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Virology 288, 379–390 (2001)doi:10.1006/viro.2001.1100, available online at http://www.idealibrary.com on

Hepatitis C Virus Core Protein Modulates the Interferon-Induced Transacting Factors of Jak/Stat Signaling Pathway But Does Not Affect the Activation of Downstream IRF-1 or 561 Gene

Arnab Basu,* Keith Meyer,* Ratna B. Ray,*,† and Ranjit Ray*,‡,1

*Departments of Internal Medicine, †Pathology, and ‡Molecular Microbiology and Immunology,Saint Louis University, St. Louis, Missouri 63110

Received May 8, 2001; returned to author for revision July 10, 2001; accepted July 18, 2001

Hepatitis C virus (HCV) has a propensity to cause chronic infection, with a low proportion of patients exhibiting a sustainedresponse to interferon-a (IFNa) therapy. An earlier report suggested that HCV inhibits IFNa-induced signal transductionthrough the Jak/Stat pathway by preventing the formation of the transacting factor ISGF3 complex, although the effect ondownstream pathway and the specific viral protein responsible for inhibition of IFNa-mediated signal transduction were notelucidated. HCV core protein displays a number of intriguing functional properties and has been implicated in virus-mediatedpathogenesis. In this study, we have analyzed the effect of core protein upon IFNa- or IFNg-induced regulation of the Jak/Statsignaling pathway. HCV core protein expression exhibited a reduced Stat1 expression in IFN-treated mammalian cells. A gelretardation assay suggested a reduced level of formation of the transacting factors, GAF and ISGF3, in IFN-treated cells.Further studies from protein expression and RNase protection assay revealed that the reduced level of GAF or ISGF3formation could be attributed to modulation of Stat1 protein expression, an important player for innate immunity in hostdefense mechanism. However, these modulatory effects did not interfere with the activation of the downstream effectorgenes, IRF-1 and 561, in IFN-treated cells. Stable transfectants of cells after introduction of a plasmid DNA encoding both thestructural and the nonstructural proteins of HCV also exhibited a similar effect. Taken together, these results suggest thatalthough expression of the core protein alone or with other HCV proteins modulate transacting factors of Jak/Stat signaling

pathway, expression of the downstream effector genes IRF-1 and 561 remains unaffected upon IFN treatment and maycontribute to host defense mechanism. © 2001 Academic Press

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INTRODUCTION

Hepatitis C virus (HCV) often causes a prolonged andpersistent infection in humans. The most important fea-ture of persistent HCV infection is the development ofchronic hepatitis in half of the infected individuals, andthe potential for disease progression to hepatocellularcarcinoma (Saito et al., 1990; DiBisceglie et al., 1998;Hayashi et al., 1999; Jeffers, 2000). This has been attrib-uted in part to the effect of viral gene product(s) on theexpression of host cellular genes. Unfortunately, a num-ber of important issues related to HCV persistence anddisease progression are unknown at this time. Further-more, neither a vaccine nor any other means of aneffective chemotherapy is available to eradicate HCVinfection. HCV genotypes 1a and 1b are predominant inpatients with chronic hepatitis C in the United States(Zein et al., 1996), and interferon-a (IFNa) alone or in

ombination with ribavirin is the current available ther-py (Martin et al., 1998). However, a significant number ofases do not respond well to therapeutic treatment, and

1 To whom correspondence and reprint requests should be ad-ressed at Division of Infectious Diseases and Immunology, Saint

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ouis University, 3635 Vista Avenue, St. Louis, MO 63110. Fax: (314)71-3816. E-mail: rayr@slu.edu.

379

he reason is not clear (Moradpur and Blum, 1999;uidotti and Chishari, 2000).Signal transducers and activators of transcription

STATs) have drawn considerable attention due to theirnique mode of action, and diversity of biological effects,hich include the ability to control an immune responsend an antiviral effect and to participate in cell transfor-ation (Ihle, 1996; O’Shea, 1997). IFNa and IFNg bind to

heir respective cell surface receptors and activate dis-inct but related signal transduction pathways, culminat-ng in the activation of an overlapping set of interferon-ensitive genes (ISGs). Upon binding of IFNa/b to recep-

tors, their associated protein kinases (Jak1 and Tyk2)phosphorylate Y701 of Stat1 and Y690 of Stat2. Stat1 andStat2 then heterodimerize, translocate to the nucleus,and associate with p48 to form ISG factor 3 (ISGF3).ISGF3 activates the transcription of genes containingIFN-stimulated response elements (ISREs) within theirpromoters. The full activity of Stat1 also depends uponthe phosphorylation of S727 via the mitogen-activatedprotein kinase pathway. On the other hand, IFNg upon

inding to specific receptors will similarly phosphorylate701 of Stat1 through their associated protein tyrosineinases (Jak1 and Jak2). Phosphorylated Stat1 ho-

odimerizes to form g-activated factor (GAF), which ac-

ivates the transcription of genes containing g-activated

0042-6822/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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380 BASU ET AL.

sequence (GAS) elements within their promoters. Phos-phorylation of Stat1 on S727 is also required for the fullactivity of GAF (Darnell et al., 1994; Stark et al., 1998).

