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Journal of Virology, February 2005, p. 1569-1580, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1569-1580.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Hepatitis C Virus (HCV) Constitutively Activates STAT-3 via Oxidative Stress: Role of STAT-3 in HCV Replication

Gulam Waris,1 James Turkson,2 Tarek Hassanein,3 and Aleem Siddiqui1*

Department of Microbiology and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado,1 Molecular Oncology Program, Moffitt Cancer Center, Tampa, Florida,2 Department of Clinical Medicine and Surgery, School of Medicine, University of California, San Diego, California3

Received 10 June 2004/ Accepted 17 September 2004


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ABSTRACT
 
The hepatitis C virus (HCV) causes chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. We have previously shown that HCV nonstructural proteins induce activation of STAT-3 via oxidative stress and Ca2+ signaling (G. Gong, G. Waris, R. Tanveer, and A. Siddiqui, Proc. Natl. Acad. Sci. USA 98:9599-9604, 2001). In this study, we focus on the signaling pathway leading to STAT-3 activation in response to oxidative stress induced by HCV translation and replication activities. Here, we demonstrate the constitutive activation of STAT-3 in HCV replicon-expressing cells. The HCV-induced STAT-3 activation was inhibited in the presence of antioxidant (pyrrolidine dithiocarbamate) and Ca2+ chelators (BAPTA-AM and TMB-8). Previous studies have shown that maximum STAT-3 transactivation requires Ser727 phosphorylation in addition to tyrosine phosphorylation. Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of STAT-3 is mediated by oxidative stress and influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2, and Src. Our results also suggest a potential role of STAT-3 in HCV RNA replication. We also observed the constitutive activation of STAT-3 in the liver biopsy of an HCV-infected patient. These studies provide an insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with the viral infection.


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INTRODUCTION
 
The hepatitis C virus (HCV) is one of the leading causes of chronic liver disease, afflicting more than 170 million individuals worldwide (17). Persistent HCV infection often leads to a risk of end-stage cirrhosis and hepatocellular carcinoma (17). HCV is an enveloped single-stranded positive-sense RNA virus, approximately 9.6 kb in length, and encodes a polyprotein of about 3,000 amino acids (18). This polyprotein is posttranslationally cleaved by a combination of host cell signal peptidases and two viral proteinases into structural (core, E1, and E2) and nonstructural (NS2 and NS3-NS5A/B) proteins (2, 36). A new protein termed F is thought to be produced by –2/+1 ribosomal frameshift during translation (55). The single open reading frame is flanked at the 5' end by a noncoding region (NCR), which harbors an internal ribosome entry site (46, 49) and at the 3' end by a highly conserved sequence essential for initiating RNA replication (44).

Despite the availability of infectious cDNA clones (27), molecular studies of HCV replication and pathogenesis have been hampered by the lack of a reliable and efficient cell culture system. To overcome these restrictions recently, Lohmann et al. reported the development of HCV subgenomic replicons (29). These bicistronic replicons are composed of an HCV 5' NCR fused to 12 amino acids of the capsid coding region, the neomycin phosphotransferase gene (Neor), which confers resistance to G418, and the internal ribosome entry site from encephalomyocarditis virus, controlling the translation of the HCV proteins NS3 to NS5B, followed by the 3' NCR. Several adaptive mutations were identified scattered throughout the NS proteins of the replicon, which conferred a high level of replication of subgenomic replicons (30). Viral proteins are found exclusively in the cytoplasm in close association with endoplasmic reticulum (ER) membrane, suggesting this as the site of RNA replication (3). A recent study has described the association of RNA replication with lipid rafts (39). The HCV nonstructural proteins form a ribonucleoprotein complex which is localized in the ER membrane (2, 18, 53). This association induces ER stress, exhibiting an unfolded protein response (45). Depletion of Ca2+ stores in the ER and its uptake by mitochondria lead to generation of reactive oxygen species (ROS) (24). Several HCV proteins, including core, NS3, and NS5A, have been shown to induce oxidative stress in cultured cells (7, 24, 34). ROS, which act as second messengers, activate cellular kinases, although the mechanism of this activation is unclear. Some of these kinases can activate transcription factors that are in a latent state in the cytoplasm. These include STAT-3, NF-{kappa}B, NF-AT, and others (10, 24).

STAT-3 is an oncogenic transcription factor that is activated upon tyrosine phosphorylation in response to extracellular signals, such as cytokines and growth factors (6, 62). Binding of cytokines such as interleukin-6 or growth factors to their cognate receptors leads to receptor dimerization and activation of receptor-associated Janus kinases (JAKs), resulting in recruitment of STAT-3 protein (57). Activated STAT-3 then translocates to the nucleus to regulate gene expression. STAT-3 activation by other nonreceptor tyrosine kinases has also been demonstrated. Transformation of mammalian cells by viral Src (v-Src) specifically induces constitutive activation of STAT-3 (47, 58). In addition to STAT-3 activation by tyrosine phosphorylation, Ser727 phosphorylation mediated by mitogen-activated protein kinases (MAPKs) contributes to its maximal transcriptional activity (48, 54). Recently, the functional importance of STAT-3 Ser727 phosphorylation has been demonstrated in mice lacking STAT-3 serine phosphorylation (38). STAT-3 is frequently overactivated in a wide variety of solid tumors and blood malignancies (59).

In the present study, we investigated the mechanism(s) of STAT-3 activation in response to oxidative stress induced by HCV gene expression in hepatoma cell lines expressing the HCV subgenomic replicon. Our results show that p38 MAPK, JNK, JAK2, and Src kinases are involved in the activation of STAT-3. We further demonstrate that both ROS and STAT-3 activation contribute to the stimulation of HCV RNA replication. These data collectively suggest a novel mechanism(s) of an ER-nucleus signal transduction pathway by the HCV subgenomic replicon. The intracellular events triggered by the HCV translation-replication activities in the cells are relevant to the mechanism(s) of liver disease pathogenesis associated with HCV infection.


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MATERIALS AND METHODS
 
Plasmids and oligonucleotides. The STAT-3 reporter plasmid pLucTKS3 is driven by the thymidine kinase promoter with seven copies of upstream STAT-3 binding sites from the human C-reactive protein gene (47). The human cyclin D1 promoter linked to a luciferase reporter (–1745 CD1 Luc) was a generous gift of K. G. Pestell (Lombardi Cancer Center, Georgetown University, Washington, D.C.). The plasmid expressing a dominant negative form of JNK (p54JNK2{alpha}) was provided by L. Heasely (University of Colorado Health Sciences Center, Denver). Construction of plasmids expressing dominant negative forms of STAT-3 (pSG5hSTAT-3ß and STAT-3S727A) and Src kinase (pM5Hmet295) were described previously (47, 48).

