Hepatitis C Virus Core Protein Suppresses NF-{kappa}B Activation and Cyclooxygenase-2 Expression by Direct Interaction with I{kappa}B Kinase {beta}

In addition to hepatocytes, hepatitis C virus (HCV) infectsimmune cells, including macrophages. However, little is knownconcerning the impact of HCV infection on cellular functionsof these immune effector cells. Lipopolysaccharide (LPS) activatesI ${kappa}$ B kinase (IKK) signalsome and NF- ${kappa}$ B, which leads to the expressionof cyclooxygenase-2 (COX-2), which catalyzes production of prostaglandins,potent effectors on inflammation and possibly hepatitis. Here,we examined whether expression of HCV core interferes with IKKsignalsome activity and COX-2 expression in activated macrophages.In reporter assays, HCV core inhibited NF- ${kappa}$ B activation in RAW264.7 and MH-S murine macrophage cell lines treated with bacterialLPS. HCV core inhibited IKK signalsome and IKKß kinaseactivities induced by tumor necrosis factor alpha in HeLa cellsand coexpressed IKK ${gamma}$ in 293 cells, respectively. HCV core wascoprecipitated with IKKß and prevented nuclear translocationof IKKß. NF- ${kappa}$ B activation by either LPS or overexpressionof IKKß was sufficient to induce robust expressionof COX-2, which was markedly suppressed by ectopic expressionof HCV core. Together, these data indicate that HCV core suppressesIKK signalsome activity, which blunts COX-2 expression in macrophages.Additional studies are necessary to determine whether interruptedCOX-2 expression by HCV core contributes to HCV pathogenesis.

INTRODUCTION

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Introduction

Hepatitis C virus (HCV), a flavivirus, causes hepatitis, cirrhosis,and hepatocellular carcinoma (18). Currently, almost 3% of theworld population is infected by HCV, and these numbers seemto be increasing (3). One of the most remarkable features ofHCV infection is that more than 85% of acutely infected patientsbecome chronically infected (4). Although CD4⁺ and CD8⁺ T-cellresponses are crucial for controlling HCV infection in acuteHCV patients, these T-cell responses are significantly impairedin chronic HCV patients (16). Thus, this suggests that HCV evadeshost immune responses. While hepatocytes are a major targetof HCV infection, recent studies showed that HCV can replicatein immune cells such as B and T lymphocytes and monocytes thatexpress HCV receptors, such as CD81 and low-density lipoproteinreceptor (1, 2, 52). Thus, it is possible that HCV infects immuneeffector cells, which contributes to evasion of host immunesurveillance.

The HCV core protein, a nucleocapsid protein, binds to the cytoplasmicdomain of tumor necrosis factor receptor (TNFR) and lymphotoxin-betareceptor to regulate apoptosis (16, 17, 45, 81). This viralprotein is also involved in oncogenesis, as evidenced by thedevelopment of hepatocellular carcinoma in transgenic mice expressingHCV core in the liver (47). In addition, HCV core has been shownto affect diverse cellular and viral gene expressions (53, 55,60) and, depending on subtypes, to activate or suppress NF- ${kappa}$ Bfunction that is involved in both innate and adaptive immunity(27, 44, 61, 79).

NF- ${kappa}$ B is a homo- or heterodimer of five proteins: c-Rel, RelA(p65), RelB, p50, and p52. Under unstimulated conditions, NF- ${kappa}$ Bresides in the cytoplasm by forming complexes with inhibitory ${kappa}$ B (I ${kappa}$ B) (27). Proinflammatory stimuli, such as TNF- ${alpha}$ and lipopolysaccharide(LPS), induce signal cascades through their cognate receptors,TNFR and Toll-like receptor 4 (TLR4), to activate I ${kappa}$ B kinase(IKK) signalsome and subsequently NF- ${kappa}$ B that is required forinnate and adaptive immune responses to pathogens (13, 38).IKK signalsome is composed of at least two kinases, IKK ${alpha}$ and-ß, and a regulatory factor, IKK ${gamma}$ (26, 36). Among theseproteins, IKKß is known as a major kinase and IKK ${gamma}$ is required for the full activation of IKKß upon proinflammatorystimulation (41-43, 56, 57, 59, 66, 78). Treatment of cellswith TNF- ${alpha}$ results in IKK signalsome recruitment to the type1 TNF- ${alpha}$ receptor (TNFR1), which induces phosphorylation at keyresidues in the activation loop of the kinase domain of IKKßto be an active kinase (21). Activated IKK signalsome phosphorylatesI ${kappa}$ B, which subsequently undergoes ubiquitination and degradation,liberating NF- ${kappa}$ B to translocate into the nucleus and activatetarget genes (37).