MHC class II expression is controlled predominantlyat the level of transcription (Boss, 1997). IFNa/b andIFNg has been shown to enhance the expression of

HC class I and MHC class II molecules (Taniguchi etl., 2001). The expression of MHC class II molecules islso uniquely regulated by IRF-1, via an indirect mecha-ism. The expression of CIITA, a critical transcription

actor for MHC class II gene induction by IFNg, requirestat1 and IRF-1 (Muhlethaler-Mottet et al., 1998;

Piskurich et al., 1999). IFNg can induce transcription ofgenes through many different cis elements (Leonard and

en, 1996; Muhlethaler-Mottet et al., 1998). GAS hasbeen found in the regulatory regions of the high-affinityIgG receptor gene, the IRF-1 gene, and the Ly-6A/E gene.The GAS element is both necessary and sufficient toconfer transcriptional activation of IRF-1 gene by IFNg.

The genomic region encoding the HCV core protein isocated between amino acids 1 and 191 and is likely toe the first gene product synthesized in virus-infectedells due to its localization at the 59 end of the HCVolyprotein transcript. In addition to its ability to interactith the viral genomic region to form nucleocapsids

Shimoike et al., 1999), the presence of a putative DNA-inding motif, nuclear localization signals, phosphoryla-

ion sites, and a nucleocytoplasmic localization of theore protein suggest its possible function as a geneegulatory protein (Shih et al., 1993). A role for HCV corerotein in the transcriptional regulation of cellular pro-oters has been observed (Kim et al., 1994; Ray et al.,

995, 1997, 1998b; Chang et al., 1998; Shrivastava et al.,998; Lu et al., 1999; Ray et al., 2000; Otsuka et al., 2000;

Bergqvist and Rice, 2001). In the course of these studieson transcriptional regulation, we have analyzed a num-ber of core protein-associated functional properties, in-cluding the regulation of apoptotic cell death and thepromotion of cell growth (Ray et al., 1996a,b, 1998a, 1997,2000). While all of these functions may be important inensuring the survival of HCV in the human host, theprecise mechanism of core protein-mediated functionshas yet to be delineated.

An earlier report using an inducible system expressingthe entire HCV open reading frame in human osteosar-coma cells suggested that HCV inhibits IFNa-inducedsignal transduction through the Jak/Stat pathway by pre-venting the formation of the transacting factor ISGF3complex (Heim et al., 1999). However, the specific viralprotein responsible for an inhibition of IFNa-mediatedsignal transduction and the effect on downstream path-way was not examined. In this study, we have investi-gated whether the expression of HCV core protein aloneand together with other HCV proteins may modulate the

cytokine-inducible gene expression of the Jak/Stat signaltransduction pathway in IFNa- or IFNg-treated cells. Our

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results suggest that expression of HCV protein(s) mod-ulates the formation of the GAF and ISGF3 complexesbut does not interfere with the IFN-stimulated activationof the downstream IRF-1 or 561 gene.

RESULTS

Effect of HCV protein on Stat1 expression in IFN-treated cells

Interferon treatment of cells causes immediate activa-tion of the transacting factor GAF and ISGF3. Once acti-vated they translocate from cytoplasm to nucleus, bind tothe ISRE or GAS element of IFN inducible genes, andactivate their transcription. Since Stat1 is a component ofboth the activation factors GAF and ISGF3 complex, thedistribution of Stat1 was investigated by an indirect im-munofluorescence using mock transfected control, orstable transfectants of HeLa cells expressing core pro-tein alone or together with other HCV proteins in un-treated and IFN-treated cells. Additionally, stable trans-fectants of HeLa cells expressing CD4 antigen (HeLaT4)was included in this experiment to verify the effect of anunrelated ectotopic protein expression upon transloca-tion of Stat1 following IFN treatment. Untreated cellsexhibited a weak intracellular localization of the Stat1protein (Figs. 1a, 1d, 1g, and 1j). However, a bright nu-clear fluorescence of Stat1 was observed upon treat-ment of control HeLa or HeLa T4 cells with IFNa or IFNg(Figs. 1b, 1c, 1k, and 1l). In contrast, a diffuse and pre-dominantly perinuclear localization of Stat1 was ob-served in IFN-treated HeLa cells expressing core proteinalone or together with other HCV proteins (Figs. 1e, 1f,1h, and 1i). IFNa or IFNg treatment of HCV proteinexpressing HT1080 or HepG2 cells exhibited a relativelyweak nuclear translocation of Stat1 when compared tothe mock transfected control (data not shown). Thus,HCV core protein is probably modulating the Jak/Statpathway at a stage prior to GAF or ISGF3 activation.