The STAT-3 consensus oligonucleotide and an antiphosphotyrosine monoclonal antibody were obtained from Santa Cruz Biotechnology. Pyrrolidine dithiocarbamate (PDTC) and TMB-8 were purchased from Sigma Chemical Co. Anti-p38 MAPK polyclonal, anti-Bcl-XL polyclonal, anti-phospho-p38 MAPK (Thr180/Tyr182) monoclonal, anti-STAT-3 polyclonal, anti-phospho-STAT-3 (Tyr705) monoclonal, anti-JNK polyclonal, anti-phospho-JNK (Thr183/Tyr185) monoclonal, anti-Src polyclonal, and anti-phospho-Src416 antibodies SB203580, SP600125, SU6656, PP2, AG490, and BAPTA-AM were purchased from Cell Signaling Technology and Calbiochem-Novabiochem Corp. (San Diego, Calif.), respectively. The anti-phospho-STAT-3 antibody (Ser727) was obtained from Upstate Biotechnology, Lake Placid, N.Y.

Cell culture. The human hepatoma cell lines Huh-7 and FCA4 were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U of penicillin/ml, and 100 µg of streptomycin sulfate/ml. FCA4 and GS4.3 cells were a generous gift of C. Seeger (Fox Chase Cancer Center, Philadelphia, Pa.) and were grown in 500 µg of G418 (Geneticin; Invitrogen)/ml. FCA4 cells are a Huh-7 cell line stably expressing an HCV subgenomic replicon with a single adaptive mutation, a deletion of serine residue 1176 (25).

Liver tissues. Liver biopsy specimens were obtained from patients with chronic hepatitis C. Frozen samples were thawed in radioimmune precipitation assay (RIPA) buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM sodium orthovanadate, 1 mM sodium formate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg of aprotinin/ml, 10 µg of leupeptin/ml) and gently crushed with a glass rod, followed by sonication and incubation on ice for 30 min. Samples were centrifuged at 4°C (13,400 x g) for 5 min. The supernatant was collected, and the activity of STAT-3 was analyzed by Western blotting.

Preparation of nuclear extracts. Nuclear lysates were prepared from FCA4 and Huh-7 cells transfected with BM4-5 RNA. Cells were lysed in hypotonic buffer (20 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM Na3VO4, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 3 mg of aprotinin/ml, 1 mg of pepstatin/ml, 20 mM NaF, and 1 mM dithiothreitol [DTT] with 0.2% NP-40) on ice for 10 min. After centrifugation at 4°C (13,000 rpm) for 1 min, the nuclear pellet was resuspended in high-salt buffer (hypotonic buffer with 20% glycerol and 420 mM NaCl) at 4°C by rocking for 30 min following centrifugation. The supernatant was collected and stored at –80°C in aliquots.

Bacterial expression of unphosphorylated STAT-3 proteins. Bacterial cultures were grown in the presence of ampicillin and tetracycline and induced in the presence of 1 mM isopropyl-ß-D-thiogalactopyranoside for the expression of STAT-3. The cells were harvested and suspended in ice-cold extraction buffer (20 mM HEPES [pH 7.6], 0.1 M KCl, 10% glycerol, 1 mM EDTA, 10 mM MnCl2, 20 mM DTT, 0.5 mM PMSF). The lysis was performed by sonication followed by centrifugation. To remove nucleic acids, polyethyleneimine (0.1%, wt/vol) was added to the ice-cooled stirred supernatant, stirred for another 15 min, and then centrifuged. Ammonium sulfate was added at 35% saturation. The precipitated protein was collected by centrifugation, suspended in a buffer consisting of 20 mM HEPES (pH 7.0), 200 mM NaCl, 10 mM MgCl2, 5 mM DTT, and 0.5 mM PMSF, and dialyzed overnight at 4°C against the same buffer.

EMSA. The STAT-3 consensus sequence was radiolabeled at the 5' end with [{gamma}-32P]ATP by T4 polynucleotide kinase. About 20,000 cpm of gel-purified probe was incubated, with nuclear lysates prepared from FCA4 and Huh-7 cells, in electrophoretic mobility shift assay (EMSA) buffer [20 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 50 mM KCl, 16.7 µg of poly(dI-dC)/ml, 1 mM EDTA, 1 mM DTT and 1 µM leupeptin] for 20 min on ice. The DNA-protein complexes were resolved by 5% polyacrylamide gel electrophoresis (PAGE) in 0.5x Tris-borate-EDTA buffer. The gels were dried and subjected to autoradiography.

Immunoprecipitation and Western blot analysis. Exponentially growing FCA4 and Huh-7 cells transfected with BM4-5 RNA were harvested, and cell extracts were prepared by incubating in RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM sodium formate, 1 mM PMSF, 10 µg of aprotinin/ml, 10 µg of leupeptin/ml) for 30 min on ice. Immunoprecipitation was performed with anti-STAT-3 serum for 4 h. The immune complexes were incubated with protein A-Sepharose, washed three times with RIPA buffer, and boiled for 5 min in SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE. Gels were electroblotted onto polyvinylidene difluoride membrane (Amersham) in 25 mM Tris, 192 mM glycine, and 20% methanol by electrophoresis. Membranes were treated for 1 h in blocking buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.3% polyvinyl pyrrolidone, 0.5% Tween 20 [wt/vol]), probed with antiphosphotyrosine monoclonal antibody overnight, and washed twice for 10 min with blocking buffer, followed by incubation with secondary antibody for 45 min. After an additional washing step with blocking buffer, immunoblots were visualized using the ECL detection system (Amersham).

In vitro kinase assays. To assay for Src kinase activity, Huh-7 and HCV replicon-expressing cells (FCA4 and GS4.3) were lysed in RIPA buffer at 4°C for 30 min. Equal amounts of cellular lysates from Huh-7 and FCA4 cells were immunoprecipitated with anti-Src serum at 4°C overnight. The immune complexes were captured on protein A-Sepharose and washed three times with RIPA buffer and once with kinase buffer (20 mM HEPES [pH 7.4], 10 mM MnCl2). Immunoprecipitates were then resuspended in kinase buffer containing 5 µg of recombinant unphosphorylated STAT-3 (substrate), 20 µCi of [{gamma}-32P]ATP, and 10 µM ATP and incubated at 30°C for 30 min. The kinase reactions were stopped with 2x sample loading buffer, boiled for 5 min, and resolved by SDS-PAGE.

