B-fosp-1783:3.2Luc) (kindly provided by H. Wajant, University of Stuttgart, Stuttgart, Germany) under the control of the B response element by transient transfection to HuH-7 or HeLa cells with or without the expression of core protein. Additionally, to provide a stringent control and to ensure the measured luciferase reporter plasmid activity mainly reflecting the NF-B-specific transcriptional activity, an NF-B-independent control plasmid (pCH110) (Pharmacia) containing -galactosidase reporter gene under simian virus 40 early promoter control was also cotransfected into cells, and its reporter activity was used for normalization. As shown in Fig. 3, relative to the level in the parental HeLa cells, about twofold enhancement of luciferase activity for both basal and cytokine-treated HeLa/C190 cells was noted (Fig. 3A). Likewise, the presence of core protein in HuH-7 cells exerted about 2.4-fold increase of basal NF-B-dependent luciferase activity (Fig. 3B). However, treatment with either cytokine elicited a more than fivefold increase of luciferase activity in HuH-7/C190 cells relative to that of HuH cells, indicating a stronger potentiation of core-mediated NF-B transcriptional activity in cytokine-stimulated HuH-7 cells than in cells without cytokine treatment (Fig. 3B). Therefore, coupled with the data from the EMSA (Fig. 1 and 2), our results clearly indicated that the HCV core protein can enhance the basal and cytokine-stimulated NF-B transcriptional activities in both HeLa and HuH-7 cells, although the strength or kinetics of NF-B induction may vary between cell lines.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Analysis of cytokine-stimulated NF-B-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-B-dependent luciferase reporter plasmid and an internal control plasmid carrying the -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- (20 ng/ml) or LT-12 (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 -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 -galactosidase expression. The NF-B-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.

HCV core protein does not alter the expression levels of NF-B but affects their nuclear translocation. To understand the molecular basis of the enhancement of NF-B activation in HCV core protein-producing cells, the total expression levels of NF-B in different cell lines were examined by immunoblotting using antibodies specific for the subunits of NF-B. Figures 4 reveals that although the expression levels of NF-B family proteins p50, p52, and p65 in HuH-7 cells were more abundant than those in HeLa cells, the core protein did not affect their expression levels. Furthermore, following stimulation with TNF- or LT-12 for 30 min or 1 h, there was little difference in the total expression levels of NF-B family proteins in core-producing cells of HeLa and HuH-7 compared to those of their parental cells (data not shown). However, in response to 1 h of cytokine treatment, a marked enhancement of nuclear retention of p50 and p65, but not p52, was noted in HuH-7/C190 cells relative to that in parental HuH-7 cells (Fig. 5B and D). This enhancement of p50 or p65 nuclear retention by the core protein was not so evident in cytokine-treated HeLa cells, although a slight enhancement of the nuclear level of p50 was also noted in TNF--treated HeLa/C190 cells (compare Fig. 5A and C with Fig. 5B and D). Moreover, following the cytokine treatment a different kinetics of nuclear translocation of p50 and p65 between the LT-₁₂-stimulated HeLa and HeLa/C190 cells was found: while the nuclear level of p50 or p65 in LT-₁₂-treated (1 h) HeLa/C190 cells declined to the steady level, their levels in the nuclear fractions of HeLa cells within the same period remained elevated compared to pretreatment levels (Fig. 5A). Based on these results, it appears that the molecular mechanism for NF-B activation in core-producing cells relative to that of their parental cells may differ between cell lines or cytokines. Additionally, a slight enhancement of the nuclear level of p50 was noted in both core-producing cells of HeLa or HuH-7 even without the cytokine treatment (Fig. 5). This suggested a constitutive activation of NF-B by the HCV core protein, which is in accordance with the data from the EMSA (Fig. 1 and 2) and the reporter plasmid assay (Fig. 3).

View larger version (34K): [in this window] [in a new window]   FIG. 4.   Western blot analysis of NF-B/Rel and IB 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-B p65, p52, and p50 subunit antibodies (Upstate Biotechnology Inc.) and IB- and IB- 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.

View larger version (28K): [in this window] [in a new window]   FIG. 5.   Subcellular distribution of NF-B family proteins in HCV core protein-producing cells after LT-12 or TNF- stimulation. Cells were treated with 500 ng of LT-12 ligand/ml (panels A and B) or 20 ng of TNF-/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-B family proteins (p65, p50, and p52) by immunoblotting using the ECL detection system.