NF- ${kappa}$ B influences gene expression of cyclooxygenase-2 (COX-2)along with other transcription factors, including CREB and C/EBP-ß(15, 20, 33, 34, 48, 50, 63, 67, 72, 75). Accordingly, proinflammatorystimuli induce the expression of COX-2 to catalyze the productionof prostaglandins, which promote inflammation through a varietyof mechanisms.

Our hypothesis is that HCV evades innate immunity by directlyinfecting immune cells and altering gene expression of key inflammatorymolecules. Here we tested whether the HCV core protein affectsNF- ${kappa}$ B activity and COX-2 production in macrophages. We demonstratethat the HCV core protein interacts with IKKß, suppressesits kinase activity, and interferes with nuclear translocationof IKKß, which are correlated with inhibition of NF- ${kappa}$ Bactivity. Furthermore, we show that the HCV core protein suppressesNF- ${kappa}$ B-dependent COX-2 expression. These data indicate that theHCV core protein interrupts diverse IKKß functions,which results in blunted COX-2 expression.

MATERIALS AND METHODS

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Materials and Methods

Cell lines. Murine macrophage cell line RAW 264.7, murine alveolar macrophagecell line MH-S, and HEK 293 and HeLa cells were all obtainedfrom the American Type Culture Collection (Rockville, MD). Celllines were maintained according to the recommendations of theAmerican Type Culture Collection.

Plasmids. The coding sequence of HCV core (Hutchinson strain, genotype1a) was inserted into the polylinker site of pCI:neo (Promega).The resultant construct, named pCI-HCV core, was engineeredto express the 192-amino-acid-long HCV core protein. To generatea construct that encodes FLAG-tagged HCV core, pCI-HCV corewas digested with restriction enzymes NheI and EcoRI (New EnglandBioLabs) and inserted with a linker containing the FLAG sequence.The coding sequence of HCV core and E1 (Hutchinson strain, genotype1a) was amplified by Pfu polymerase (Stratagene). The forwardprimer binds to the first 20 nucleotides of the 5' end of theHCV core sequence and contains NheI restriction site, Kozak,and FLAG sequences (5'-GCGGCTAGCCACCATGGACTACAAAGACGATGACGATAAAGGGATGAGCACGAATCCTAAACC-3';underlined is the core sequence). The reverse primer binds to18 nucleotides of the 3' end of the HCV E1 sequence and containsHindIII (5'-GCAAGCTTGCCGGCAAATAGCAGCAG-3'). The PCR productwas digested with restriction enzymes NheI and HindIII (NewEngland BioLabs) and inserted into the corresponding sites ofpcDNA3(-)-Myc/His (Invitrogen). Candidate clones encoding HCVcore and E1 were analyzed by sequencing (Vanderbilt DNA SequencingCore Facility). Expression of the FLAG-tagged HCV core and Myc-taggedprotein was determined by Western blotting with M2 antibody(Sigma) and anti-Myc antibody (Santa Cruz Biotechnology). Plasmidsencoding a FLAG-tagged IKK ${alpha}$ and IKKß were kindly providedby R. Carter (Vanderbilt University) and F. Mercurio (SignalResearch Division, Celgene Corp., New Jersey), respectively,and a plasmid expressing T7-tagged NEMO/IKK ${gamma}$ was a gift fromE. S. Alnemri (Thomas Jefferson University). A plasmid encodingsuperrepressor I ${kappa}$ B ${alpha}$ ^S32,36A was a gift from K. Pennington (VanderbiltUniversity). The luciferase reporter plasmid NF- ${kappa}$ B-Luc was purchasedfrom Stratagene (La Jolla, CA).

