Hepatitis C Virus Core Protein Differently Regulates the JAK-STAT Signaling Pathway under Interleukin-6 and Interferon-{gamma} Stimuli*

Atsushi Hosui {ddagger}, Kazuyoshi Ohkawa {ddagger}, Hisashi Ishida §, Aki Sato §, Fumihiko Nakanishi {ddagger}, Keiji Ueda ¶, Tetsuo Takehara §, Akinori Kasahara ||, Yutaka Sasaki §, Masatsugu Hori {ddagger} and Norio Hayashi § **

From the {ddagger}Department of Internal Medicine and Therapeutics, §Department of Molecular Therapeutics, Department of Microbiology, and ||Department of General Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Received for publication, October 13, 2002 , and in revised form, May 14, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We established hepatitis C virus (HCV) core-expressing cells and investigated whether HCV core would modify the Janus kinase (JAK)-signal transducer and activator transcription factor (STAT) pathway under interleukin-6 (IL-6) and interferon (IFN)-{gamma} stimuli. Phosphorylation of JAK1/2 and STAT3, and STAT3-mediated transcription, were prevented by HCV core under IL-6 stimulation. In contrast, HCV core increased phosphorylation of JAK1/2 and STAT1 and STAT1-mediated transcription under IFN-{gamma} stimulation. Immunoprecipitation/Western blot analysis showed that HCV core could bind to JAK1/2. The PGYPWP sequences at codons 79–84 within HCV core were important for interaction with JAKs by in vitro binding analysis. In the reporter gene assay, HCV core-mediated suppression of JAK-STAT pathway under IL-6 stimulation was not observed by abrogation of PGYPWP sequence, suggesting that HCV core/JAK interaction may directly affect the signal transduction. In contrast, augmentation of JAK-STAT pathway was still seen by HCV core without functional PGYPWP sequence under IFN-{gamma} stimulation. Flow cytometric analysis revealed that HCV core up-regulated of IFN-{gamma} receptor 2 expression, which may be responsible for HCV core-mediated enhancement of JAK-STAT pathway under IFN-{gamma} stimulation. In conclusion, HCV core has different effects on the JAK-STAT pathway under IL-6 and IFN-{gamma} stimuli. This may be exerted by these two independent mechanisms.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis C virus (HCV)1 often causes persistent infection and leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 2). HCV has a positive-stranded RNA genome composed of ~9,400 nucleotides and contains a large open reading frame (ORF) (3). The precursor protein encoded in the ORF is cleaved by viral and cellular proteases and results in production of at least 10 viral proteins (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (4, 5). Recent experimental evidence suggests that the HCV core protein affects many biological functions in the host cell, such as cellular growth, malignant transformation, and apoptosis, which may be involved in HCV-related liver diseases. Overexpression of the HCV core protein has been reported to lead to malignant transformation of rat fibroblasts in cooperation with H-ras (6, 7). Moriya et al. (8) also revealed that hepatocellular carcinoma was induced in HCV core protein-expressing transgenic mice. In addition, the HCV core protein has been shown to alter the cellular apoptotic process under various stimuli (913).

To understand the molecular mechanisms through which such biological functions would be exhibited, the modulation effect of HCV core protein on various cellular signaling pathways has been studied by several investigators. Tsuchihara et al. (14) showed that the HCV core protein enhanced the mitogen-activated protein kinase pathway under epidermal growth factor stimulation. It has also been demonstrated by Aoki et al. (15) that the activation of Raf-1 kinase, a member of the mitogen-activated protein kinase kinase family, was exerted by binding of the HCV core protein to 14-3-3{epsilon} protein. Zhu et al. (13) reported that the HCV core protein became bound to the tumor necrosis factor receptor 1, resulting in enhanced tumor necrosis factor-{alpha}-induced apoptosis by modulating the tumor necrosis factor-{alpha}-mediated signal transduction pathways. Furthermore, it has recently been shown by Yoshida et al. (16) that the HCV core protein modifies the NF-{kappa}B-mediated signal transduction pathway.

The Janus kinase (JAK)-signal transducer and activator transcription factor (STAT) signaling pathway is known to be a major cascade associated with the signal transduction for many cytokines and growth factors. The interactions of these cytokines with specific surface receptors trigger the activation of JAK and then STAT. The activation of JAK and STAT depends on the phosphorylation at the specific tyrosine residues. There are four JAK proteins (JAK1–3 and Tyk2) and seven STAT proteins (STAT1–4, -5a, -5b, and -6). After phosphorylation of JAK and STAT, the STAT is dimerized, resulting in nuclear translocation. Most STAT dimers recognize the DNA element with the consensus sequence 5'-TT(N4–6)AA-3' and regulate transcription of many STAT-responsive genes (reviewed in Refs. 1722). For example, IL-6 stimulation leads to the phosphorylation of JAK1, JAK2, and STAT3, followed by nuclear translocation of the STAT3 homodimer (23, 24). In the case of IFN-{gamma}, JAK1, JAK2, and STAT1 phosphorylation occurs, followed by STAT1 homodimerization and its nuclear translocation (25). As for IFN-{alpha}/{beta}, the stimulation results in binding of the interferon stimulatory response element (ISRE) with the sequence 5'-AGTTTN3TTTCC-3', through the activation of JAK1, Tyk2, STAT1, and STAT2 and the conformation of interferon-stimulated gene factor 3 composed of STAT1, STAT2, and interferon regulatory factor 9 (26, 27). A few investigators have so far studied the influence of HCV expression on the JAK-STAT signaling pathway in the host cell. Naganuma et al. (28) revealed the enhancement of the IFN-{alpha}-inducible 2',5'-oligoadenylate synthetase gene expression by HCV core protein. It has also been shown by Markus et al. (29) that expression of the full-length of HCV ORF suppressed the IFN-{alpha}-mediated STAT/DNA binding activity. However, the detailed molecular mechanisms of HCV core-mediated modification on the JAK-STAT signaling pathway have not been fully elucidated.

In the present study, we investigated the influence of the HCV core protein on the JAK-STAT signaling pathway under IL-6 and IFN-{gamma} stimuli using murine cells constitutively expressing HCV core protein. The results of this study demonstrated that the expression of HCV core protein has different effects on the JAK-STAT signaling pathway; it causes inhibition in the case of IL-6 treatment or enhancement in the case of IFN-{gamma} treatment of JAK-STAT activation. We also showed that such HCV core-mediated modulation of JAK-STAT signaling pathway was exerted by the two mechanisms; its binding to the JAK protein and the up-regulation of the IFN-{gamma} receptor 2 (IFNGR2) expression.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs—The mammalian expression vector pc/3EFpro was constructed from pcDNA3 (Invitrogen, Tokyo, Japan) by replacing the cytomegalovirus promoter sequence with the elongation factor (EF)-1 promoter sequence in front of the multicloning site. The HCV core expression plasmid pc/3EF{Delta}NCTH carried the entire core region and the part of the E1 region of the genotype 1b HCV strain downstream of the EF-1 promoter (30). Plasmid pCore (1–191)-V5 was made up from the plasmid pcDNA3.1/V5-His A (Invitrogen) by inserting the entire HCV core region (amino acid positions 1–191) downstream of the cytomegalovirus and T7 promoters. Both plasmid constructs, pCoreMut-V5 and pCoreDel-V5, were generated from pCore (1–191)-V5 by site-directed mutagenesis. pCoreMut-V5 possessed the AGYAWA sequences instead of the PGYPWP ones at amino acid positions 79–84, whereas pCoreDel-V5 lacked the PGYPWP amino acid residues of the HCV core protein. Plasmids pCore (1–173)-V5 and pCore (1–122)-V5 carried the part of the HCV core gene (amino acid positions 1–173 and 1–122, respectively) downstream of the cytomegalovirus and T7 promoters. Plasmids pAPRELuci and pISRELuci contained the three repeats of the acute phase response elements (APREs) and ISRE upstream of the minimal mouse junB promoter and luciferase gene (31, 32). Plasmid pGASLuci, which included the two repeats of the {gamma}-interferon activation site (GAS) and the minimal promoter from the herpes simplex virus thymidine kinase gene upstream of the luciferase gene, was purchased from Clontech (Palo Alto, CA). Plasmid pRLtk, the seapansy luciferase expression plasmid, was obtained from Promega Co. (Madison, WI).