HCV core protein expression reduces the level ofIFN-induced GAF and ISGF3 transacting factors

To investigate the possible role of HCV core protein inGAF or ISGF3 activation, a gel retardation assay wasperformed. IFN-treated or untreated HeLa, HepG2, andHT1080 cells stably expressing core alone or togetherwith other HCV proteins were used for this purpose.Mock transfected cells were separately used as control.Formation of a DNA–protein complex with the mobilityand specificity of GAF was observed in both HeLa andcore expressing HeLa cells following IFNg treatment.

he image of the separation profile is shown in Fig. 2A.he specificity of GAF complex was verified using an

nti-Stat1 antibody which disrupted the intensity of theAF–DNA complex. While use of an anti-Stat2 antibody

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381HCV CORE PROTEIN AND ISGF3/GAF MODULATION

or unrelated antibody (data not shown in figure) as con-trol did not inhibit GAF–DNA complex formation. In somecases, addition of antibody disrupts the formation of thecomplex rather than further changing its mobility (Sam-brook and Russell, 2001). This may be due to high affinityof the antibody to Stat1, resulting in competition of theDNA binding sites for complex formation. Our observa-tion of a decreased intensity of the GAF complex usingan antibody to Stat1 is similar to those reported earlier byother investigators (Li et al., 1998, 1999). The uncharac-terized band below GAF appeared in extracts of bothuntreated and IFN-treated cells, as observed earlier(Levy et al., 1990; Li et al., 1998). This complex was

FIG. 1. Effect of HCV protein on Stat1 expression in IFN-treated HeLrotein alone (d, e, and f), or together with other HCV proteins (g, h, and

j, k, and i) were either left untreated (a, d, g, and j) or treated with IFNtemperature. Cells were incubated with a monoclonal antibody to Sisothiocyanate was added for cell staining. Treatment with IFNg or IFNacells. In contrast, cells expressing core protein alone or together with otin the perinuclear regions. Arrows indicate the distribution of Stat1 in

specifically inhibited by antibody to Stat1 or Stat2; how-ever, anti-Stat2 antibody did not inhibit GAF formation.

These complexes may represent inactivated heteromericform of the Stats and awaits further characterization.

Densitometric scanning of the autoradiogram sug-gested that the GAF–DNA complex is ;4.8-fold higher inmock transfected control cells as compared to the coreprotein transfected HeLa cells (Figs. 2A and 2B). TheGAF–DNA complex formation was also examined inHeLa cells stably transfected with HCV1–2962 plasmid.Mock transfected control HeLa cells exhibited an ;2.5-fold higher level of GAF–DNA complex formation thanHeLa cells expressing core along with other HCV pro-teins (Figs. 2A and 2B). A similar finding was observed inHT1080 and HepG2 cells. The GAF–DNA complex for-

Mock transfectants (a, b, and c), stable transfectants expressing coreHeLa cells expressing unrelated CD4 protein as an additional control

U/ml (b, e, h, and k) or 500 U/ml IFNa (c, f, i, and j) for 30 min at roomA second antibody, goat anti-mouse Ig, conjugated to fluorescein

ited a strong and distinct nuclear immunofluorescence in control HeLaV proteins displayed a weak Stat1 expression, predominantly localizedated control and HCV gene transfected cells.

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382 BASU ET AL.

FIG. 2. Inhibition of GAF–DNA complex formation by HCV protein(s) in IFNg-treated cells. Mock transfected control or experimental cells (as markedn top) were either treated with IFNg (500 U/ml for 90 min) or left untreated. Nuclear extracts from cells were examined for GAF–DNA complex

formation by a gel retardation assay with a probe corresponding to the GAS element of the IRF-1 gene (a, c, and e). The addition of an antibody to

Stat1 in the nuclear extract–DNA reaction mixture inhibited GAF–DNA complex formation. The amount of complex formation was quantitated bydensitometric scanning and presented as a bar diagram (b, d, and f).