Plasmid transfection, LPS treatment, and luciferase assay. Cells were transfected with Superfect (QIAGEN), as specifiedby the manufacturer. Each transfection was normalized with appropriateempty vector plasmids. At 48 h posttransfection, transfectedcells were harvested for the experiment. For the LPS treatment,after incubation for 14 to 16 h in a 37°C, CO₂ incubator,transfected cells were further maintained without serum overnightand subsequently treated with LPS (1 µg/ml in phosphate-bufferedsaline; Sigma) for 4 h to induce COX-2 expression. For the luciferaseassay, cells were cotransfected with luciferase reporter plasmidsNF- ${kappa}$ B-Luc and tk-Renilla Luc. Luciferase activity was measuredby a dual-luciferase assay system (Promega) according to theprotocol recommended by the manufacturer. NF- ${kappa}$ B-mediated luciferaseactivity was normalized with Renilla luciferase activity. Theluciferase assay was done in triplicate.

Viruses and infection. Adenoviruses encoding IKKß, superrepressor IkB ${alpha}$ ^S32,36A,and ß-galactosidase (ß-Gal) were describedpreviously (58). Viruses were propagated and virus titer wasdetermined by standard protocols. RAW 264.7 cells were infectedwith adenoviruses at multiplicity of infection of 0.2 for 1h. After inocula were removed, cells were placed in fresh mediumand incubated overnight at 37°C in a CO₂ incubator. At 24h postinfection, cells were harvested for the experiment.

Immunoprecipitation and Western blotting. Total cell lysate was prepared with RIPA (radioimmunoprecipitationassay) cell lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl,2 mM EDTA, 1% sodium orthovanadate, 1% Triton X-100, 0.5% deoxycholate,0.1% sodium dodecyl sulfate [SDS]) supplemented with phenylmethylsulfonylfluoride (1 mM), aprotonin (2 µg/ml), leupeptin (2 µg/ml),and soybean trypsin inhibitor (37.5 µg/ml). Proteins wereseparated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)and transferred to a polyvinylidene difluoride membrane (Bio-Rad).Specific bands were revealed using enhanced chemiluminescence(ECL plus; Amersham). For immunoprecipitation analysis, 1 to2 µg of appropriate antibodies was added to preclearedcell lysate that was normalized by protein content and incubatedovernight at 4°C. Immune complexes were captured with 30µl of protein A-Sepharose (Zymed) for 30 min at 4°Cand washed five times with RIPA buffer. Antibodies used in thisstudy were as follows: polyclonal anti-murine COX-2 (CaymanChemical); monoclonal anti-FLAG M2 antibody (Sigma); polyclonalanti-IKK ${alpha}$ /ß and anti-IKK ${gamma}$ (Santa Cruz Biotechnology);monoclonal anti-IKK ${alpha}$ and -ß (PharMingen); monoclonalanti-HCV core antibody (Affinity Bioreagents, Inc.).

IKK assay. Transfected HeLa cells were treated with 10 ng/ml of TNF- ${alpha}$ (R&DSystems Inc.) for 15 min before lysis. Cytoplasmic extractsfrom HeLa or HEK 293 cells were prepared as described previously(49). To immunoprecipitate IKK signalsome, ELB buffer (49) wasadded to the prepared cytoplasmic extract. Kinase activity wasmeasured in a reaction buffer containing 10 mM HEPES (pH 7.4),5 mM MgCl₂, 1 mM MnCl₂, ATP (10 µM), [ ${gamma}$ -³²P]ATP (5 µCi),and recombinant glutathione S-transferase (GST)-I ${kappa}$ B ${alpha}$ , GST proteinfused to amino acids 1 to 54 of I ${kappa}$ B ${alpha}$ , at 30°C for 30 min.Radiolabeled products were separated by SDS-PAGE, transferredto polyvinylidene difluoride membrane, and revealed by autoradiography.

Preparation of cytoplasmic and nuclear fractions. Cytoplasmic and nuclear fractions were prepared as describedelsewhere (9). In brief, cells were washed three times withice-cold phosphate-buffered saline and added to hypotonic buffer(10 mM HEPES [pH 7.9], 5 mM KCl, 1.5 mM MgCl₂, 1 mM NaF, and1 mM Na₃VO₄) supplemented with 1 mM dithiothreitol, phenylmethylsulfonylfluoride (1 mM), aprotonin (2 µg/ml), leupeptin (2 µg/ml),and soybean trypsin inhibitor (37.5 µg/ml). After lysisfor 15 min on ice, the cytoplasmic fraction was prepared bycentrifugation at 3,000 rpm for 5 min. The cytoplasmic fractionwas cleared by centrifugation at 13,000 rpm for 15 min beforefurther analysis. To prepare the nuclear fraction, the cellpellet generated after initial centrifugation was washed withthe hypotonic buffer three times and suspended in high-saltbuffer (20 mM HEPES [pH 7.9], 1.5 mM MgCl₂, 0.2 mM EDTA [pH8.0], 420 mM NaCl, 25% glycerol [vol/vol], 50 mM ß-glycerophosphate,1 mM NaF, and 1 mM Na₃VO₄) supplemented with 1 mM dithiothreitol,phenylmethylsulfonyl fluoride (1 mM), aprotonin (2 µg/ml),leupeptin (2 µg/ml), and soybean trypsin inhibitor (37.5µg/ml). The suspended cell pellet was incubated for 30min on ice with occasional vortexing, and the nuclear fractionwas collected after centrifugation at 13,000 rpm for 10 min.Protein content from each fraction was quantified by the Bradfordassay (Bio-Rad) as specified by the manufacturer to ensure equalloading.