Cell Culture and Transfection—A murine normal liver cell line, BNL CL. 2 (CL2) (33), was maintained in the Dulbecco's modified Eagle's medium (Sigma, Tokyo, Japan) supplemented with 10% fetal calf serum, 100 µg/ml streptomycin sulfate, 100 units/ml penicillin G sodium, and 0.25 µg/ml of amphotericin B in 5% CO2 at 37 °C. For the establishment of cells constitutively expressing HCV core protein, CL2 cells were transfected with pc/3EF{Delta}NCTH using Lipofectin (Invitrogen) (34). Next, the cells were cultured in the presence of 800 ng/ml G418 (Invitrogen). After 14 days, G418-resistant colonies were isolated. Finally, three clones of HCV core-expressing cells were established (designated CL2 core-I, -II, and -III). In the present study, the CL2 core-I cell was mainly used for subsequent experiments. In some experiments, the results were confirmed in CL2 core-II, and -III cells. As a negative control, CL2 cells were transfected with pc/3EFpro and cultured in the presence of 400 ng/ml G418 without the colony isolation (designated CL2 mock). In this study, a human hepatoma cell line, HepG2 (35), was also maintained in the same condition as CL2 cells. For the transient transfection study, 2 x 106 cells were seeded on a 10-cm culture dish and transfected with pc/3EF{Delta}NCTH using Lipofectin. Next, the cells were incubated in serum-free medium (Opti-MEM, Invitrogen), containing the DNA-Lipofectin complex. Twenty-four hours after transfection, the medium was removed, followed by further culture in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 24 h. Finally, the cells were lysed and subjected to the subsequent experiments. Cells transfected with pc/3EFpro were also used as a negative control.

Reporter Gene Assay—For the reporter gene assay using cells constitutively expressing HCV core protein, 8.0 x 104 CL2 mock or CL2 core-I cells were seeded in the 6-well culture dishes and transfected with 1.5 µg of the reporter plasmid (pAPRELuci for IL-6 stimulation, pGASLuci for IFN-{gamma} stimulation, or pISRELuci for IFN-{alpha}/{beta} stimulation) and 0.1 µg of pRLtk using Lipofectin. Next, the cells were incubated in Opti-MEM (Invitrogen) containing the DNA-Lipofectin complex. Twelve hours after transfection, the medium was removed, followed by further culture in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 24 h. For the last 6 h, the cells were stimulated with 10 or 100 ng/ml of murine IL-6 (Pepro Tech, London, UK), 20 or 200 units/ml of murine IFN-{gamma} (Pepro Tech), 50 or 500 units/ml of murine IFN-{alpha}/{beta} (Sigma), or left unstimulated. In the cotransfection experiment using CL2 and HepG2 cells, 8.0 x 104 CL2 or 2.0 x 105 HepG2 cells were seeded in the 6-well culture dishes and cotransfected of 0.75 µg of the effector plasmid (pCore (1–191)-V5, pCoreMut-V5, pCoreDel-V5, pCore (1–173)-V5, pCore (1–122)-V5, or pcDNA3.1/V5-HisA), with 0.75 µg of the reporter plasmid (pAPRELuci or pGASLuci) and 0.1 µg of pRLtK. For the last 6 h, the cells were stimulated with 100 ng/ml murine or human IL-6 (Pepro Tech), 200 units/ml of murine or human IFN-{gamma} (Pepro Tech), or left unstimulated. Finally, the cells were lysed and subjected to the dual luciferase assay (Toyo Ink Co., Ltd., Tokyo, Japan). Both firefly and seapansy luciferase activities were measured with a luminometer (Lumat LB9507, EG&G Berthold, Bad Wildbad, Germany), and the firefly luciferase activity was normalized for transfection efficiency based on the seapansy luciferase activity. The relative light unit of the unstimulated sample was regarded as 1, and the fold activity of each sample was calculated. All assays were done in triplicate.

Western Blot Analysis—For the detection of HCV core protein, CL2 mock and core-I cells at a confluent state were lysed in low salt buffer (10 mM HEPES (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 mM aprotinin, leupeptin, and pepstatin) at 4 °C for 5 min. After centrifugation for 5 min at 5,000 rpm, the supernatant was used as the cytoplasmic fraction for subsequent experiments. On the other hand, the pellet was suspended in high salt buffer (50 mM HEPES (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 mM aprotinin, leupeptin, and pepstatin, 5 mM MgCl2, and 2% glycerol) at 4 °C for 30 min. Next, the sample was centrifuged at 4 °C for 15 min at 15,000 rpm. The supernatant was collected as a nuclear function. Both cytoplasmic and nuclear fractions were used for the Western blot analysis with mouse monoclonal HCV core antibody (East Coast Biologics, Inc., North Berwick, MN).

For the detection of JAK1, JAK2, STAT1, STAT3, tyrosine phosphorylated JAK1 (pY1022,1023JAK1; pJAK1), JAK2 (pY1007,1008JAK2; pJAK2), STAT1 (pY701STAT1; pSTAT1), and STAT3 (pY705STAT3; pSTAT3), the CL2 mock and core-I cells were stimulated with 100 ng/ml murine IL-6 or 200 units/ml of murine IFN-{gamma} and then lysed with buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na2P2O7, 0.1% 2-mercaptoethanol, 1% Triton X-100, 10 mM {beta}-glycerophosphate, 0.5 mM sodium vanadate, 1 mM aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride. Antibodies against JAK1, JAK2, and pSTAT1 were purchased from Upstate Biotechnology (Lake Placid, NY), and antibodies against pJAK1 and pJAK2 were from BioSource (Camarillo, CA). Antibodies against pSTAT3 were obtained from Cell Signaling Technology (Beverly, MA), and antibodies against STAT1 and STAT3 were from Santa Cruz Biotechnology (Santa Cruz, CA). As for the Western blot analysis, the sample was mixed with 5x sample buffer containing 15% 2-mercaptoethanol, 15% sodium dodecyl sulfate (SDS), 1.5% bromphenol blue, and 50% glycerol, separated with SDS-polyacrylamide gel electrophoresis (PAGE), and blotted onto polyvinylidene difluoride membrane (Hybond P; Amersham Biosciences, Buckinghamshire, UK). After blocking with milk, the membrane was incubated with the first antibody, followed by incubation with horse-radish peroxidase-labeled immunoglobulin as a second antibody. Finally, the immune complex was detected by an enhanced chemiluminescent assay (Super Signal, Pierce).