HCV core protein enhances the degradation of IB- and IB- in a cell line- and cytokine-specific manner. Since both LT-₁₂ and TNF- may elicit transient NF-B activation by affecting the degradation of NF-B inhibitor IB, the expression levels of IB inhibitors in HeLa and HeLa/C190 cell lines before or after the cytokine treatment were examined by immunoblot analysis using the IB-- and IB--specific antibodies. The results shown in Fig. 4 indicate that the HCV core protein did not alter the expression of IB- and IB-. However, LT-₁₂ treatment (500 ng/ml) of HeLa/C190 but not HeLa cells caused proteolytic breakdown of IB- within 10- to 60-min time intervals and the amounts then returned to the control level by 1.5 h (Fig. 6A). This enhancement of IB- degradation did not occur in HuH-7/C190 cells. In fact, the IB- inhibitor of HuH-7 and HuH-7/C190 cells was unresponsive to LT-₁₂ stimulation (Fig. 6A). Interestingly, compared to what occurred in their parental cells, the degradation of IB- inhibitor was enhanced and/or sustained in both LT-₁₂-stimulated core-producing cells of HeLa and HuH-7 but with different kinetics. For LT-₁₂-treated HeLa/C190 cells, the IB- degradation occurred within a 0.5- to 1-h time interval, while for HuH-7/C190 cells, it occurred at a 0.5- to 2-h interval (Fig. 6B). Furthermore, similar to the case of IB-, the IB- protein in HeLa cells was unresponsive to stimulation with LT-₁₂ (Fig. 6B). However, a slight depletion of IB- was observed in LT-₁₂-treated (0.5 to 1 h) HuH-7 cells (Fig. 6B).

View larger version (40K): [in this window] [in a new window]   FIG. 6.   Degradation of IB proteins in various LT-12- or TNF--stimulated HCV core protein-producing cells. Cells were stimulated with 500 ng of LT-12 ligand/ml (panels A and B) or 20 ng of TNF-/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 IB- or IB- protein by using rabbit polyclonal antibody against human IB- or IB- (Santa Cruz) and ECL detection system.

HCV core protein does not potentiate the TNF- or LT-₁₂-stimulated JNK activity of HeLa and HuH-7 cells. Since in addition to NF-B activation, both cytokine treatments also lead to JNK activation (21, 41), we carried out experiments to determine whether the triggering of JNK activity by the cytokines was also modulated by the HCV core protein. Cytoplasmic extracts from uninduced or cytokine-treated cells were assayed for JNK activity through the immunocomplex kinase method using glutathione S-transferase-C-Jun_1-79 as the substrate (14). The results indicated that although TNF- (10 ng/ml) could induce a strong transient response (after 10 to 30 min) of JNK activation (maximum of eight- to ninefold in HeLa or HeLa/C190 cells and three- to fourfold in HuH-7 or HuH-7/C190 cells), the presence of HCV core protein in both HeLa and HuH-7 cells did not show any modulatory effect on JNK activation (data not shown). In the LT-₁₂-treated cells (500 ng/ml), the cytokine-induced JNK activity was weaker than the response induced by TNF- (maximum of 3- to fivefold in LT-₁₂-treated HeLa and HeLa/C190 cells; less than twofold in LT-₁₂-treated HuH-7 and HuH-7/C190 cells) (data not shown). Moreover, the core protein either had no effect (HuH-7/C190 cells) or slightly downregulated the LT-₁₂-induced JNK activity (HeLa/C190 cells) (data not shown). Therefore, our results suggested that unlike in NF-B activation, the HCV core protein does not potentiate JNK activation stimulated by both cytokines.

Discussion. In this study, we analyzed the mechanisms and kinetics of LT-12-stimulated NF-B activation in comparison to those of TNF-. Additionally, the NF-B signal pathways of these two stimuli in HCV core protein-producing cells (HeLa/C190 and HuH-7/C190) were also parallel to those of their parental cells. An interesting phenomenon was noted in this study. It appears that varying patterns of NF-B potentiation (in regard to the kinetics of NF-B activation, NF-B nuclear translocation, or IB degradation) between cell lines and stimuli were observed. These results suggest that the complexity of NF-B signaling pathway triggering by either stimulus and presumably the different cell type-specific pathways are responsible for this phenomenon, as has been previously noted for both the TNF- and the LT-12 system (4, 62). Despite this, our results clearly demonstrate that following either stimulus, in both cell lines expressing HCV core protein the IB- steady-state level substantially declined in parallel with the increase of NF-B-DNA binding activity and the nuclear translocation of the p65 and p50 NF-B species (Fig. 1, 2, 5, and 6). However, unlike that observed with IB-, the increase in IB- turnover appeared only in LT-12-stimulated HeLa/C190 cells; in HuH-7 cells with or without the HCV core protein, the LT-12 ligand failed to stimulate IB- turnover, and no apparent enhancement of IB- degradation was found in TNF--triggering core-producing cells relative to the level in parental cells (Fig. 6). Our results also indicated that unlike the p50 and p65 members, the nuclear translocation of p52 is inert to both cytokine stimuli (Fig. 5) and may be irrelevant to the mechanism of NF-B activation by these agents. These findings suggest that the enhancement of NF-B-dependent transcriptional activity following cytokine stimulation in both core-producing cells (Fig. 3) may be mediated by a complex mechanism involving the deregulation of various cytoplasmic inhibitors of NF-B, which may differ between the stimuli and the cell lines.