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383HCV CORE PROTEIN AND ISGF3/GAF MODULATION

HCV core or HCV1–2962 plasmid DNA (Figs. 2C and 2D).Similarly, an increased GAF–DNA complex formation(;twofold) was observed in mock transfected HepG2cells as compared to HepG2 cells transfected with coreor HCV1–2962-expressing plasmid (Figs. 2E and 2F). Fur-her analysis with an antibody to Stat1 verified inhibitionf GAF–DNA complex formation in both HT1080 andepG2 cells and identified Stat1 as a component of the

omplex.ISGF3–DNA complex formation was also analyzed us-

ng ISRE as a probe in IFNa-treated control and core-

FIG. 3. Inhibition of ISGF3–DNA complex formation by HCV proteinmarked on top) were treated with IFNa (1000 U/ml for 90 min) or left unormation by gel retardation assay with a probe corresponding to the ISn the nuclear extract–DNA reaction mixture inhibited ISGF3–DNA censitometric scanning and presented as fold difference of the ISGF3

expressing HeLa cells (Fig. 3A). The mobility and spec-ificity of the ISGF3–DNA complex was verified using

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Stat-specific antibodies which competed with the pro-tein–DNA complex formation. The ISGF3–DNA complexwas ;threefold higher in mock transfected HeLa controlas compared to core or HCV1–2962-transfected HeLa cellsFig. 3B). A similar result of ;threefold higher ISGF3–

NA complex formation was observed in the control overore-transfected HT1080 cells (Figs. 3C and 3D) and inepG2 cells (data not shown). The gel shift assays forAF and ISGF3 DNA complex formation were reproduc-

ble from at least three different experiments. Together,ur results indicated that the HCV core protein modu-

Na-treated cells. Mock transfected control or experimental cells (as. Nuclear extracts from cells were examined for ISGF3–DNA complex

ment of the 561 gene (a and c). Addition of antibodies to Stat1 or Stat2x formation. The amount of complex formation was quantitated byomplex in different cell lines (b and d).

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ates both GAF and ISGF3 protein complex formation inFN-treated cells.

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384 BASU ET AL.

Effect of IFN on expression of the signaling proteins

Our results indicated that HCV core-protein expres-sion lowers the level of GAF and ISGF3 in IFN-treatedcells. Thus, we investigated the protein expression levelof Stat1 and p48 in control and core-expressing HeLacell lysates by Western blot analysis using specific anti-bodies. Results suggested an approximately threefolddecrease in Stat1 protein expression in core-expressingHeLa cells than mock transfected control cells (Fig. 4A).However, the level of p48 remained similar in both theexperimental and the control cells (Fig. 4C).

The phosphorylated Stat1 level was also investigatedin IFNa-treated control and experimental HeLa cells bymmunoprecipitation. Cell lysates were immunoprecipi-ated by a Stat1 monoclonal antibody and subjected to

estern blot analysis using an antibody directed to thehosphotyrosine of Stat1 (Fig. 4B). The phosphorylated

orm of the Stat1 protein was barely detectable in core-xpressing HeLa cells and could be due to lower Stat1xpression in HeLa cells. In contrast, phosphorylatedtat1-a polypeptide was clearly visible in mock trans-

ected HeLa cells. A relatively lower level of Stat1 proteinxpression was also observed in HCV core-transfectedT1080 (Fig. 4E) and HepG2 cell lysates (Fig. 4H). On thether hand, expression level of the DNA binding protein48 was similar in both cell lines (Figs. 4F and 4I).ellular actin was used as an internal control for com-arison (Figs. 4D, 4G, and 4J). Further examination of

FIG. 4. Effect of core protein on Stat1 and p48 expression in IFNa-treanes) cells were treated with 1000 U/ml IFNa for 30 min. HeLa cemmunoprecipitates were separated by SDS–8% PAGE and transferredonjugated with horseradish peroxidase. Phosphorylated tyrosine moietat1-a/b and p48 were analyzed by Western blot using a monoclonal

An antibody to cellular actin was used as an internal control (d, g, and j)sizes correlated with the migration of standard molecular weight mark

tat1 mRNA expression by RNase protection assay sug-ested that the level of Stat1 increases greater than

wofold following IFNa treatment in mock transfectedHeLa cells than HCV core-expressing cells (Fig. 5). Sim-ilar observations were made from cells treated with IFNg(data not shown). All of the experiments presented inFigs. 4 and 5 are representative of at least three inde-pendent experiments exhibiting similar results. Theseresults suggested that HCV core protein modulates Stat1expression at the transcriptional level. Thus, the coreprotein appears to modulate the Jak/Stat pathways bylowering the abundance of Stat1 protein.