Protein-Protein Interaction Analysis—To examine the binding of the JAK protein to HCV core protein, the immunoprecipitation/Western blot analysis was applied in this study. From CL2 mock, CL2 core-I, and transiently transfected HepG2 cells, the cellular protein was extracted by the method described above. The lysates were precleared by the incubation with protein A-Sepharose beads (Amersham Biosciences, Uppsala, Sweden) at 4 °C for 1 h. Next, the sample was incubated with the beads coupled to the JAK1, JAK2, STAT1, or STAT3 antibody, or the rabbit nonspecific {gamma}-globulin (Dako, Glostrup, Denmark) for 18 h. The immune complex were eluted by being boiled for 5 min. Finally, the supernatant was used for Western blot to detect the HCV core protein as described above.

To confirm the binding site of HCV core protein to JAK proteins, HCV core proteins possessing the wild-type or the mutated amino acid sequences were synthesized from the plasmid pCore (1–191)-V5, pCoreMut-V5, or pCoreDel-V5 by the in vitro translation method. The reaction was performed with the TNT T7 coupled reticulocyte lysate system (Promega) and labeled with L-[35S]methionine (Amersham Biosciences). The cellular lysates from CL2 and HepG2 cells were extracted and mixed with the in vitro synthesized various types of the HCV core proteins. Then, the mixture was subjected to the immunoprecipitation using antibodies against JAK1/2 as above. Finally, the immunoprecipitate was separated with SDS-PAGE, and the gel was dried and autoradiographed.

Northern Blot Analysis—The expression levels of interferon regulatory factor (IRF)-1, fibrinogen {beta}-chain, and IFNGR2 mRNAs were analyzed by Northern blot analysis. The total cellular RNA was extracted from CL2 mock and core-I cells using an Isogen kit (Nippon Gene Co., Toyama, Japan) based on the guanidine-isothiocyanate method. The RNA sample were denatured and electrophoresed, followed by the transfer to the nylon membrane (Hybond N; Amersham Biosciences). The membrane was then hybridized with the 32P-labeled cDNA probe at 65 °C for 24 h, washed, and autoradiographed. The band intensity was quantitated with an image analyzer (BAS 2500, Fuji Film, Tokyo, Japan).

Flow Cytometry—CL2 mock and core-I cells were suspended in 100 µl of phosphate-buffered saline supplemented with 2% bovine serum albumin and 0.1% sodium azide and incubated in the presence or absence (for negative control) of the specific antibody at 4 °C for 1 h. Antibodies against mouse gp-80, gp-130, IFN-{gamma} receptor 1 (IFNGR1), and IFNGR2 were purchased from Santa Cruz. Then, cells were washed and resuspended in 100 µl of phosphate-buffered saline with 10 µl of fluorescein isothiocyanate-labeled anti-rabbit IgG antibody (Santa Cruz, CA). Finally, samples were fixed with 1% parafolmaldehyde and subjected to the flow cytometry. The expression level of the proteins on the cell surface was quantitated by calculating the mean fluorescein intensities.

Statistical Analysis—Statistical analysis was performed using the non-paired t test as appropriate. p values less than 0.05 were considered to be statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
HCV Core Protein Expression in Stably Transfected Cells—To investigate the influence of HCV core protein expression on host cellular phenotype, we tried to establish cell lines constitutively expressing HCV core protein. We first used the human hepatoma cell lines Huh-7 (36), Hep3B (35), HepG2 (35), but HCV core-expressing cells could not be well established from these cells. Therefore, we next chose CL2 cells, a murine normal liver cell line, as the parent cell. The expression of HCV core protein was stable within at least 20 passages (R60 days) in these cells. Fig. 1 shows the expression of HCV core protein in CL2 mock and core-I cells. In the CL2 core-I cells, the 21-kDa HCV core protein was observed mainly in the cytoplasm and a small portion in the nucleus. HCV core protein was also clearly detectable in CL2 core-II and -III cells (data not shown). On the other hand, the HCV core protein was not detected in the CL2 mock cells.



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FIG. 1.
Expression of HCV core protein in stably transfected CL2 cells. The HCV core protein was not detected in the CL2 mock cell (lanes 1 and 3), whereas the 21-kDa of HCV core protein was detectable in both cytoplasmic (lane 2) and nuclear (lane 4) fractions of the CL2 core-I cell.

 

Modulation of STAT-mediated Transcriptional Activity under IL-6 and IFN Stimuli by HCV Core Protein—To examine whether the expression of HCV core protein would affect the activation of JAK-STAT signaling cascade under various types of cytokines stimulation, we first performed the reporter gene assay by transient transfection of the plasmids pAPRELuci (for IL-6 stimulation), pGASLuci (for IFN-{gamma} stimulation), or pISRELuci (for IFN{alpha}/{beta} stimulation). The induction level of APRE-dependent transcription activity under 100 ng/ml of IL-6 was 2.6-fold lower in CL2 core-I cells than in the mock cells (Fig. 2A). On the other hand, 200 units/ml of IFN-{gamma} stimulation led to 2.2-fold higher induction of GAS-dependent transcription activity in the CL2 core-I cells than in the mock cells (Fig. 2B). In the case of IFN-{alpha}/{beta} stimulation, no significant differences in the induction of ISRE-dependent transcription activity between CL2 mock and core-I cells (Fig. 2C). CL2 core-II and -III cells displayed the same tendency for the luciferase activities as CL2 core-I cells (data not shown). Next, the expression levels of the fibrinogen {beta}-chain and the IRF-1 mRNAs were studied as IL-6/APRE-responsive and IFN-{gamma}/GAS-responsive genes, respectively. As shown in Fig. 2D, the expression of fibrinogen {beta}-chain was almost absent in both cells before IL-6 stimulation. But its induction level was ~2.5-fold lower in the CL2 core-I cells than in the mock cells. As for expression of IRF-1 mRNA, the induction level after IFN-{gamma} stimulation was 2.1-fold higher in the CL2 core-I cells than in the mock cells (Fig. 2E). Taken together, the activation of JAK-STAT signaling cascades was altered significantly by HCV core protein under stimulation with IL-6 and IFN-{gamma}, but not with IFN-{alpha}/{beta}.



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FIG. 2.
Reporter gene assay and Northern blot analysis to examine JAK-STAT-mediated transcription activity under stimulation with IL-6, IFN-{gamma}, or IFN-{alpha}/{beta} in CL2 mock and core-I cells. A–C, reporter gene assay. CL2 mock and core-I cells were cotransfected of pAPRELuci, pISRELuci, or pGASLuci with pRLtK. The cells were then stimulated with IL-6, IFN-{gamma}, IFN-{alpha}/{beta}, or left unstimulated and subjected to the dual luciferase assay. The firefly luciferase activity was normalized for transfection efficiency based on the seapansy luciferase activity. The relative light unit of the unstimulated sample was considered as 1, and the fold activity of each sample was calculated. The data were expressed as mean ± S.D. A, luciferase activity controlled by APRE after IL-6 stimulation. B, luciferase activity controlled by GAS after IFN-{gamma} stimulation. C, luciferase activity controlled by ISRE after IFN-{alpha}/{beta} stimulation. *, p < 0.05 by the non-paired t test. D and E, Northern blot analysis. D (upper), changes in the mRNA level of the IL-6/APRE-responsive fibrinogen {beta}-chain in CL2 mock and core-I cells before and after IL-6 stimulation. The relative band intensity determined by an image analyzer is also shown. E (upper), changes in the mRNA level of the IFN-{gamma}/GAS-responsive IRF-1 in CL2 mock and core-I cells before and after IFN-{gamma} stimulation. The relative band intensity determined by an image analyzer is also shown. D and E (lower), ribosomal 18 (18S) and 28 S (28S) indicate similar amounts of RNA loaded on gels.