For JNK and p38 MAPK assays, cellular extracts were immunoprecipitated with anti-phospho-JNK and p38 MAPK antisera at 4°C overnight. Immunoprecipitates were washed twice with RIPA buffer and once with kinase buffer (25 mM Tris [pH 7.5], 5 mM ß-glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2). Immunoprecipitates were resuspended in 20 µl of kinase buffer supplemented with 10 µCi of [{gamma}-32P]ATP and 5 µg of recombinant STAT-3 as substrate, at 30°C for 30 min. The kinase reactions were stopped with 2x sample loading buffer, boiled for 5 min, and resolved by SDS-10% PAGE.

Luciferase assays. For transient transfections, Huh-7 cells were plated at a density of ~5 x 105 cells/60-mm dish and maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and penicillin (75 U/ml) and streptomycin (50 U/ml) at 37°C. Cells (~50% confluent) were transfected with 200 ng of luciferase reporter plasmid by using Lipofectin reagent (Life Technologies). Thirty-six hours posttransfection, cells were serum starved overnight before the addition of PDTC, BAPTA-AM, TMB-8, SB203580, SP600125, SU6656, PP2, and AG490 at various times. Cells were harvested, and cellular lysates were analyzed for luciferase expression by using a luminometer (16). All transfections included a ß-galactosidase expression vector to serve as an internal control.

BM4-5 plasmid DNA (SP1/DS-BM4-5; gift of C. Seeger) was linearized and transcribed into RNA by using the Ampliscribe T7 transcription kit (Epicenter Technology). BM4-5 RNA (5 µg) was transfected into Huh-7 cells by using Lipofectin reagents. At 15 h posttransfection, G418-resistant cells were harvested.

Flow cytometric analysis of cellular ROS levels. Intracellular ROS levels were measured by using an oxidative-sensitive fluorescent probe, dihydroethidium (DHE; Molecular Probes), as described elsewhere (15) with some modifications. Briefly, FCA4 or Huh-7 cells (~2.5 x 105) in 100-mm plates were transfected with BM4-5 RNA by using Lipofectin (Gibco). About 20 h after transfection, cells were incubated with 4 µM DHE for 45 min at 37°C. Cells were harvested and washed with phosphate-buffered saline, and the ROS levels were analyzed by using an XL00W42322 flow cytometer. The fluorescence from oxidized DHE was detected at a wavelength of 630 nm.

Quantitative, real-time RT-PCR. Total RNA was extracted from HCV replicon-containing cells (FCA4) as well as Huh-7 cells by using RNA STAT-60 (Tel-Test, Inc., Friendswood, Tex). HCV RNA was quantified by real-time reverse transcription-PCR (RT-PCR) by using an ABI Prism 7000 sequence detector (Perkin-Elmer/Applied Biosystems). Amplifications were conducted in duplicate using the following primers and 6-carboxyfluorescein (6FAM)- and tetrachloro-6-carboxyfluorescein (TAMRA)-labeled probes (Perkin-Elmer): HCV replicon Taqman probe, 5'-6FAM-CCT TCA TCT CCT TGA GCA CGT CCC G-TAMRA-3'; HCV replicon RNA-FWD, CTT TGA CAG ACT GCA GGT CCT G; HCV replicon RNA-REW, GCC TTA ACT GTG GAC GCC TTC; 18S rRNA Taqman probe, 5'-6FAM-TGC TGG CAC CAG ACT TGC CCT C-TAMRA; 18S rRNA-FWD, 5'-CGG CTA CCA CAT CCA AGG AA-3'; and 18S rRNA-REW, 5'-GCT GGA ATT ACC GCG GCT-3'. The sequences for the primers and probes were designed using Primer Express software (Perkin-Elmer/Applied Biosystems). Amplification reactions were performed in a 25-µl mix containing 8% glycerol, 1x TaqMan buffer A (500 mM KCl, 100 mM Tris-HCl, 0.1 M EDTA, 600 nM passive reference dye ROX; pH 8.3), 300 µM (each) dATP, dGTP, and dCTP, 600 µM dUTP, 5.5 mM MgCl2, 900 nM forward primer, 900 nM reverse primer, 200 nM probe, 1.25 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer), 12.5 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen), and the template RNA. Reactions were performed in a 96-well spectrofluorometric thermal cycler under the following conditions: 30 min at 50°C (RT reaction); 10 min at 95°C (heat inactivation of reverse transcriptase and activation of TaqGold polymerase); and 40 cycles of 15 s at 95°C and 1 min at 60°C (PCR amplification). Fluorescence was monitored during every PCR cycle at the annealing step. At the termination of each PCR run, the data were automatically analyzed by the system and amplification plots were generated.


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RESULTS
 
HCV replicon induces tyrosine phosphorylation of STAT-3. We have previously shown that expression of HCV nonstructural proteins activated STAT-3 (24). In the present study, we sought to investigate the molecular mechanism(s) of STAT-3 activation in the context of HCV translation-replication activities. Cellular lysates from cells stably expressing HCV subgenomic replicons (FCA4 and GS4.3 cells), or those transiently transfected with BM4-5 replicon RNA in Huh-7 cells, were immunoprecipitated with anti-STAT-3 serum, fractionated by SDS-PAGE, and electroblotted onto a nitrocellulose membrane. The membrane was then incubated with antiphosphotyrosine monoclonal antibody. This analysis showed that the cells expressing subgenomic replicons contained tyrosine-phosphorylated STAT-3 (Fig. 1A, lanes 2, 3, and 5). The untransfected Huh-7 cells did not induce tyrosine phosphorylation of STAT-3 (Fig. 1A, lanes 1 and 4). Next, the nuclear translocation of activated STAT-3 induced by the HCV replicon was demonstrated by gel mobility shift assay. The cytoplasmic and nuclear lysates from Huh-7, FCA4, and Huh-7 cells transiently transfected with BM4-5 RNA were incubated with [{gamma}-32P]ATP-labeled STAT-3 cognate nucleotide probe. The results showed the activated STAT-3 in the nucleus of cells expressing HCV replicons (Fig. 1B, lanes 3 and 5), whereas cytoplasmic extracts did not contain activated STAT-3 (lanes 2 and 4). The specificity of the DNA-protein interaction was confirmed by supershift of DNA-protein complex in the presence of anti-STAT-3 serum (Fig. 1C, lane 4) and by using unlabeled competitor oligonucleotides representing the STAT-3 sequences (Fig. 1C, lane 5). The involvement of ROS and Ca2+ signaling in the STAT-3 tyrosine phosphorylation induced by the HCV replicon was evaluated by using the antioxidant PDTC and Ca2+ chelators BAPTA-AM and TMB-8. Stably expressing HCV replicon (FCA4) (Fig. 1D) or Huh-7 cells transiently transfected with BM4-5 RNA treated with these reagents effectively inhibited activation of STAT-3 (Fig. 1E, lanes 4, 5, and 6). These results together demonstrate that HCV subgenomic replicon expression leads to constitutive activation of STAT-3 in the absence of a cytokines.