Statistical analysis. For comparison among groups, paired or unpaired t tests andone-way analysis of variance tests were used (with the assistanceof InStat; Graphpad Software, Inc., San Diego, CA). P valuesof <0.05 are considered significant.

RESULTS

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Results

HCV core suppresses NF- ${kappa}$ B activity in LPS-treated macrophages. HCV core differentially modulates NF- ${kappa}$ B activity, depending onthe subtype of HCV (54). To examine whether the HCV core usedin this study (genotype 1a) interferes with NF- ${kappa}$ B activity inLPS-treated macrophages, RAW 264.7, a murine macrophage cellline, and MH-S, a murine alveolar macrophage cell line, weretransfected with a plasmid encoding HCV core along with theNF- ${kappa}$ B-luciferase reporter construct in the presence or absenceof LPS. The results in Fig. 1 showed that HCV core, by itself,did not activate NF- ${kappa}$ B but rather suppressed NF- ${kappa}$ B activity elicitedby LPS treatment of RAW 264.7 (Fig. 1A) and MH-S (Fig. 1B) cells.A similar experiment using TNF- ${alpha}$ produced similar results (datanot shown). These results demonstrate that HCV core suppressesNF- ${kappa}$ B activity in macrophages.

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FIG. 1. HCV core inhibits NF- ${kappa}$ B activated by LPS. RAW 264.7 (A) and MH-S (B) cells were transfected with 0.5 µg of NF- ${kappa}$ B-Luc and 0.2 µg of tk-Renilla-Luc along with the indicated amounts of the HCV core-expressing plasmid. The total amount of transfected plasmid was adjusted with an empty vector plasmid. NF- ${kappa}$ B activity elicited by LPS was measured after treating cells with LPS for 4 h (lanes 1 and 2). To examine an effect of the HCV core on NF- ${kappa}$ B-mediated transcriptional activity, the plasmid encoding HCV core was transfected without or with treatment with LPS (1 µg/ml) for 4 h (lanes 3 and 4).

HCV core inhibits IKK signalsome kinase activity. To investigate the mechanism by which HCV core inhibits NF- ${kappa}$ Bactivity, we tested whether HCV core interferes with IKK kinaseactivity. For these studies, we employed HeLa and 293 cellsbecause of the ability to achieve high transfection efficienciesin these cell lines. First, to test whether these cell lineswere able to recapitulate the results shown in Fig. 1, we performeda similar experiment. As shown in Fig. 2A, when HeLa cells weretreated with TNF- ${alpha}$ , NF- ${kappa}$ B-mediated transcriptional activity wasincreased (lanes 1 and 2). However, the presence of HCV coresuppressed NF- ${kappa}$ B activity elicited by TNF- ${alpha}$ (lanes 2 and 4). Similarly,HCV core suppressed NF- ${kappa}$ B activated by TNF- ${alpha}$ in HEK 293 cells(Fig. 2B, lanes 2 and 3). These results suggest that HCV corealso represses NF- ${kappa}$ B activity in these cell lines.