 

Influence of HCV Core Protein on Tyrosine Phosphorylation of JAK and STAT Proteins by IL-6 Stimulation—The reporter gene assay and Northern blot analysis revealed that the expression of HCV core protein modulated the JAK-STAT signaling cascade differently under stimulation with IL-6 or IFN-{gamma}. We thus examined the change in the level of tyrosine-phosphorylated JAK and STAT proteins before and after IL-6 stimulation in CL2 mock and core-I cells. As shown in Fig. 3A (upper panel), the degree of JAK1 phosphorylation after the stimulation was reduced in the CL2 core-I cells, compared with the mock cells. The induction level of JAK2 phosphorylation after IL-6 stimulation was also lower in the CL2 core-I cells than in the mock cells (data not shown). As for the phosphorylation of STAT3, it was also lower in the CL2 core-I cells than in the mock cells (Fig. 3A, lower panel). This tendency of phosphorylation status of JAK and STAT proteins under IL-6 stimulation was also seen in CL2 core-II and -III cells (data not shown). When the expression levels of the whole JAK1, JAK2, and STAT3 proteins were compared between CL2 mock and core-I cells (Fig. 3B), their levels were the same irrespective of IL-6 stimulation in both cells. Also, the expression of HCV core protein did not considerably affect the expression levels of JAK1, JAK2 and STAT3. We also studied the STAT3/DNA binding activity in the nuclear extracts from CL2 mock and core-I cells after IL-6 stimulation using the double-stranded DNA probe corresponding to the APRE. The STAT3/DNA binding activity was considerably reduced in the CL2 core-I cells, compared with the CL2 mock cells at both 1 and 6 h after IL-6 stimulation (data not shown).



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FIG. 3.
Detection of tyrosine phosphorylated or whole JAK and STAT proteins under IL-6 stimulation in CL2 mock and core-I cells. A, changes in the pJAK1 and pSTAT3 levels in CL2 mock and core-I cells before and after IL-6 stimulation as detected by Western blot analysis. B, expression of whole JAK1 and STAT3 proteins in CL2 mock and core-I cells before and after IL-6 stimulation as detected by Western blot analysis.

 

Influence of HCV Core Protein on Tyrosine Phosphorylation of JAK and STAT Proteins by IFN-{gamma} Stimulation—The changes in the pJAK1, pJAK2, and pSTAT1 levels before and after the IFN-{gamma} stimulation were further examined in both CL2 mock and core-I cells. As shown in Fig. 4A, the induction levels of the JAK1 (upper panel) and JAK2 (middle panel) phosphorylation after IFN-{gamma} stimulation were stronger in the CL2 core-I cells than in the mock cells. As for changes in the STAT1 phosphorylation, its induction level was higher in the CL2 core-I cells than in the mock cells (Fig. 4A, lower panel). CL2 core-II and -III cells displayed the same tendency for the phosphorylation status of JAK and STAT proteins under IFN-{gamma} stimulation as CL2 core-I cells (data not shown). We also investigated the expression levels of the whole JAK1, JAK2, and STAT1 proteins in CL2 mock and core-I cells (Fig. 4B), resulting in the same expression levels of these proteins irrespective of IFN-{gamma} stimulation. As for the influence of HCV core on the expression levels of these proteins, it did not substantially affect their expression levels. In the nuclear extracts from CL2 mock and core-I cells after IFN-{gamma} stimulation, the EMSA was also performed to examine the STAT1/DNA binding activity using the double-stranded DNA probe corresponding to the GAS. The STAT1/DNA binding activity was considerably enhanced in the CL2 core-I cells, compared with the CL2 mock cells at both 1 and 6 h after IFN-{gamma} stimulation (data not shown).



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FIG. 4.
Detection of tyrosine phosphorylated or whole JAK and STAT proteins under IFN-{gamma} stimulation in CL2 mock and core-I cells. A, changes in the pJAK1, pJAK2, and pSTAT1 levels in CL2 mock and core-I cells before and after IFN-{gamma} stimulation as detected by Western blot analysis. B, expression of whole JAK1, JAK2, and STAT1 proteins in CL2 mock and core-I cells before and after IFN-{gamma} stimulation as detected by Western blot analysis.

 

Binding of HCV Core Protein to JAK Proteins—Our results showed that the constitutive expression of HCV core protein caused inhibition (in the case of IL-6 treatment) or enhancement (in the case of IFN-{gamma} treatment) of tyrosine phosphorylation of JAK and STAT proteins, resulting in modification of the STAT-mediated transcription. This would be responsible for the direct effect of JAK activity by the HCV core protein, because the tyrosine phosphorylation of JAK has been shown to be a primary event of the JAK-STAT signaling cascade (1722). Therefore, we examined the binding of HCV core protein to the JAK and STAT proteins in the CL2 core-I cells by means of the immunoprecipitation/Western blot analysis (Fig. 5A). The HCV core protein was not found in the negative control sample using nonspecific {gamma}-globulin (lane 1). On the other hand, the HCV core protein was detected in the immunoprecipitates using antibodies against JAK1 (lanes 2–4), JAK2 (lanes 5–7), and Tyk2 (data not shown), but not detected in those using antibodies against STAT1 (lanes 8–10) and STAT3 (lanes 11–13). This finding indicates the possible binding of HCV core protein to murine JAK proteins. Similar results were observed in cells stimulated with IL-6 (lanes 3, 6, 9, and 12) or IFN-{gamma} (lanes 4, 7, 10, and 13), suggesting that the binding of HCV core protein to JAK proteins may occur in a phosphorylation-independent manner. To confirm the interaction between HCV core protein and human JAK proteins, we further carried out the assay using HepG2 extracts transiently transfected with HCV core-expressing plasmid (Fig. 5B). The HCV core protein was found in the immunoprecipitates using antibodies against JAK1 (lane 4), JAK2 (lane 5), and Tyk2 (data not shown), but not detected in those using nonspecific {gamma}-globulin (lane 3) and antibodies to STAT1 and STAT3 (data not shown).



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FIG. 5.
Binding of murine or human JAK proteins to HCV core protein in CL2 core-I or HepG2 cells. A, the cellular lysates from CL2 core-I cells were immunoprecipitated with the nonspecific {gamma}-globulin (lane 1) and antibodies against JAK1 (lanes 2–4), JAK2 (lanes 5–7), STAT1 (lanes 8–10), and STAT3 (lanes 11–13), and the immunoprecipitates were subjected to the Western blot analysis to detect the HCV core protein. Lanes 2, 5, 8, and 11, cells without cytokine stimulation; lanes 3, 6, 9, and 12, cells stimulated with IL-6 for 1 h; lanes 4, 7, 10, and 13, cells stimulated with IFN-{gamma} for 1 h. B, lanes 1 and 2, HepG2 cells were transiently transfected with pc/3EFpro (lane 1, negative control) or pc/3EF{Delta}NCTH (lane 2, positive control), and cellular lysates were subjected to Western blot analysis to detect HCV core protein without immunoprecipitation. Lanes 3–5, HepG2 cells were transiently transfected with pc/3EF{Delta}NCTH, and cellular lysates were immunoprecipitated with nonspecific {gamma}-globulin (lane 3) and antibodies against JAK1 (lane 4) and JAK2 (lanes 5). The immunoprecipitates were subjected to Western blot analysis to detect HCV core protein.