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  		FIG. 1. HCV replicons induce tyrosine phosphorylation of STAT-3. (A) Whole-cell lysates from cells expressing HCV replicons were immunoprecipitated with anti-STAT-3 serum, fractionated by SDS-PAGE, and immunoblotted with antiphosphotyrosine monoclonal antibody. Lanes 1 and 4, untransfected Huh-7 lysates; lanes 2 and 5, FCA4 and GS4.3 cells expressing HCV subgenomic replicons; lane 3, Huh-7 cells transfected with in vitro-synthesized BM4-5 RNA. The bottom panel represents the expression of HCV NS5A in HCV replicon-expressing cells. (B) An EMSA was carried out in the presence of {gamma}-32P-labeled STAT-3 cognate nucleotide probe, and the nuclear lysates were prepared from FCA4 cells and Huh-7 cells transiently transfected with in vitro-synthesized BM4-5 RNA. Lane 1, STAT-3 probe incubated with Huh-7 nuclear lysates; lanes 2 and 3, equal amounts of FCA4 cytoplasmic and nuclear lysates, respectively; lanes 4 and 5, equal amounts of BM4-5 RNA-transfected cytoplasmic and nuclear lysates, respectively. (C) EMSA. Lane 1, probe alone; lanes 2 and 3, equal amounts of Huh-7 and FCA4 nuclear lysates; lane 4, DNA-protein complex incubated with anti-STAT-3 serum; lane 5, DNA-protein complex treated with a 100-fold excess of unlabeled STAT-3 oligonucleotide. (D) Luciferase reporter gene assay. FCA4 cells were transfected with STAT-3-responsive pLucTKS3 and pLucTK (without STAT-3 binding sites) luciferase plasmids. At 36 h posttransfection, cells were treated with PDTC (6 h), BAPTA-AM (2 h), and TMB-8 (4 h) at various times before preparing the lysates for luciferase activity. (E) EMSA. Lane 1, probe alone; lanes 2 and 3, equal amounts of untransfected and BM4-5 replicon RNA-transfected nuclear lysates; lanes 4, 5, and 6, BM4-5 RNA-transfected lysates treated with PDTC (6 h), BAPTA-AM (2 h), and TMB-8 (4 h) at various times.

Tyrosine kinase mediates activation of STAT-3. It has been demonstrated that autophosphorylation of JAKs and transformation of mammalian fibroblasts by v-Src specifically induces activation of STAT-3 (47, 62). It was therefore of particular interest to investigate the role of Src and JAK tyrosine kinases in the HCV replicon-induced STAT-3 activation. To determine whether Src and Janus kinases, which activate STAT-3, are involved in the STAT-3 activation induced by the HCV replicon, known inhibitors of Src (SU6656 and PP2) and JAK (AG490) kinases were employed in cell-based reporter assays. The results showed that the STAT-3-controlled reporter gene activity was considerably reduced in the presence of these tyrosine kinase inhibitors (Fig. 2A). To confirm the effect of c-Src kinase on STAT-3 transcriptional activity, the STAT-3-responsive luciferase reporter construct was cotransfected along with a c-Src kinase dominant negative (pM5Hmet295) expression vector in cell-based reporter assays. As indicated in Fig. 2B, the magnitude of STAT-3-mediated reporter gene expression was reduced in FCA4 cells expressing kinase-defective c-Src. These results implicate a potential role of tyrosine kinases such as Src and JAK2 in the HCV replicon-induced STAT-3 activation.



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  		FIG. 2. HCV replicon-induced activation of STAT-3 is mediated by JAK2 and Src kinases. (A) Huh-7 and FCA4 cells were transfected with STAT-3-responsive pLucTKS3 luciferase plasmid. At 36 h posttransfection, cells were treated with inhibitors of Src kinase (10 µM SU6656 for 2 h and 20 µM PP2 for 2 h) and JAK2 (40 µM AG490 for 4 h) before preparing the lysates for luciferase activity determinations. (B) FCA4 cells were transfected with a Src dominant negative (pM5Hmet295) expression vector along with the STAT-3-responsive pLucTKS3 luciferase plasmid. At 36 h posttransfection, cellular lysates were prepared for luciferase activity determinations. (C) HCV replicon activates Src kinase. Equal amounts of cellular lysates from Huh-7 and FCA4 cells were subjected to SDS-PAGE and immunoblotted with anti-Src serum. Lane 1, Huh-7 lysates; lane 2, FCA4 lysates. (D) In vitro c-Src kinase assay. Equal amounts of cellular lysates were immunoprecipitated with anti-Src serum, and Src activity was measured in an in vitro kinase assay using [{gamma}-32P]ATP and unphosphorylated STAT-3 as a substrate. Labeled STAT-3 was resolved by SDS-PAGE and visualized by autoradiography. Lane 1, Huh-7 lysates; lane 2, FCA4 lysates. The bottom panel represents the total Src activity in Huh-7 and FCA4 lysates.

HCV replicon activates c-Src kinase. To determine whether c-Src played a role in HCV replicon-mediated STAT-3 activation, we analyzed the phosphorylation status of c-Src kinase in HCV replicon-expressing cells. The results showed that HCV replicon-expressing cells (FCA4) contained both phosphorylated and unphosphorylated forms of c-Src during Western blot assays (Fig. 2C, lane 2), whereas Huh-7 cells contained the unphosphorylated form of Src (lane 1). Next, we examined the in vitro kinase activity of c-Src enzyme in the immunoprecipitates derived from Huh-7 and FCA4 cells. Huh-7 and FCA4 cellular lysates were immunoprecipitated with anti-c-Src serum and subjected to in vitro kinase assays in the presence of an exogenous unphosphorylated STAT-3 substrate. The results in Fig. 2D, lane 2, show that FCA4 cells contained c-Src kinase activity as measured by phosphorylation of STAT-3; however, Huh-7 lysates did not exhibit any tyrosine kinase activity (lane 1). These lysates contained equivalent levels of endogenous c-Src protein (Fig. 2D, bottom panel).