HCV protein(s) does not inhibit induction of IFNstimulated genes

ISRE-mediated signaling was examined from the levelof gene induction of two alternative IFN signaling path-ways. IRF-1 mRNA is induced by IFNg with GAS as a ciselement and GAF as the transcription factors. On theother hand, 561 mRNA is induced by IFNa using ISRE as

cis element and ISGF3 as cognate transcription factor.ellular levels of the IFNg-inducible IRF-1 mRNA and

IFNa-inducible 561 mRNA were analyzed by RNase pro-ection assay. Activation of the IRF-1 mRNA upon IFNreatment (500 U/ml) was observed at a similar level fort least up to 12 h in both mock transfected and core orCV protein expressing HeLa cells (Figs. 6A and 6B),nd a similar result was observed using a lower dose

100 U/ml) of IFNg (data not shown). Activation of IRF-1mRNA was also observed following IFNg treatment of

lls. Mock transfected control (left lanes) or core gene transfected (rights were immunoprecipitated with a monoclonal antibody to Stat1-a.

rocellulose for probing with a monoclonal antibody to phosphotyrosineStat1-a were detected by chemiluminescence (b). Expression levels ofy to Stat1-a (a, e, and h) and a polyclonal antibody to p48 (c, f, and i).

s on the right indicate the respective polypeptides, and their molecularbco BRL).

ated cell lysateonto nitties ofantibod

HepG2 cells (Figs. 6C and 6D). Investigation for 561 geneinduction suggested an increase in mRNA level within

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385HCV CORE PROTEIN AND ISGF3/GAF MODULATION

6 h of IFNa treatment, which then decreased slightlyetween 6 and 12 h in HepG2 cells (Fig. 6E). The reason

or this decline of 561 mRNA is unclear at present.ime-dependent activation of 561 was also observed ineLa cells (data not shown). These results suggested

hat even a reduced level of GAF or ISGF3 complex isufficient to activate the interferon-stimulated down-tream effector genes of Jak/Stat signaling pathways,nd this effect appeared to be dependent upon IFNtimulation.

DISCUSSION

Results from this study indicate that expression of corerotein alone or together with other HCV proteins mod-lates the transacting factors GAF or ISGF3 of the IFN-

nduced Jak/Stat signaling pathway. This may be relatedo a decreased abundance of the Stat1 protein and/or a

eak translocation of Stat1 to the cell nucleus. However,hese modulatory effects do not interfere with IFN-in-uced downstream activation of the IRF-1 and 561 gene

FIG. 5. Analysis for Stat1 mRNA expression in HeLa cells upon IFNatreatment by RNase protection assay. The mRNA expression level of Stat1and L32 (internal control) were determined in untreated (marked U on top)or IFNa (1000 U/ml) treated cells for 6 or 12 h (a). The relative abundance

f the Stat1 mRNA level was estimated by densitometric scanning afterormalization against L-32 and shown as bar diagram (b).

xpression. We have used ectopic expression of HCVrotein in this study as that will be more relevant to the

ature of chronic HCV infection, when IFNa treatment isnormally offered. Our results from expression of HCVproteins agree in part with the reduced GAF or ISGF3formation observed following inducible expression of theentire HCV open reading frame in osteosarcoma cells(Heim et al.,1999), although a difference in Stat1 expres-sion level was observed in our study. This difference inresults could be attributed to inducible vs stable expres-sion system and the nature of the cell lines used. Ourstudy also suggests a difference in expression levelbased on the cell types used. However, our results indi-cate that even a reduced level of GAF or ISGF3 complexin IFN-treated cells is sufficient to activate IS genes,IRF-1 and 561. Thus, we find evidence to suggest a rolefor the HCV core protein in the inhibition or impairment ofthe Stat1 expression, which can be augmented by IFNtreatment for induction of the downstream effector geneof Jak/Stat pathway in HeLa, HT1080, or HepG2 cells.

IFNa regulates the function of cytokines, their recep-tors, and other molecules of immune importance (Good-bourn et al., 2000). A large number of genes are inducedby IFNa, but only a few of these gene products havebeen associated with an intrinsic antiviral activity. Thoseidentified include the p68 protein kinase (PKR) and 29-59-oligoadenylate synthetase (29-59-OAS), which are bothdependent upon double-stranded RNA for activity, cer-tain MxA family proteins, and the most recently identifiedpromyelocyte leukemia protein (Goodbourn et al., 2000).While IFN-treated cells are resistant to many differentviruses, individual overexpression of these intrinsicallyantiviral proteins confers host resistance only againstsome viruses. HCV E2 and NS5A proteins have beenimplicated in IFN resistance through inhibition of PKR(Gale et al., 1997, 1998; Taylor et al., 1999). Inhibitoryproperties of HCV E2 glycoprotein may occur by mimick-ing the autophosphorylation site via a pseudosubstratemechanism (Taylor et al., 2001). PKR inhibition by E2 orNS5A protein may contribute but do not solely explainthe resistance to sustained IFN treatment (Polyak et al.,2000; Francois et al., 2000; Gerotto et al., 2000). Inchronic HCV patients, the expression of 29-59-OAS andMxA are not significantly different at the hepatic mRNAlevel when compared with chronic HBV or other liverdiseases of different etiology (Yu et al., 2000). HCV coreprotein also activates 29-59-OAS gene in hepatocyte celllines, which is further enhanced upon treatment withinterferon (Naganuma et al., 2000). Unfortunately, thelack of a suitable virus-cell culture system for study ofHCV growth does not allow the study the contribution ofIFN induced proteins for antiviral response at this time.