 

Detection of the Interaction Site of HCV Core Protein to JAK Proteins—It has been shown that various authentic JAK-binding proteins have the proline-rich PXXPXP sequence, designated box 1, as an interaction site with the JAK protein (3741). HCV core protein also bears the box 1-like PGYPWP sequence at amino acid positions 79–84. Therefore, this region would be important for the binding of HCV core protein to JAKs. To confirm this possibility, we synthesized the mutant HCV core protein having three proline to alanine amino acid substitutions within the box 1-like region, and the deleted HCV core protein lacking the 6 amino acid residues of box 1-like region, as well as the wild-type HCV core protein, by means of the in vitro translation method (Fig. 6A). Similar levels of wild-type, mutant and deleted HCV core proteins were synthesized in vitro from plasmids pCore (1–191)-V5, pCoreMut-V5 and pCoreDel-V5, respectively (Fig. 6B). Then, these HCV core proteins were mixed with the cellular lysate and immunoprecipitated with the antibody against JAK1 or JAK2. As shown in Fig. 6C, only wild-type HCV core protein was detected, whereas mutant and deleted HCV core proteins were not detectable in the immunoprecipitates using the JAK1 (lanes 1–3) or JAK2 (lanes 4–6) antibody. This result was obtained in both experiments using the murine CL2 lysate (Fig. 6C, upper panel) and the human HepG2 lysate (Fig. 6C, lower panel). According to these findings, the PGYPWP sequence of the HCV core protein may play a crucial role in its binding to both murine and human JAK proteins.



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FIG. 6.
Detection of the interaction site between HCV core and JAK proteins. A, schematic presentation of wild-type, mutant, and deleted HCV core-expressing plasmid constructs, pCore (1–191)-V5, pCoreMut-V5, and pCoreDel-V5. B, aliquots of in vitro translation products from pCore (1–191)-V5 (lane 1), pCoreMut-V5 (lane 2), and pCoreDel-V5 (lane 3) were fractionated by SDS-PAGE. The reaction was labeled by L-[35S]methionine. C, in vitro synthesized HCV core proteins were mixed with the cellular lysate and immunoprecipitated with antibodies against JAK1 (lanes 1–3) and JAK2 (lanes 4–6). Then, the immunoprecipitates were fractionated by SDS-PAGE. Upper, murine CL2 lysate. Lower, human HepG2 lysate.

 

Direct Effect on JAK-STAT Signaling Pathway Caused by the HCV Core-JAK Interaction under IL-6 and IFN-{gamma} Stimuli—To examine whether the interaction between HCV core protein and JAKs would directly affect the activation of the JAK-STAT signaling cascade under IL-6 and IFN-{gamma} stimuli, we conducted the reporter gene assay in both murine CL2 and human HepG2 cells by means of cotransfection of various effector plasmids with the reporter plasmid. As effector plasmids, pCoreMut-V5 and pCoreDel-V5 encoding the HCV core protein without the functional JAK-binding site, as well as pCore (1–191)-V5 and the empty vector pcDNA3.1/V5-HisA, were used for this experiment. In the CL2 cells under the stimulation with 100 ng/ml of IL-6 (Fig 7A), the APRE-dependent transcription activity was 1.8-fold lower by transfection with pCore (1–191)-V5 than by that with pcDNA3.1/V5-HisA, as was the case for the experiment using CL2 mock and core-I cells (see Fig. 2A). In the case of transfection with pCoreMut-V5 or pCoreDel-V5, however, the APRE-dependent transcription activity was restored to the level of transfection with pcDNA3.1/V5-HisA. HepG2 cells also displayed the same tendency for the APRE-dependent transcription activity under IL-6 stimulation as the CL2 cells (Fig. 7B). These results indicate that HCV core-mediated inhibition of JAK-STAT signaling pathway under IL-6 stimulation may be directly caused by the binding of HCV core to the JAK proteins.



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FIG. 7.
Reporter gene assay to examine the direct effect of HCV core-JAK interaction on JAK-STAT-mediated transcription activity under IL-6 stimulation. CL2 or HepG2 cells were cotransfected of various effector plasmids (pcDNA3.1/V5-His A, pCore (1–191)-V5, pCoreMut-V5, and pCoreDel-V5), with pAPREluci and pRLtk. The cells were then stimulated with 100 ng/ml murine or human IL-6, or left unstimulated, and subjected to the dual luciferase assay. The firefly luciferase activity was normalized for transfection efficiency based on the seapansy luciferase activity. The relative light unit of the unstimulated sample was considered as 1, and the fold activity of each sample was calculated. The data were expressed as mean ± S.D. A, CL2 cells. B, HepG2 cells. *, p < 0.05 by the non-paired t test.

 

As for the experiment using the CL2 cells under 200 units/ml of IFN-{gamma} stimulation (Fig. 8A), the GAS-dependent transcriptional activity was 2.2-fold higher by transfection with pCore (1–191)-V5 than by that with pcDNA3.1/V5-HisA, as was the case for the experiment using CL2 mock and core-I cells (see Fig. 2B). Unlike the case of IL-6 stimulation, transfection with pCoreMut-V5 or pCoreDel-V5 revealed the same level of the GAS-dependent transcription activity as that with pCore (1–191)-V5. This tendency of the transcription activity under IFN-{gamma} stimulation was also seen in the HepG2 cells (Fig. 8B). Thus, the mutated or deleted HCV core protein, which was not capable of binding to the JAKs, could still activate the JAK-STAT signaling pathway under IFN-{gamma} stimulation. Therefore, HCV core-mediated enhancement of JAK-STAT signaling pathway under IFN-{gamma} stimulation may not be responsible for the HCV core-JAK interaction. To determine the region of HCV core protein contributing to the augmentation of the JAK-STAT signaling pathway under IFN-{gamma} stimulation, we further constructed two plasmids encoding the C-terminal deletion mutant forms of HCV core protein, pCore (1–173)-V5 and pCore (1–122)-V5. Transfection with these two plasmids lost the ability of the enhancement of GAS-mediated transcription activity in both CL2 and HepG2 cells (data not shown), suggesting that the C-terminal region (amino acids positions 174–191) of HCV core protein may be one of the important regions for the activation of JAK-STAT signaling pathway under IFN-{gamma} stimulation.



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FIG. 8.
Reporter gene assay to examine the direct effect of HCV core-JAK interaction on JAK-STAT-mediated transcription activity under IFN-{gamma} stimulation. CL2 or HepG2 cells were cotransfected of various effector plasmids (pcDNA3.1/V5-His A, pCore (1–191)-V5, pCoreMut-V5, and pCoreDel-V5), with pGASLuci and pRLtK. The cells were then stimulated with 200 units/ml murine or human IFN-{gamma}, or left unstimulated, and subjected to the dual luciferase assay. The firefly luciferase activity was normalized for transfection efficiency based on the seapansy luciferase activity. The relative light unit of the unstimulated sample was considered as 1, and the fold activity of each sample was calculated. The data were expressed as mean ± S.D. A, CL2 cells. B, HepG2 cells. *, p < 0.05 by the non-paired t test.