Role of MAPKs on HCV-dependent activation of STAT-3. Previous studies have suggested that maximal activation of STAT-3 requires serine phosphorylation mediated by mitogenic signaling cascades in addition to tyrosine phosphorylation (48). Here, we sought to determine whether HCV-dependent STAT-3 activation depends on serine phosphorylation mediated by mitogenic signaling for its maximal transactivation. Cellular lysates from cells stably expressing HCV subgenomic replicons (FCA4 and GS4.3 cells) or those transiently transfected with BM4-5 replicon RNA were immunoprecipitated with anti-STAT-3 serum and immunoblotted with antiserum specific to STAT-3 Ser727. The results showed that the cells expressing subgenomic replicons contained serine-phosphorylated STAT-3 (Fig. 3A, lanes 2, 3, and 5). The untransfected Huh-7 cells did not induce serine phosphorylation of STAT-3 (Fig. 3A, lanes 1 and 4). Importantly, addition of the inhibitors of p38 MAPK (SB203580) and JNK (SP600125) pathways eliminated the HCV-induced serine phosphorylation of STAT-3 (Fig. 3B, lanes 3 and 4). Similar results were obtained in cell-based reporter assays (Fig. 3D). To confirm the effect of JNK on STAT-3 activation, FCA4 cells were transiently transfected with a JNK dominant negative expression vector (p54 JNK2{alpha}). The results showed that the expression of the JNK dominant negative mutant significantly reduced STAT-3 serine phosphorylation (Fig. 3C, lane 3). Similarly, transient transfection of a STAT-3-responsive luciferase reporter gene along with a JNK dominant negative mutant displayed reduced levels of luciferase activity in FCA4 cells (Fig. 3D). Next, we tested the functional importance of phosphorylation of STAT-3 on Ser727 by using the dominant negative STAT-3S727A mutant, in which alanine was replaced with serine at residue 727. Transient transfection of the plasmid encoding a dominant negative STAT-3 (S727A) along with a luciferase reporter gene containing STAT-3 binding sites reduced the luciferase activity by 50% (Fig. 3C). Since both Ser/Thr and tyrosine kinases are needed for maximal activation of STAT-3, individual Ser/Thr and tyrosine kinase inhibitors were unable to maximally reduce the levels of activated STAT-3. These results suggest that phosphorylation of STAT-3 at Ser727 is important for HCV-induced STAT-3 activation.



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  		FIG. 3. HCV replicon stimulates phosphorylation of STAT-3 at Ser727. (A) Whole-cell lysates from cells expressing HCV replicons were immunoprecipitated with anti-STAT-3 serum, fractionated by SDS-PAGE, and immunoblotted with anti-STAT-3 Ser727 serum. Lanes 1 and 4, untransfected lysates; lanes 2 and 3, FCA4 and GS4.3 cells expressing HCV subgenomic replicons; lane 5, transfected with in vitro-synthesized BM4-5 RNA. The bottom panel represents the total STAT-3 in Huh-7 and HCV replicon-expressing cells. (B) FCA4 cells were treated with inhibitors of p38 MAPK (10 µM SB203580 for 6 h) and JNK (30 µM SP600125 for 2 h). Equal amounts of cellular lysates were immunoprecipitated with anti-STAT-3 serum and immunoblotted with anti-STAT-3 Ser727 serum. Lanes 1 and 2, equal amounts of Huh-7 and FCA4 lysates; lanes 3 and 4, FCA4 lysates treated with SB203580 and SP600125, respectively. (C) FCA4 cells were transfected with a JNK dominant negative (p54JNK2{alpha}) expression vector. Equal amounts of cellular lysates were immunoprecipitated with anti-STAT-3 serum and immunoblotted with anti-STAT-3 Ser727 serum. Lanes 1 and 2, equal amounts of Huh-7 and FCA4 lysates; lane 3, FCA4 lysates expressing dominant negative JNK. (D) Huh-7 and FCA4 cells were transfected with the STAT-3-responsive pLucTKS3 luciferase plasmid along with dominant negative mutants of JNK (p54JNK2{alpha}) and STAT-3 (STAT-3S727A). At 36 h posttransfection, FCA4 cells expressing pLucTKS3 alone were treated with inhibitors of p38 MAPK (SB203580) and JNK (SP600125) at various times before preparing the lysates for luciferase activity assays.

Since the inhibitors of JNK and p38 MAPK pathways significantly inhibited the HCV-induced STAT-3 activation, we directly performed the in vitro kinase assays of JNK and p38 MAPK in HCV replicon-expressing cells. Cellular extracts from Huh-7 and FCA4 cells were immunoprecipitated with anti-phospho-JNK and anti-p38 MAPK antibodies. The resulting immunokinase complexes were assayed in vitro using unphosphorylated STAT-3 as an exogenous substrate. The results illustrated in Fig. 4 show that FCA4 cells contained activated p38 MAPK and JNK enzymes (Fig. 4A and B, lanes 2). Huh-7 lysates did not exhibit any kinase activity (lanes 1). The activities of these MAPKs involve monitoring the enzymatic incorporation of [{gamma}-32P]ATP in the STAT-3 protein. These results support the notion that HCV-induced activation of STAT-3 is regulated by both JNK and p38 MAPK.



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  		FIG. 4. HCV replicon activates MAPKs. Equal amounts of cellular lysates were immunoprecipitated with anti-p38 MAPK and anti-phospho-JNK antibodies, and the kinase activities were measured in an in vitro kinase assay using [{gamma}-32P]ATP and unphosphorylated STAT-3 as substrate. Labeled STAT-3 was resolved by SDS-PAGE and visualized by autoradiography. Lanes 1, Huh-7 lysates; lanes 2, FCA4 lysates. The bottom panel represents the total p38 MAPK and phospho-JNK activities in Huh-7 and FCA4 lysates, respectively.