The function of Stat1 is to regulate a set of genes thatcollectively provide innate immunity (Ihle, 1996). The ho-mozygous mutant cells and animals devoid of functionalStat1 protein revealed that Stat1 is indispensable for the

IFN pathway, but may not be necessary for other signal-ing systems for normal development (Durbin et al., 1996).

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386 BASU ET AL.

However, mutant animals demonstrated the essentialnature of Stat1-dependent system for survival in the faceof otherwise innocuous viral pathogens. This may resultfrom the combined loss of both type I and type II IFNresponses, which has been shown to impair some anti-viral responses (van den Broek et al., 1995). Stat1-depen-

FIG. 6. Analysis for IFN-induced IRF-1 and 561 gene expression byan internal control (a and c) were determined in IFNg (500 U/ml) treaabundance of IRF-1 mRNA level (b and d) at 6 and 12 h of IFN treanormalized with respect to actin used as an internal control. The results

epG2 cells for 6 and 12 h are also shown (e).

ent innate immunity, presumably produced in responseo IFN, appears to be a crucial host defense mechanism

combating mouse hepatitis virus (MHV) disease. A ma-jority of humans fail to develop protection against HCVinfection and Stat1 modulation by core protein may con-tribute to this defect.

Although Stats are translocated to the nucleus afteractivation, neither the mechanism underlying this trans-

protection assay. The mRNA expression level of IRF-1 and g-actin asuntreated (marked as U on top) HeLa and HepG2 cells. The relativewas estimated by densitometric scanning of the autoradiogram andsimilar analysis for 561 mRNA expression in IFNa (1000 U/ml) -treated

RNaseted ortment

location nor the reason for their retention in the cyto-plasm prior to stimulation is well understood (O’Shea,

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u(vA

387HCV CORE PROTEIN AND ISGF3/GAF MODULATION

1997). Two mechanisms, ubiquitination and dephosphor-ylation, have been proposed for attenuating Stat activa-tion. However, the relative importance of these twomechanisms and the identity of the Stat phosphatase arenot known. A recent report suggests a complex of un-phosphorylated Stat1 and IRF-1 transcribes the LMP2gene, which would involve them in the processing of viraland tumor antigens for presentation to CD81 T cells inthe context of MHC class I molecules (Chatterjee-Kishore et al., 2000). IFNg is a potent inducer of MHCclass II expression and is activated through Jak/Statpathway. Regulation of MHC class II gene expression byIFNg and other modulators occur primarily at the mRNAranscription, although posttranscriptional modulationas been described. The results from our study are ingreement with the observations that HCV core proteinoes not disrupt the expression of MHC class II mole-ules on cell surface (Hiasa et al., 1998; Large et al.,999). Our data suggest that the core protein when con-titutively expressed in cells decreases Stat1 expres-ion, the level of which differs depending on the cell typesed. However, this effect can be overcome by IFN

reatment to induce the downstream effector gene ex-ression of the Jak/Stat signaling pathways. Stat1 alsolays an important role in growth arrest, in promotingpoptosis, and is implicated as a tumor suppressor

Bromberg and Darnell, 2000). Cross talk between Statnd other cellular signaling pathways can be achievedy the action of certain Stat-associated proteins. Forxample, an interaction between ERK and Stat may con-ect the MAPK and Stat signaling pathways (Shuai,000). IFNg, which activates large amounts of Stat1,

decreases the AP1- and ETS-mediated transcriptionalincrease, possibly by competing for CBP, p300, or both(Darnell, 1997). Stats may have direct effects on inhibitingproliferation by induction of cyclin-dependent kinase in-hibitors, such as p21/Waf1 (Chin et al., 1996). This mayexplain the antiproliferative effects of interferons. Inter-estingly, a reduced level of p21/Waf1 protein expressionin HCV-related hepatocellular carcinoma has been re-ported (Shi et al., 2000). Our present results coupled withthe previous observations made on the effect of HCVcore protein mediated modulation of AP-1, MAPKK, andp21/Waf1 (Hayashi et al., 2000; Ray et al., 1998b; Shriv-astava et al., 1998; Tsuchihara et al., 1999) suggest thatcore protein may target a number of cellular genes thatcould have a concerted action on normal cell growthcheckpoint control. HCV has a uniquely adapted mech-anism to inhibit host antiviral effector pathways, ensuringsurvival in the human host. Studies on expression ofsome of IFN-regulated genes in the liver of chronic HCVpatients (Meier et al., 2000; Patzwahl et al., 2001; Yu et al.,2000) does not suggest a clear picture for their contribu-tion in anti-HCV response. A complex gene network is

involved in the regulation of the IFN system. In fact, the59 regulatory region of the IRF-1 gene has been shown to

contain highly GC-rich sequences and consensus bind-ing sequences for several known transcription factors,including NF-kB (Harada et al.,1994). However, our ob-servations do not support such an alternative effect,especially when results from IFNa and IFNg are consid-

red, since both of these pathways are partially overlap-ing through Stat1. Even though our results suggest that