 

Influence of HCV Core Protein on Cytokine Receptor Expression—To examine the expression level of each cytokine receptor on the cell surface, we further carried out the flow cytometric analysis. The IL-6 receptor subunits, gp-80 and gp-130, and IFN-{gamma} receptors, IFNGR1 and IFNGR2, were examined in cytokine-untreated CL2 mock and core-I cells. The gp-80, gp-130, and IFNGR1 were expressed with no substantial differences between the CL2 mock and core-I cells (Fig. 9, A–C). However, the surface protein level of IFNGR2 was ~3.3-fold higher in the CL2 core-I cells than in the CL2 mock cells (Fig. 9D). The enhanced expression of IFNGR2 by HCV core protein was also seen in cells stimulated with 200 units/ml of IFN-{gamma} for 1 h (data not shown), suggesting that HCV core-mediated up-regulation of IFNGR2 expression can occur irrespective of the presence or absence of IFN-{gamma} stimulation. When the IFNGR2 mRNA level was investigated by Northern blot analysis, it was also 2.2-fold higher in the CL2 core-I cells than in the CL2 mock cells (Fig. 9E). These results showed that HCV core protein up-regulated both mRNA and surface protein levels of IFNGR2. On the other hand, it did not affect the expression levels of the IL-6 receptor subunits.



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FIG. 9.
Flow cytometric and Northern blot analyses to examine the expression levels of IL-6 and IFN-{gamma} receptor in CL2 mock and core-I cells. A–D, flow cytometric analysis to examine IL-6 receptor subunits, gp-80 and gp-130, and IFN-{gamma} receptors, IFNGR1 and IFNGR2, expression on the cell surface. Representative histograms of fluorescence intensities in CL2 mock and core-I cells stained with the specific antibody are shown by the thick line, whereas those stained only with the fluorescein isothiocyanate-conjugated anti rabbit IgG antibody without the primary antibody are shown by the thin line. E, total RNA samples were extracted from CL2 mock and core-I cells and subjected to Northern blot analysis to detect IFNGR2 mRNA. The relative band intensity was determined by an image analyzer (upper). Ribosomal 18 (18S) and 28 (28S) S indicate similar amounts of RNA loaded on gels (lower).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
HCV core protein has been shown to possess various biological activities that substantially affect host cellular functions (68). In particular, the modulation effect of HCV core protein on various cellular signal transduction pathways has been documented (1416). In the present study, we carried out a detailed investigation of the influence by HCV core protein on the JAK-STAT signaling pathway, which is known to be activated under various cytokine stimuli, using murine normal liver cells constitutively expressing HCV core protein. We first conducted the reporter gene assay and Northern blot analysis to clarify the STAT-mediated transcription activity in the absence or presence of HCV core protein under various cytokine stimuli. We found that STAT3/APRE-mediated transcription activity was suppressed by the expression of HCV core protein under IL-6 stimulation. On the other hand, expression of HCV core protein resulted in enhancement of STAT1/GAS-mediated transcription activity under IFN-{gamma} stimulation. As for IFN-{alpha}/{beta} administration, ISRE-mediated transcription in HCV core-expressing cells showed the same level as that in the mock cells.

We next examined the level of the tyrosine-phosphorylated JAK and STAT proteins in both mock and HCV core-expressing cells under IL-6 and IFN-{gamma} stimuli. The phosphorylation levels of JAK1, JAK2, and STAT3 proteins were suppressed by expression of HCV core protein under IL-6 stimulation. On the other hand, the phosphorylation levels of JAK1, JAK2, and STAT1 were increased by expression of HCV core protein under IFN-{gamma} stimulation. The HCV core protein appeared to directly affect the kinase activity of JAK protein, because the whole protein levels of JAK and STAT proteins were not substantially influenced by its expression. Taken together, this demonstrated that the HCV core protein regulates the JAK-STAT signaling pathway differently under IL-6 and IFN-{gamma} stimuli.

To further assess the influence of HCV core protein on JAK proteins, its binding to JAK proteins was examined in HCV core-expressing cells. The result strongly suggests that HCV core protein may interact with JAK1 and JAK2, but not with STAT1 and STAT3 in cells. The binding of HCV core protein to JAK proteins was observed in both the cytokine-untreated cells and those treated with IL-6 and IFN-{gamma}, indicating that it may occur irrespective of the phosphorylation status of JAK proteins.

The authentic JAK-binding proteins have been shown to possess the proline-rich sequence, PXXPXP, designated box 1, which serves as a docking site for the JAK protein (3741). For example, PNVPDP for human and mouse gp130, the IL-6 receptor subunit (37, 41), PGIPSP for human erythropoietin receptor (38), and PSVPDP for granulocyte colony-stimulating factor receptor (39) have been identified. As for the HCV core protein, it also includes one box 1-like sequence, PGYPWP, at amino acid positions 79–84. The in vitro protein-protein interaction analysis revealed that both the mutant HCV core protein having the AGYAWA sequence instead of the PGYPWP one and deleted HCV core protein lacking the PGYPWP sequence lost the binding activity to JAK proteins. This result indicates that this box-1 like PGYPWP sequence within the HCV core protein may play a major role in its direct interaction with JAK proteins. HCV is known to be susceptible to mutations and can be divided into six major genotypes (42). The rate of amino acid divergence of HCV has been shown to be 22.8–34.0% among the different genotypes (4346). Nevertheless, this PGYPWP box 1 motif of HCV core protein is well conserved among HCV strains (47). This suggests that the binding of the HCV core protein to JAK proteins may be a general event throughout all genotypes of HCV strains.

In the present study, the mouse cell line was mainly used, because cells constitutively expressing HCV core protein could not be established in the human liver-derived cell lines HepG2, Huh-7, and Hep3B. Therefore, the binding of HCV core protein to JAK proteins was also investigated using HepG2 cells transiently transfected with HCV core-expressing plasmid. The interaction of HCV core protein with JAK1 and JAK2 proteins was also found in human HepG2 cells. In addition, it was shown that the mutation and the deletion of the box 1-like PGYPWP sequence resulted in the loss of the binding activity to JAK proteins in the experiment using the human HepG2 lysate. Thus, the HCV core protein was shown to have binding activity for human JAK proteins, as well as murine JAK ones.

To assess the direct contribution of the HCV core-JAK interaction to the HCV core-mediated modulation of JAK-STAT signaling pathway under IL-6 and IFN-{gamma} stimuli, we further carried out the reporter gene assay by means of the cotransfection method. The results showed that the mutated or deleted HCV core protein, which could not bind to the JAK proteins, lost the inhibitory effect on the JAK-STAT pathway under IL-6 stimulation in both murine CL2 and human HepG2 cells. Our finding strongly suggests that HCV core-mediated inhibition of JAK-STAT pathway under IL-6 stimulation may be directly caused by the interaction between HCV core and JAK. Such suppressive effect of HCV core protein on the JAK-STAT pathway may be exhibited by its competitive inhibition of interaction between the gp-130 and JAK proteins, because the binding manner of HCV core protein to JAK proteins may be similar to that of gp-130. Otherwise, the binding of HCV core protein to JAKs may lead to the conformational change of JAK protein, resulting in decrease in the JAK activity.