ROS activates both tyrosine and MAP kinases. Previous studies have shown that ROS can stimulate the phosphorylation of several kinases, such as Src and MAPK (1, 20). We have previously shown that HCV gene expression induces ROS production (24). To examine the status of tyrosine and serine kinases under conditions of oxidative stress in replicon-expressing cells, FCA4 cells were treated with antioxidant (PDTC) and cellular lysates were subjected to Western blot analysis. The results showed that the activities of Src, p38 MAPK, and JNK were inhibited in the presence of the antioxidant PDTC in FCA4 cells (Fig. 5, compare lanes 2 and 3). Huh-7 cellular lysates did not contain activated kinases. These results demonstrate a role for ROS in mediating the activation of these kinases in HCV replicon-expressing cells.



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  		FIG. 5. HCV replicon-induced c-Src, p38 MAPK, and JNK are sensitive to antioxidants. Huh-7 and FCA4 cells were treated with the antioxidant PDTC (100 µM) for 6 h. Equal amounts of cellular lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-Src, anti-phospho-p38 MAPK, and anti-phospho-JNK antibodies. Lanes 1, Huh-7 lysates; lanes 2, FCA4 lysates; lanes 3, FCA4 lysates treated with antioxidant (PDTC) for 6 h before harvesting the cells.

HCV replicon induces Bcl-XL and cyclin D1 expression. It has been previously shown that STAT-3 activation results in induction of cell growth regulatory genes (reviewed in reference 4). To demonstrate that activated STAT-3 in HCV replicon-expressing cells (FCA4) induces the expression of STAT-3-responsive genes, we examined the STAT-3-dependent cyclin D1 promoter-luciferase (–1745 CD1 Luc) activity in FCA4 cells. The results showed that the cyclin D1 luciferase reporter activity was elevated to ~2.5-fold in FCA4 cells and the expression of dominant negative mutants of Src, JNK, and STAT-3 in FCA4 cells significantly reduced the luciferase reporter activity (Fig. 6A). We also examined the expression of antiapoptotic protein Bcl-XL in FCA4 cells. The results of this analysis showed that the elevated levels of Bcl-XL expression during Western blot analysis were inhibited in FCA4 cells expressing dominant negative mutants of STAT-3 (Fig. 6B, lanes 3 and 4). These results collectively demonstrate that HCV translation-replication activities modulate STAT-3-responsive cellular gene expression.



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  		FIG. 6. HCV replicon induces cyclin D1 and Bcl-XL expression. (A) Huh-7 and FCA4 cells were transiently transfected with cyclin D1 luciferase reporter along with the dominant negative mutants of Src (pM5Hmet295), JNK (p54JNK2{alpha}), and STAT-3 (pGS5hSTAT-3ß; STAT-3S727A). At 36 h posttransfection, cellular lysates were prepared for luciferase activity assays. (B) Equal amounts of Huh-7 and FCA4 lysates expressing dominant negative mutants of STAT-3 were subjected to SDS-PAGE and immunoblotted with anti-Bcl-XL serum. Lane 1, Huh-7 lysates; lane 2, FCA4 lysates; lanes 3 and 4, FCA4 lysates expressing STAT-3 dominant negative mutants.

Constitutive activation of STAT-3 in HCV-infected liver. A growing number of tumor-derived cell lines as well as samples from human cancers are reported to contain constitutively activated STAT-3 (reviewed in reference 6). In this study, we utilized the liver biopsy specimens from an HCV-infected patient and analyzed the constitutive activation of STAT-3. Equal amounts of cellular lysates from HCV-infected and uninfected liver biopsies were subjected to SDS-PAGE and immunoblotted with anti-phospho-STAT-3 Tyr705 antibody. The result displayed the phosphorylated form of STAT-3 in HCV-infected liver biopsy compared to uninfected liver tissues (NASH) (Fig. 7).



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  		FIG. 7. Constitutive activation of STAT-3 in the liver of HCV-positive patients. Cellular extracts were prepared from liver biopsies of HCV-infected and uninfected (NASH) patients as described in Materials and Methods. Equal amounts of cellular extracts were subjected to SDS-PAGE and electroblotted onto a nitrocellulose membrane. The membrane was then immunoblotted with anti-phospho-STAT-3 antibody. Lane 1, cellular lysates from NASH; lane 2, cellular lysates from HCV-infected patients.

HCV replicon induces ROS generation. Next, we examined whether HCV replicon expression triggers the production of ROS. Cells stably expressing replicon (FCA4) (Fig. 8A) or Huh-7 cells transiently transfected with BM4-5 RNA (Fig. 8B) were stained with dihydroethidium (DHE) and subjected to flow cytometry. Results described in Fig. 5 demonstrated the production of ROS as evidenced by an increase in ethidium staining of DNA. DHE is oxidized to ethidium by superoxide radicals. Once oxidized, ethidium is free to intercalate with DNA in the nucleus, where it emits fluorescence at 605 nm. We have previously shown that HCV NS5A alone or in the context of other nonstructural proteins induces ROS in cultured cells (24). These data implicate a direct role of HCV translation-replication activities in triggering the production of ROS.



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  		FIG. 8. HCV replicon induces intracellular production of ROS. (A) Huh-7 and FCA4 cells were treated with 4 µM DHE for 45 min. Cells were harvested, and ROS levels were measured by flow cytometry with excitation emission at 605 nm. The bars show the fold increase in oxidized DHE fluorescence. (B) Untransfected (control) or in vitro-synthesized BM4-5 RNA-transfected Huh-7 cells were treated with 4 µM DHE. ROS levels were assessed in untransfected cells (blue) and cells transfected with BM4-5 RNA (red).