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FIG. 2. HCV core inhibits NF- ${kappa}$ B activated by TNF- ${alpha}$ in other cell types. HeLa (A) and HEK 293 (B) cells were transfected with 0.2 µg of the NF- ${kappa}$ B-Luc and 0.1 µg of the tk-Renilla-Luc reporter construct, along with the indicated amount of the HCV core-expressing plasmid. The total amount of transfected plasmid was adjusted with an empty vector plasmid. NF- ${kappa}$ B-mediated transcriptional activity was measured after treating cells with TNF- ${alpha}$ (10 ng/ml) for 6 h. (C) A possible effect of HCV E1 on HCV core was examined. HeLa cells were transfected with two reporter constructs, indicated above, along with either the plasmid encoding HCV core or a plasmid encoding HCV core and E1, and treated with TNF- ${alpha}$ as described above. NF- ${kappa}$ B-mediated transcription activity was measured by assaying luciferase activity. The result shown here is a representative of three independent experiments.

We tested whether coexpression of HCV core and E1 alters theeffect of HCV core on NF- ${kappa}$ B activity. HeLa cells were transfectedwith a plasmid encoding HCV core and E1 along with the NF- ${kappa}$ Breporter construct and treated with TNF- ${alpha}$ . As shown in Fig. 2C,HCV core was able to suppress NF- ${kappa}$ B activity elicited by TNF- ${alpha}$ in the presence of HCV E1 (lane 4). A similar result was obtainedin HEK 293 cells (data not shown). These results indicate thatsuppression of NF- ${kappa}$ B activity by HCV core is not significantlyaffected by E1.

Next, we tested whether HCV core interferes with IKK signalsomekinase activity. HeLa cells were transfected with increasingamounts of a plasmid encoding HCV core and treated with TNF- ${alpha}$ (10 ng/ml) for 15 min to induce IKK signalsome activity. TheIKK signalsome was immunoprecipitated with anti-IKKßantibody, extensively washed, and incubated with GST-I ${kappa}$ B ${alpha}$ and[ ${gamma}$ -³²P]ATP for kinase assays. As shown in Fig. 3A, HCV core reducedIKK kinase activity elicited by TNF- ${alpha}$ treatment in a dose-dependentfashion.

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FIG. 3. The HCV core protein inhibits IKK kinase activity. (A) HeLa cells were transfected with either an empty vector (lanes 1 and 2) or increasing amounts of the HCV core-expressing plasmid (lanes 3 and 4). The total amount of transfected plasmid was normalized as indicated in Materials and Methods. At 48 h posttransfection, the cells were treated with TNF- ${alpha}$ (10 ng/ml) for 15 min and the cytoplasmic fraction was prepared for immunoprecipitation of IKK with anti-IKKß antibody. Immune complex, after being extensively washed, was incubated with GST-I ${kappa}$ B ${alpha}$ and [ ${gamma}$ -³²P]ATP to measure IKK kinase activity. (B) HEK 293 cells were transfected with 0.1 µg of the FLAG-tagged IKKß-expressing plasmid along with 0.5 µg of a plasmid encoding IKK ${gamma}$ (lanes 2 and 3) and 0.5 µg of the HCV core-expressing plasmid (lane 3). At 48 h posttransfection, total cell lysate was prepared for immunoprecipitation with M2 antibody to capture IKKß. Immune complexes were incubated with GST-I ${kappa}$ B ${alpha}$ and [ ${gamma}$ -³²P]ATP to measure IKKß kinase activity (top panel). Expression levels of IKKß and the HCV core were determined by Western blotting with M2 antibody and anti-HCV core antibody, respectively (middle and bottom panels).

Since IKKß is known as a major kinase in inflammatorygene expression and overexpression of IKKß resultsin kinase activity that is markedly increased by IKK ${gamma}$ (24), weexamined whether HCV core inhibits IKKß kinase activityin the absence of an activating stimulus (Fig. 3B). HEK 293cells were transfected with plasmids expressing FLAG-taggedIKKß and T7-tagged IKK ${gamma}$ in the presence or absenceof HCV core. For IKKß kinase assays, IKKßwas precipitated with M2 antibody. As shown in Fig. 3B, IKKßkinase activity was markedly enhanced by IKK ${gamma}$ (lane 2 comparedto lane 1); however, this was strongly suppressed by HCV core(lane 3). Western blot analysis also revealed that the phosphorylatedform of IKKß, an indicator of kinase activation, wasinduced by overexpression of IKK ${gamma}$ . The phosphorylation of IKKßwas blocked by ectopic expression of the HCV core protein (middlepanel, lanes 2 and 3). These results indicate that HCV corespecifically suppresses IKKß kinase activity.