In contrast, HCV core-mediated augmentation of JAK-STAT pathway under IFN-{gamma} stimulation was not so much affected by introducing the mutation or deletion of the box 1-like PGYPWP sequence within HCV core. This result indicates that the interaction between HCV core and JAK may have no or minimal effect on this pathway under IFN-{gamma} stimulation, unlike the case of IL-6 stimulation. To further investigate how HCV core protein would contribute to the activation of JAK-STAT pathway under IFN-{gamma} stimulation, we examined the differences in the expression levels of cytokine receptors between the mock and HCV core-expressing cells. HCV core protein was found to be considerably up-regulated the mRNA and cell surface expression levels of IFNGR2. It is known that IFNGR1 can bind the ligand with high affinity and that IFNGR2 is a crucial factor for signal transduction (4850). It has also been reported that elevated expression of IFNGR2 by external stimuli increased sensitivity to IFN-{gamma} and JAK-STAT activation (51). Thus, regulation of IFNGR2 expression has been shown to be one of important factors in determining IFN-{gamma} responsiveness. According to these, the enhancement of JAK-STAT pathway by HCV core protein under IFN-{gamma} stimulation may be mainly due to elevated expression of IFNGR2. Under IFN-{gamma} stimulation, HCV core-mediated up-regulation of IFNGR2 may have a stronger effect on the JAK-STAT signal transduction than the suppressive action caused by the interaction of HCV core with JAK proteins.

The influence of HCV expression on the JAK-STAT signaling pathway has been studied. Naganuma et al. (28) showed that the HCV core protein enhanced the IFN-{alpha}-inducible 2'-5'-oligoadenylate synthetase gene expression in the human hepatocyte-derived cells transiently transfected with the HCV core gene. This does not agree with our findings that persistent expression of HCV core protein caused no effects on the ISRE-mediated transcription activity under IFN-{alpha}/{beta} stimulation in mouse normal liver cells. It has also been suggested by Markus et al. (29) that the expression of full-length HCV ORF strongly suppressed the IFN-{alpha}- or leukemia inhibitory factor-mediated STAT/DNA binding activity without affecting the STAT phosphorylation status in a human osteosarcoma cell line permanently transfected with HCV cDNA. Such mutually conflicting results may have arisen due to different expression levels of HCV protein and/or different kinds of cultured cells. In addition, HCV proteins, other than the core protein, may also play a substantial role in the modification of the JAK-STAT signaling pathway. Further studies should offer better understandings of this problem.

In summary, this is the first report indicating that the JAK protein is one of the target molecules of HCV core protein. Our findings also showed that HCV core protein modulates the JAK-STAT signaling pathway differently under IL-6 and IFN-{gamma} stimuli, which may be due to the total effects of its binding to the JAK proteins and the enhanced expression of the IFN-{gamma} receptor. It is known that IL-6-induced STAT3 activation stimulates hepatocytes to produce a variety of acute-phase proteins, such as C reactive protein and fibrinogen (52). Furthermore, liver regeneration after partial hepatectomy has been shown to be delayed in IL-6-deficient mice, suggesting that IL-6 may play an important role in hepatocyte proliferation (53). As for the role of IFN-{gamma} in hepatocytes, IFN-{gamma} has also been shown to contribute to the inhibition of hepatitis B virus expression in hepatitis B virus transgenic mice (54). In a clinical setting, the clearance of HCV viremia has been reported to be associated with the enhanced antigen-specific IFN-{gamma} production in chronic hepatitis C patients who underwent the antiviral treatment (55). In addition, severe hepatitis has been shown to arise in IFN-{gamma} transgenic mice (56). Thus, IFN-{gamma} may be closely associated with both virus eradication from the liver and the development of hepatitis. Taken together, HCV core-mediated modulation of the JAK-STAT signaling pathway under IL-6 and IFN-{gamma} stimuli may have a substantial role in the pathogenesis of HCV-related liver diseases.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Dept. of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3440; Fax: 81-6-6879-3449; E-mail: hayashin{at}moltx.med.osaka-u.ac.jp.

1 The abbreviations used are: HCV, hepatitis C virus; ORF, open reading frame; JAK, Janus kinase; STAT, signal transducer and activator transcription factor; IL, interleukin; IFN, interferon; ISRE, interferon stimulatory response element; IFNGR, IFN-{gamma} receptor; EF, elongation factor; APRE, acute phase response element; GAS, {gamma}-interferon activation site; IRF, interferon regulatory factor. Back