Effect of STAT-3 on HCV replication. To evaluate the effect of STAT-3 activation on HCV RNA replication in HCV replicon-containing cells, FCA4 cells were treated with the inhibitors of tyrosine and MAP kinases, which are known to activate STAT-3. To determine the levels of HCV RNA, total cellular RNA was extracted from Huh-7 and FCA4 cells and quantified by real-time RT-PCR. The results showed an approximate 35 to 50% decline in HCV RNA levels in FCA4 cells that were incubated with each tyrosine and MAP kinase inhibitor (AG490, PP2, SB203580, and SP600125) (Fig. 9A). Since overall activation of STAT-3 requires several kinases, each making a specific impact, a maximal reduction of HCV RNA was not observed by the treatment of a given specific kinase inhibitor. Next, we examined the collective effect of tyrosine and serine kinase inhibitors in the STAT-3 activation process, FCA4 cells were treated with the inhibitors of JAK, Src, JNK, and p38 MAPK. The results showed a significant reduction (~66%) in HCV RNA levels (Fig. 9A). To further confirm the role of Src kinase and STAT-3 on HCV replication, FCA4 cells were transiently transfected with the expression vectors encoding a Src dominant negative mutation (Src dn) and STAT-3 dominant negative mutations (STAT-3 Tyr705 and STAT-3 Ser727). The quantitative real-time RT-PCR results showed a significant decrease in the levels of HCV RNA in FCA4 cells transfected with Src dn and STAT-3 dn vectors (Fig. 9A). To demonstrate that the effect of STAT-3 on HCV replication is not specific to the FCA4 stable cell line, we also performed transient transfections of Huh-7 cells with an in vitro-synthesized subgenomic replicon BM4-5 RNA (25) along with the Src dominant negative mutant (Src dn) and STAT-3 dominant negative mutants (Tyr705 and Ser727). In addition, the BM4-5 RNA transfected cells were also treated with the inhibitors of tyrosine and MAP kinases. These transient-transfection results showed a similar decrease of HCV RNA levels by these treatments (Fig. 9B). These results clearly indicate that STAT-3 positively regulates HCV replication. Because HCV nonstructural proteins play a critical role in HCV replication (29), we examined whether the expression of HCV NS5A, a representative gene encoded by HCV subgenomic replicon RNA, is altered by tyrosine and MAP kinase inhibitors. The results showed that the FCA4 cells treated with tyrosine and MAP kinase inhibitors displayed reduced levels of NS5A protein expression similar to the RNA levels observed in Fig. 9A and B (Fig. 9C, lanes 3 to 6).



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  		FIG. 9. Effect of STAT-3 on HCV RNA replication. (A, B, and D) FCA4 cells and Huh-7 cells transiently transfected with in vitro-synthesized BM4-5 RNA were first incubated with the inhibitors of tyrosine and MAP kinases (AG490, PP2, SB203580, and SP600125) and the antioxidant PDTC overnight. The replicon-expressing cells were also transiently transfected with the dominant negative expression vectors of Src (pM5Hmet295), STAT-3 Tyr705 (pSG5hSTAT-3ß), and STAT-3 S727A. The total RNA was extracted and subjected to quantitative RT-PCR analysis. The data are expressed as relative HCV RNA levels in relation to the RNA levels in the control cells. (C and E) FCA4 cells were treated with the inhibitors of tyrosine and MAP kinases (AG490, PP2, SB203580, and SP600125) and the antioxidant PDTC overnight. Equal amounts of cellular lysates were subjected to SDS-PAGE and Western blotted with anti-NS5A serum. (C) Lanes 1 and 2, equal amounts of Huh-7 and FCA4 lysates; lane 3, 4, 5, and 6, FCA4 lysates treated with PP2, AG490, SP600125, and SB203580, respectively. (E) Lanes 1 and 2, equal amounts of Huh-7 and FCA4 lysates; lane 3, FCA4 lysates treated with PDTC.

Since ROS induced the activation of STAT-3 in HCV replicon-expressing cells, we also analyzed the role of ROS in HCV replication. FCA4 cells were treated with the antioxidant PDTC and the levels of cellular RNA were quantified by real-time RT-PCR as described above. These results showed decreased expression of HCV RNA in FCA4 cells treated with PDTC (Fig. 9D). Similarly, the expression of HCV NS5A protein was reduced in the presence of PDTC (Fig. 9E, lane 3). Similar results were observed with FCA4 cells transiently transfected with an Mn-superoxide dismutase expression vector (data not shown). These data collectively implicate a potential role of ROS and activated STAT-3 in HCV RNA translation and replication.


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DISCUSSION
 
The activities of RNA translation and replication associated with the HCV life cycle cause induction of the ER stress activating unfolded protein response and an ER overload response (24, 45). One of the consequences of the ER overload response is the activation of STAT-3 via Ca2+ signaling and induction of ROS, triggering an ER-to-nucleus signal transduction pathway (24, 35). STAT-3 protein controls fundamental cellular processes, including survival, proliferation, and differentiation (4). The exact signal HCV gene expression triggers in these intracellular events remains to be characterized. STAT-3-responsive motifs are found in several cellular and viral genes (4, 51), and elevated levels of STAT-3 activity have been observed in various human cancers as well as in cell lines transformed with v-Abl and v-Src (8, 23). This constitutive activation of STAT-3 is not due to mutations in STAT-3 but occurs due to deregulations of protein tyrosine kinases or constitutive release of growth factors that activate STAT-3 (22). Once STAT-3 is activated in HCV-infected cells, it may regulate gene expression of survival factors to ensure an antiapoptotic environment in the cells, a situation favorable for oncogenesis (6, 11). STAT-3 exerts its growth-deregulating activity by activating the expression of cellular genes that are involved in cell cycle progression, such as fos, cyclin D1, myc, and pim-1, and by activating antiapoptotic proteins, such as Bcl-2 and Bcl-XL (5, 6, 11, 41). Inactivation of STAT-3 function in transformed or tumor cells can reverse cell transformation and/or induce apoptosis in vitro (33). The mechanisms that block initiation of immune responses during cancer development are poorly understood. Recently, it has been demonstrated that STAT-3 activation in tumors negatively regulates induction of innate and adaptive immunity (50).

It is well established that the activation of STAT-3 requires tyrosine phosphorylation, which occurs in response to growth factors and cytokine signaling (62). Previous studies have shown that activation of STAT-3 is associated with alpha interferon antiviral activity (56, 63). In addition to these agonists, STAT-3 can also be activated by oxidative stress (10, 24). In this study, we investigated the molecular mechanism(s) of STAT-3 activation in response to oxidative stress induced by HCV gene expression. One possibility is that the alteration of their redox status could directly alter their conformation in such a way that their interaction with cytosolic proteins responsible for nuclear targeting is triggered. The other likely possible explanation is the ability of oxidants to act as inhibitors of tyrosine phosphatases, thereby inducing STAT-3 nuclear translocation by enhanced tyrosine phosphorylation.