FN-stimulated genes of the Jak/Stat pathway are notmpaired or blocked by HCV proteins, the relative weak-ess of IFNa therapy may arise from virus-mediated

interruption of other gene products with antiviral activityor from the nature of poor antiviral activity associatedwith human hepatocytes.

MATERIALS AND METHODS

Cell lines, plasmid construct, and protein expression

Human cervical carcinoma (HeLa) and human hepa-toma (HepG2) cells were obtained from the AmericanType Culture Collection (Rockville, MD). The human fi-brosarcoma epithelial-like HT1080 cells were kindly pro-vided by G. R. Stark (Cleveland Clinic Foundation, OH).Cells were grown in Dulbecco’s modified Eagle’s me-dium (DMEM) supplemented with 10% heat-inactivatedfetal bovine serum. A full-length HCV clone (kindly pro-vided by C. M. Rice, Washington University, St. Louis,MO) was digested with suitable restriction enzymes andthe genomic region representing the structural and non-structural proteins (amino acids 1–2962) was subclonedinto a pCIneo mammalian expression vector, introducinga stop codon at the translational end. Cloning of the HCVgenomic region representing the core protein (aminoacids 1–191) has been described earlier (Ray et al., 1995).Subconfluent cell monolayers were transfected with amammalian expression vector containing HCV (pCIneo/HCV1–2962) or HCV core (pcDNA3/Core1–191) gene usinglipofectamine (Gibco BRL, MD). An empty vector DNAwas transfected into cells for use as a control. Neomy-cin-resistant stable transfectants were pooled to avoidclonal selection and tested for integration of HCV geneand protein expression. Stable cell transfectants exhib-ited NS5A and/or core protein expression (depending onthe plasmid transfected) separately by Western blot anal-ysis and indirect immunofluorescence (Ray et al., 1996b)

sing monoclonal antibodies (mAbs) to NS5A proteinBiogenesis, Inc., MA) or core protein (C7-50, kindly pro-ided by Jack R. Wands, Harvard Medical School, MA).dditionally, cells transfected with the pCIneo/HCV1–2962

gene exhibited distinct immunofluorescence with mono-clonal antibody to E1 or E2. Transfected cells did notexhibit unusual growth characteristics and were ana-

lyzed for the role of viral protein(s) in IFN signalingpathways.

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388 BASU ET AL.

Immunofluorescence

Stable cell transfectants were examined for subcellu-lar localization of Stat 1 expression by a specific mAb(Santa Cruz Biotechnology, CA). Briefly, cells grown over-night on coverslips were left untreated or treated withIFNg (500 U/ml) or IFNa (1000 U/ml) for 30 min. Cellswere washed and then fixed with methanol/acetone (1:1)for 2 min. After washing, cells were incubated with themAb at room temperature for 1 h, washed, and stainedwith a goat anti-mouse IgG conjugated to fluoresceinisothiocyanate. A human CD4 gene stably transfectedinto HeLa cells was also included as an unrelated con-trol in similar immunofluorescence study.

Immunoprecipitation and Western blot analysis

Cells were treated with 1000 U/ml IFNa or 500 U/ml ofIFNg for 90 min, washed, and lysed in 13 lysis buffer(0.5% NP-40, 50 mM Tris–HCl, pH 8.0, 10% glycerol,0.1mM EDTA, 300 mM NaCl, 1mM DTT, and a cocktail ofprotease inhibitors) for 1 h on ice. Cell lysates wereincubated with a monoclonal antibody to Stat1-a at 4°Cand the immunoprecipitate was immobilized onto proteinA–Sepharose CL-4B (Pharmacia, NJ). The sample wassubjected to electrophoresis on a SDS–polyacrylamidegel. Proteins were transferred onto a nitrocellulose mem-brane and blocked with 5% low-fat milk. HRP-conjugatedanti-human phosphotyrosine monoclonal antibody(Santa Cruz Biotechnology) was used for detection of thephosphorylated form of the Stat1 polypeptide. Stat1 andp48 protein expression was also determined directlyfrom cell lysates by Western blot analysis using specificmonoclonal and polyclonal antibodies, respectively(Santa Cruz Biotechnology). An antibody to cellular actin(Oncogene Research, CA) was used as a control inWestern blot for analyzing protein load in each lane. AHRP-conjugated second antibody to mouse Ig was usedto visualize the protein bands by enhanced chemilumi-nescence (ECL; Amersham, IL).