    ACKNOWLEDGMENTS
 
We are grateful to Dr. T. Wakita (Department of Microbiology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) for providing pc/3EFpro and pc/3EF{Delta}NCTH and Dr. T. Hirano (Division of Molecular Oncology, Osaka University Medical School, Osaka, Japan) for providing pAPRELuci and pISRELuci.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kiyosawa, K., Sodeyama, T., Tanaka, E., Gibo, Y., Yoshizawa, K., Nakano, Y., Furuta, S., Akahane, Y., Nishioka, K., Purcell, R. H., and Alter, H. J. (1990) Hepatology 12, 671–675[Medline] [Order article via Infotrieve]
  2. Ikeda, K., Saitoh, S., Koida, I., Arase, Y., Tsubota, A., Chayama, K., Kumada, H., and Kawanishi, M. (1993) Hepatology 18, 47–53[Medline] [Order article via Infotrieve]
  3. Choo, Q. L., Richman, K. H., Han, J. H., Berger, K., Lee, C., Dong, C., Gallegos, C., Coit, D., Medina-Selby, A., Barr, P. J., Weiner, A. J., Bradley, D. W., Kuo, G., and Houghton, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2451–2455[Abstract]
  4. Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M., and Shimotohno, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5547–5551[Abstract]
  5. Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K., and Shimotohno, K. (1993) J. Virol. 67, 4665–4675[Abstract]
  6. Chang, J., Yang, S. H., Cho, Y. G., Hwang, S. B., Hahn, Y. S., and Sung, Y. C. (1998) J. Virol. 72, 3060–3065[Abstract/Free Full Text]
  7. Ray, R. B., Lagging, L. M., Meyer, K., and Ray, R. (1996) J. Virol. 70, 4438–4443[Abstract]
  8. Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., Matsuura, Y., Kimura, S., Miyamura, T., and Koike, K. (1998) Nat. Med. 4, 1065–1067[CrossRef][Medline] [Order article via Infotrieve]
  9. Ray, R. B., Meyer, K., and Ray, R. (1996) Virology 226, 176–182[CrossRef][Medline] [Order article via Infotrieve]
  10. Ray, R. B., Meyer, K., Steele, R., Shrivastava, A., Aggarwal, B. B., and Ray, R. (1998) J. Biol. Chem. 273, 2256–2259[Abstract/Free Full Text]
  11. Marusawa, H., Hijikata, M., Chiba, T., and Shimotohno, K. (1999) J. Virol. 73, 4713–4720[Abstract/Free Full Text]
  12. Ruggieri, A., Harada, T., Matsuura, Y., and Miyamura, T. (1997) Virology 229, 68–76[CrossRef][Medline] [Order article via Infotrieve]
  13. Zhu, N., Khoshnan, A., Schneider, R., Matsumoto, M., Dennert, G., Ware, C., and Lai, M. M. C. (1998) J. Virol. 72, 3691–3697[Abstract/Free Full Text]
  14. Tsuchihara, K., Hijikata, M., Fukuda, K., Kuroki, T., Yamamoto, N., and Shimotohno, K. (1999) Virology 258, 100–107[CrossRef][Medline] [Order article via Infotrieve]
  15. Aoki, H., Hayashi, J., Moriyama, M., Arakawa, Y., and Hino, O. (1999) J. Virol. 74, 1736–1741[CrossRef]
  16. Yoshida, H., Kato, N., Shiratori, Y., Otsuka, M., Maeda, S., Kato, J., and Omata, M. (2001) J. Biol. Chem. 276, 16399–16405[Abstract/Free Full Text]
  17. Boehm, U., Klamp, T., Groot, M., and Howard, J. C. (1997) Annu. Rev. Immunol. 15, 749–795[CrossRef][Medline] [Order article via Infotrieve]
  18. Bach, E. A., Aguet, M., and Schreiber, R. D. (1997) Annu. Rev. Immunol. 15, 563–591[CrossRef][Medline] [Order article via Infotrieve]
  19. Heim, M. H. (1996) Eur. J. Clin. Invest. 26, 1–12[Medline] [Order article via Infotrieve]
  20. Ihle, J. N. (1996) Cell 84, 331–334[Medline] [Order article via Infotrieve]
  21. Darnell, J. E. (1997) Science 277, 1630–1635[Abstract/Free Full Text]
  22. David, S. A., and Curt, M. H. (2001) Science 296, 1653–1655[CrossRef]
  23. Zhong, Z., Wen, Z., and Darnell, J. E., Jr. (1994) Science 264, 95–98[Medline] [Order article via Infotrieve]
  24. Murakami, M., Hibi, M., Nakagawa, N., Nakagawa, T., Yasukawa, K., Yamanishi, K., Taga, T., and Kishimoto, T. (1993) Science 260, 1808–1810[Medline] [Order article via Infotrieve]
  25. Pestka, S. (1997) Semin. Oncol. 24, S9–S40
  26. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415–1421[Medline] [Order article via Infotrieve]
  27. Velazquez, L., Fellous, M., Stark, G. R., and Palletrini, S. (1992) Cell 70, 313–322[Medline] [Order article via Infotrieve]
  28. Naganuma, A., Nozaki, A., Tanaka, T., Sugiyama, K., Takagi, H., Mori, M., Shimotohno, K., and Kato, N. (2000) J. Virol. 74, 8744–8750[Abstract/Free Full Text]
  29. Markus, H. H., Darius, M., and Hubert. E. B. (1999) J. Virol. 73, 8469–8475[Abstract/Free Full Text]
  30. Tokushige, K., Moradpour, D., Wakita, T., Geissler, M., Hayashi, N., and Wands, J. R. (1997) J. Virol. Methods 64, 73–80[CrossRef][Medline] [Order article via Infotrieve]
  31. Fujitani, Y., Nakajima, K., Kojima, H., Nakae, K., Takeda, T., and Hirano, T. (1994) Biochem. Biophys. Res. Commun. 202, 1181–1187[CrossRef][Medline] [Order article via Infotrieve]
  32. Muraoka, O., Tanaka, H., Itoh, M., Ishikawa, K., and Hirano, T. (1996) Immunol. Lett. 54, 1–4[CrossRef][Medline] [Order article via Infotrieve]
  33. Patek, P., Collins, J., and Cohn, M. (1978) Nature 276, 510–511[Medline] [Order article via Infotrieve]
  34. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7413–7417[Abstract]
  35. Aden, D. P., Fogel, A., Plotkin, S., Damjanov, I., and Knowles, B. B. (1979) Nature 282, 615–616[Medline] [Order article via Infotrieve]
  36. Nakabayashi, H., Takeda, K., Miyano, K., Yamane, T., and Sato, J. (1982) Cancer Res. 42, 3858–3863[Abstract]
  37. Hibi, M., Murakami, M., Saito, M., Hirano, T., Taga, T., and Kishimoto, T. (1990) Cell 63, 1149–1157[Medline] [Order article via Infotrieve]
  38. Yoshimura, M., and Jessell, T. M. (1990) J. Physiol. (Lond.) 430, 315–335[Abstract]
  39. Larsen, A., Davis, T., Curtis, B. M., Gimpel, S., Sims, J. E., Cosman, D., Park, L., Sorensen, E., March, C. J., and Smith, C. A. (1990) J. Exp. Med. 172, 1559–1570[Abstract]
  40. Tanner, J. W., Chen, W., Young, R. L., Longmore, G. D., and Shaw, A. S. (1995) J. Biol. Chem. 270, 6523–6530[Abstract/Free Full Text]
  41. Murakami, M., Narazaki, M., Hibi, M., Yawata, H., Yasukawa, K., Hamaguchi, M., Taga, T., and Kishimoto, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11349–11353[Abstract]
  42. Donald, B. S., Fiona, D., and Peter. S. (1995) J. Hepatol. 23, Suppl. 2, 26–31[Medline] [Order article via Infotrieve]
  43. Adams, N. J., Chamberlain, R. W., Taylor, L. A., and Davidson, F. (1997) Biochem. Biophys. Res. Commun. 234, 393–396[CrossRef][Medline] [Order article via Infotrieve]
  44. Okamoto, H., Kurai, K., Okada, S., Yamamoto, K., Lizuka, H., Tanaka, T., Fukuda, S., Tsuda, F., and Mishiro, S. (1992) Virology 188, 331–334[Medline] [Order article via Infotrieve]
  45. Sakamoto, M., Akahane, Y., Tsuda, F., Tanaka, T., Woodfield, D. G., and Okamoto, H. (1994) J. Gen. Virol. 75, 1761–1768[Abstract]
  46. Chamberlain, R. W., Adams, N. J., Taylor, L. A., Simmonds, P., and Elliott, R. M. (1997) Biochem. Biophys. Res. Commun. 236, 44–49[CrossRef][Medline] [Order article via Infotrieve]
  47. Mori, S., Kato, N., Yagyu, A., Tanaka, T., Ikeda, Y., Petchclai, B., Chiewslip, P., Kurimura, T., and Shimotohno, K. (1992) Biochem. Biophys. Res. Commun. 183, 334–342[Medline] [Order article via Infotrieve]
  48. Hemmi, S., Bohni, R., Stark, G., Di-Marco, F., and Aguet, M. (1994) Cell 76, 803–810[Medline] [Order article via Infotrieve]
  49. Soh, J., Donelly, R. J., Kotenko, S., Mariano, T. M., Cook, J. R., Wang, N., Emanuel, S., Schwartz, B., Miki, T., and Pestka, S. (1994) Cell 76, 793–802[Medline] [Order article via Infotrieve]
  50. Bach, E. A., Szabo, S. J., Dighe, A. S., Ashkenazi, A., Aguet, M., Murphy, K. M., and Schreiber, R. D. (1995) Science 270, 1215–1218[Abstract]
  51. Sakatsume, M., and Finbloom, D. S. (1996) J. Immunol. 156, 4160–4166[Abstract]
  52. Zhang, D., Sun, M., Samols, D., and Kushner, I. (1996) J. Biol. Chem. 271, 9503–9509[Abstract/Free Full Text]
  53. Cressman, D. E., Greenbaum, L. E., DeAngelis, R. A. Ciliberto, G., Furth, E. E., Poli, V., and Taub, R. (1996) Science 274, 1379–1383[Abstract/Free Full Text]
  54. Guidotti, L. G., Ando, K., Hobbs, M. V., Ishikawa, T., Runkel, L., Schreiber, R. D., and Chisari, F. V. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3764–3768[Abstract]
  55. Cramp, M. E., Rossol, S., Chokshi, S., Carucci, P., Williams, R., and Naoumov, N. V. (2000) Gastroenterology 118, 346–355[Medline] [Order article via Infotrieve]
  56. Toyonaga, T., Hino, O., Sugai, S., Wakasugi, S., Abe, K., Shichiri, M., and Yamamura, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 614–618[Abstract]