In the present analysis, we observed that HCV subgenomic replicon-induced STAT-3 activation was sensitive to antioxidants and calcium chelators (Fig. 1C and D). This is consistent with the previous observations that tyrosine phosphorylation and activation of STAT-3 occur under conditions of oxidative stress (10, 24). The evidence for the increased level of ROS in HCV replicon-expressing cells is shown in Fig. 8. Previously, the role of ROS in viral pathogenesis has been documented for influenza virus and human immunodeficiency virus (21, 32). An overwhelming number of studies support the role of ROS in the initiation and progression of multistage carcinogenesis (42). An important part of the cellular defense to oxidative stress is the specific induction of gene expression in response to specific oxidative stressors. Consistent with this idea, free radical scavengers and antioxidant enzymes are down-regulated in chronic hepatitis C patients and tumor cells (14, 31).

Previous studies have shown that ROS can stimulate the phosphorylation of several kinases, such as JAK, Src, and MAP kinases (1, 20, 40), and these kinases are known to induce the phosphorylation and subsequent activation of STAT-3 (47, 48, 57, 58). Using in vitro kinase assays and schemes of reporter gene expressions, we demonstrated that both tyrosine kinases (Src and JAK2) and MAPKs (JNK and p38 MAPK) are important for STAT-3 phosphorylation and subsequent activation. The involvement of these kinases in HCV-induced STAT-3 activation was further illustrated by the use of selective inhibitors of tyrosine and MAP kinases and dominant negative mutants of c-Src and JNK (Fig. 2 and 3). The autophosphorylation and activation of JAKs are known to stimulate the ligand-mediated activation of STAT-3 (62), but the role of JAKs in HCV-induced constitutive activation of STAT-3 is poorly understood. Previous studies have demonstrated that JAKs are constitutively activated by v-Src, and another study supported a model in which JAKs serve to recruit STAT-3 to Src, which in turn directly phosphorylates and activates STAT-3 (9, 60). Importantly, we demonstrated that the activation of STAT-3 in HCV replicon-expressing cells modulates cell growth regulatory genes Bcl-XL and cyclin D1 (Fig. 6), which may lead to HCV-mediated pathogenesis.

The biological significance of STAT-3 tyrosine phosphorylation is very well studied, but the role of STAT-3 serine phosphorylation is not well documented. Our results demonstrated that the expression of STAT-3 S727A, the dominant negative mutant of STAT-3, reduced the STAT-3-dependent reporter activity by 40% during cell-based assays, suggesting that STAT-3 serine phosphorylation plays an important role in HCV-induced STAT-3 activation. This is consistent with an earlier study, in which expression of dominant negative STAT-3 S727 suppressed STAT-3 signaling and Src transformation (48). This implies that STAT-3 is the point of convergence for tyrosine and serine kinases (54). Recently, the functional importance of STAT-3 Ser727 phosphorylation was demonstrated in mice by mutating Ser727 to alanine (38). Mice with the STAT-3 S727A mutant reduced the transcriptional potential by 50% (38). Serine phosphorylation has been reported to increase transcriptional activation by a number of different proteins (26). A possible mechanism by which serine phosphorylation generally increases transcription has been described. The coactivator protein CBP has been shown to bind to phosphorylated CREB or AP-1 protein much better than it does to unphosphorylated proteins (12). Thus, there may be a general requirement for many resident nuclear transcription factors to be phosphorylated on serine to be maximally active in the assembly of active transcription complexes. In addition to enhanced transcriptional activity induced by serine phosphorylation, repression of STAT signaling has also been associated with serine phosphorylation events under certain conditions (4).

HCV infection is characterized by elevated levels of ROS in patients (19, 43). Previous studies have shown that ROS can regulate viral replication (37, 61). In our model, HCV-induced ROS is a focal point from which signals emanate, leading ultimately to activation of STAT-3 (24) (Fig. 10). We have recently reported a similar model for the activation of NF-{kappa}B (52). It is generally believed that STAT-3 is involved in liver regeneration and growth regulation (28), but its role in HCV replication has not been reported. The establishment of the subgenomic replicon system provides an effective cellular system for the study of the dynamics of HCV RNA replication. Using this system, we examined the effect of STAT-3 on HCV replication by quantitative real-time RT-PCR. Our results showed enhanced replication of HCV RNA in replicon-containing cells, which contained activated STAT-3 (Fig. 9). The inhibition of STAT-3 activity either by specific inhibitors of tyrosine and MAP kinases or by overexpression of dominant negative c-Src and STAT-3 reduced the levels of HCV replicon RNA (Fig. 9A and B), suggesting that the constitutive activation of STAT-3 may be involved in upregulation of HCV RNA replication. In contrast to these results, a recent study described reduced levels of HCV RNA in cells incubated with an exogenous source of ROS (H2O2) (13). In our model, HCV gene expression induced ROS, and inactivation of ROS led to a decrease in HCV replication (Fig. 9C and D). These results implicate a possible role for ROS in HCV RNA replication.



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  		FIG. 10. Model illustrating the mechanism(s) of HCV replicon-induced activation of STAT-3 via oxidative stress. This pathway involves the activation of tyrosine and MAP kinases in HCV replicon-expressing cells. By a mechanism not clearly understood, STAT-3 enhances HCV RNA replication.

In summary, we have shown that constitutive activation of STAT-3 by the HCV subgenomic replicon involves activation of tyrosine (JAK and Src) and MAP (JNK and p38 MAPK) kinases (Fig. 10). Our data show that STAT-3 has the ability to potentiate HCV replication and translation. Protein kinase activation by ROS may be, at least partially, responsible for STAT-3-mediated upregulation of HCV RNA replication. These observations open avenues for future studies on the cellular and molecular mechanism(s) involved in regulation of HCV replication. Our data also provide important clues to the understanding of the mechanisms of chronic liver disease induced by oxidative stress and other intracellular events associated with HCV infection.


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ACKNOWLEDGMENTS
 
This work is supported by a grant from the National Institutes of Health (DK 61566 to A.S.).

We thank C. Seeger (Fox Chase Cancer Institute, Philadelphia, Pa.) for the generous gift of FCA4 and BM4-5, L. Heasley (University of Colorado Health Sciences Center, Denver) for the JNK dominant negative mutant, and G. Pestell (Lombardi Cancer Center, Georgetown University, Washington, D.C.) for the human cyclin D1 luciferase reporter construct.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Colorado HSC, 4200 East Ninth Ave., Denver, CO 80262. Phone: (303) 315-7016. Fax: (303) 315-8330. E-mail: aleem.siddiqui{at}uchsc.edu. Back


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Journal of Virology, February 2005, p. 1569-1580, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1569-1580.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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