HCV core physically interacts with IKKß. To understand how the HCV core protein suppresses IKKßkinase activity, we examined the physical interaction betweenthe two proteins by immunoprecipitation assay. HeLa cells weretransfected with either an empty vector or a plasmid encodingFLAG-tagged HCV core. After cell lysis, the cytoplasmic fractionwas incubated with M2 antibody to capture FLAG-tagged HCV core,and the immune complex was analyzed by SDS-PAGE and Westernblotting. First, to detect any subunits of IKK signalsome interactingwith the HCV core protein, the membrane was incubated with amixture of anti-IKK ${alpha}$ , -IKKß, and -IKK ${gamma}$ antibodies. Asshown in Fig. 4A, the HCV core protein coprecipitated with IKK ${alpha}$ and/or IKKß but not with IKK ${gamma}$ . To identify the precipitatedband, coimmunoprecipitates of the HCV core protein were furtheranalyzed by Western blotting with IKK ${alpha}$ and IKKß antibodies.The result in Fig. 4B indicated that IKKß but notIKK ${alpha}$ coimmunoprecipitated with the HCV core protein.

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FIG. 4. The HCV core protein interacts with IKKß. HeLa cells were transfected with either 2 µg of an empty vector plasmid or 1 µg of a plasmid encoding FLAG-tagged HCV core that was normalized with the empty vector plasmid to 2 µg. At 48 h posttransfection, cell lysate was prepared for immunoprecipitation of FLAG-tagged HCV core with M2 antibody. Immune complex captured by protein A-Sepharose was separated by SDS-PAGE and analyzed by Western blotting. (A) HCV core protein immunoprecipitated with IKK. Blot was incubated with anti-IKK ${alpha}$ , -ß, and - ${gamma}$ antibodies (rabbit polyclonal antibodies) to reveal subunits of IKK that interact with the HCV core protein. One-tenth of the total cell lysate used for immunoprecipitation was loaded as a positive control for IKK ${alpha}$ , -ß, and - ${gamma}$ . (B) The HCV core protein interacts with IKKß. Total cell lysate was prepared from transfected cells and incubated with M2 antibody. One-tenth of the total cell lysate used for immunoprecipitation was loaded as a positive control for IKK ${alpha}$ . Precipitated immune complex was analyzed by Western blotting after SDS-PAGE separation. The blot was incubated with either anti-IKK ${alpha}$ antibody or anti-IKKß (mouse monoclonal) antibody to reveal IKK ${alpha}$ and IKKß, respectively.

The HCV core interferes with nuclear localization of IKKß. A recent discovery that IKK ${alpha}$ and -ß are located notonly in the cytoplasm but also in the nucleus suggests additionalfunctions of IKK signalsome in regulation of gene expression(12). Indeed, IKK ${alpha}$ is recruited to the NF- ${kappa}$ B-binding DNA elementvia physical interaction with CREB-binding protein, which enhancestarget gene expression (7, 76). IKK ${gamma}$ is also directly involvedin transcription regulation by inhibiting the interaction betweenIKK ${alpha}$ and CREB-binding protein (71). Therefore, we investigatedthe possibility that the HCV core protein affects IKKßactivity by interfering with nuclear-cytoplasmic partitioningof IKKß.

To test whether HCV core selectively interferes with IKKßpartitioning within a cell, HEK 293 cells were transfected withplasmids encoding FLAG-tagged IKK ${alpha}$ and IKKß along withthe plasmid encoding HCV core. To determine partitioning ofIKK ${alpha}$ and IKKß, cytoplasmic and nuclear fractions wereprepared and analyzed by Western blotting with M2 antibody toreveal IKK ${alpha}$ and -ß. As shown in Fig. 5A, while themajority of IKK ${alpha}$ resides in the cytoplasm, IKKß wasdetected in both cytoplasmic and nuclear fractions (lanes 1,2, 5, and 6). However, the presence of the HCV core proteindecreased localization of IKKß in the nuclear fraction(lanes 6 and 8), while not significantly affecting the amountof cytoplasmic IKKß. To ensure equal loading of nuclearproteins, the membrane was stripped and probed with anti-YY1antibody (8, 29, 46).