Gel retardation assay

The gel retardation assay for GAF and ISGF3 wasperformed as previously described (Leonard and Sen,1996). Briefly, cells were treated for 30 min with1000 U/mlof IFNa or 500 U/ml of IFNg and untreated cells wereused as control. Cells were lysed with NP-40 in low-saltbuffer (10mM HEPES, pH 7.9; 10 mM KCl, 0.1 mM EDTA,1 mM DTT, 0.5mM PMSF, and a cocktail of proteaseinhibitors) on ice for 5 min. The nuclear pellet was col-lected by centrifugation and suspended in a high-saltbuffer (20 mM HEPES, pH 7.9; 0.4 mM NaCl, 0.1 mMEDTA, 1 mM DTT, and 1 mM PMSF) for 30 min at 4°C.Nuclear extracts were obtained by centrifugation at

12,000 g, and aliquots were flash frozen immediately forstorage at 270°C. The protein concentration of the nu-

bp

clear extracts were determined using protein assay re-agent (Bio-Rad, CA). The GAS element located between2129 and 2106 of the IFNg-inducible IRF1 gene, andISRE element located between 2125 and 293 of theIFNa 561 gene (Leonard and Sen, 1997), were used asprobes (kindly provided by G. C. Sen, Cleveland ClinicFoundation, OH) for our studies. The annealed double-stranded probes were end labeled with [a-32P]dCTP us-ing a DNA polymerase to fill in the ends. Gel retardationassay was initially standardized by varying the quantityof the nuclear extracts in obtaining a linear range ofdetection of the specific complexes. Finally, 10 mg ofnuclear proteins were incubated with 32P-labeled oligo-

ucleotide probe in the presence of poly[d(I-C)] in ainding buffer (50 mM HEPES, pH 7.5; 250 mM NaCl, 2.5M EDTA, 1mM MgCl2, 2.5 mM DTT, and 1 mM PMSF, 5M MgCl2, 20% glycerol, and a cocktail of protease

inhibitors) for 30 min at room temperature. The specificityof the Stat1 protein in DNA–protein complex was identi-fied by adding a murine monoclonal antibody to Stat1(Santa Cruz) to the reaction mixture before formation ofthe complex. A rabbit polyclonal antibody to Stat2 (SantaCruz), unrelated murine monoclonal antibodies, or fetalcalf serum as negative controls, were used separatelyfor determining the inhibitory effect on DNA–proteincomplex formation. The samples were electrophoreseduntil the free radiolabeled probe was removed from thenondenaturing 5% polyacrylamide separation gel andexposed to X-ray film for autoradiography. The autoradio-gram was scanned densitometrically for a comparison ofband intensity.

RNase protection assay

The induction of Stat1, IRF-1, and 561mRNAs wereanalyzed using specific probes as described earlier (Kal-vakolanu et al., 1991; Leonard and Sen, 1997). A 409-nucleotide fragment of the Stat1 gene (Pharmingen, CA),a 175-nucleotide fragment (nucleotide positions192–367) of the IRF-1 gene, or 142-nucleotide fragment(nucleotide positions 1369–1511) of the 561 gene (kindlyprovided by G. C. Sen) was used as a probe. A 113-nucleotide-long L32 and 128-nucleotide-long g-actinprobe was used for internal control. Briefly, total RNAwas isolated from cells, with or without IFN treatment,using a RNA isolation kit (Gentra Systems, MN). Anantisense RNA probe was labeled with [32P]UTP and coldNTPs using SP6 or T7 RNA polymerase. The probe washybridized in excess to target cellular RNA (2 mg) over-

ight. Free probe and single-stranded RNA were di-ested with RNAseA and RNAseT1, followed by protein-se K treatment. The protected RNA was recovered byhenol/chloroform extraction, ethanol precipitation, and

dentified by resolving on a 5% urea–polyacrylamide gel

y electrophoresis, followed by autoradiography. Therotected RNA band in the autoradiogram was densito-

B

B

C

C

C

L

L

L

389HCV CORE PROTEIN AND ISGF3/GAF MODULATION

metrically scanned to quantitate intensity after normal-ization against the L32 or g-actin as an internal control.

ACKNOWLEDGMENTS

We thank R. B. Belshe and Paula M. Pitha for helpful discussions,C. M. Rice for providing the full-length HCV clone (p90/HCV FL), G. C.Sen for providing important research materials and helpful sugges-tions, G. R. Stark for providing HT1080 cell line, J. R. Wands for mono-clonal antibody C7-50, and SuzAnn Price for preparation of the manu-script. This work was supported by Research Grants CA85486,DK56143, and AI45250 from the National Institutes of Health.

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