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FIG. 5. The HCV core protein interferes with localization of IKKß to the nucleus. (A) The HCV core protein interferes with nuclear localization of transfected IKKß. HEK 293 cells were transfected with 0.5 µg of the plasmid encoding FLAG-tagged IKK ${alpha}$ and IKKß with (lanes 3 and 4 and lanes 7 and 8) or without (lanes 1 and 2 and lanes 5 and 6) 2 µg of the plasmid encoding HCV core. The total amount of transfected plasmid was adjusted to 3 µg with an empty host plasmid. Cytoplasmic and nuclear fractions were prepared as described in Materials and Methods. Ten micrograms of cytoplasmic proteins and 50 µg of nuclear proteins were analyzed by SDS-PAGE and Western blotting with M2 antibody to reveal the location of IKK ${alpha}$ and -ß. To ensure equal loading of nuclear proteins, nuclear protein YY1 was Western blotted after stripping membrane. (B) The HCV core protein interferes with nuclear localization of endogenous IKKß. HEK 293 cells were transfected with either 4 µg of an empty host plasmid (lanes 1 and 2 and lanes 5 and 6) or a different dose of the plasmid encoding HCV core (lanes 3 and 4 and 7 and 8). The total amount of transfected plasmid was adjusted to 4 µg with an empty host plasmid. At 48 h posttransfection, transfected cells were treated with TNF- ${alpha}$ (10 ng/ml) for 30 min before preparing cytoplasmic and nuclear fractions. Fifty micrograms of each fraction was analyzed by Western blotting to reveal endogenous IKKß. To ensure equal loading of nuclear protein, the membrane was stripped and reblotted to reveal nuclear protein YY1.

To further confirm this observation, a similar experiment wasconducted to determine the distribution of endogenous IKKß(Fig. 5B). HEK 293 cells were transfected with increasing amountsof the HCV core-encoding plasmid. Transfected cells were treatedwith 10 µg/ml of TNF- ${alpha}$ to examine a possible effect ondistribution of endogenous IKKß. Western blottinganalysis in Fig. 5B showed that distribution of IKKßwas not significantly affected by TNF- ${alpha}$ treatment (compare lanes1 and 2 and lanes 5 and 6), as indicated in another study (7).However, the HCV core protein reduced nuclear localization ofIKKß (lanes 6, 7, and 8). To ensure equal loadingof nuclear protein, the membrane was treated as for Fig. 5Ato reveal nuclear protein YY1. Taken together, these resultsindicate that the HCV core protein blocks IKKß shuttlingto the nucleus.

NF- ${kappa}$ B activation elicited by IKKß is sufficient to induce COX-2. Involvement of NF- ${kappa}$ B in COX-2 expression is well documented ina variety of cell types (15, 20, 77).

To define the relationship between NF- ${kappa}$ B activation and COX-2expression in RAW 264.7 cells, we treated the cells with endotoxinand measured COX-2 expression and NF- ${kappa}$ B activation by Westernblot analysis. As indicated in Fig. 6A, COX-2 protein productionincreased in response to treatment with LPS, and this was associatedwith degradation of cytoplasmic I ${kappa}$ B ${alpha}$ and the appearance of immunoreactiveRelA in the nucleus (Fig. 6B). These data show that the macrophagecell line treated with LPS results in both activation of NF- ${kappa}$ Band COX-2 protein production.

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FIG. 6. Casual relationship between NF- ${kappa}$ B activation and COX-2 induction. (A) Murine macrophage cell line RAW 264.7 was treated with 1 µg/ml of LPS for the indicated times, and 20 µg of total cell lysate was analyzed for COX-2 expression. To ensure equal loading, membrane was stripped and Western blotted for actin. (B) After LPS treatment of RAW 264.7 cells for the indicated times, Western blotting was performed to reveal I ${kappa}$ B ${alpha}$ in the cytoplasm and RelA in the nucleus.

To assess the role of NF- ${kappa}$ B in COX-2 expression in macrophageswithout ambiguity associated with cross talk by inflammatorysignals, NF- ${kappa}$ B was activated in a highly specific manner throughectopic expression of IKKß (26), and resultant COX-2expression was analyzed by Western blotting in two separatemurine macrophage cell lines, RAW 264.7 and MH-S. As shown inFig. 7, ectopic expression of IKKß increases COX-2protein production in both macrophage cell lines in a dose-dependentfashion. These data indicate that NF- ${kappa}$ B activation by ectopicexpression of IKKß is sufficient to induce COX-2 expression.

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FIG. 7. NF- ${kappa}$ B activation confers COX-2 expression. RAW 264.7 and MH-S cells were transiently transfected with either 1.5 µg of an empty vector plasmid (lanes 1 and 2) or an increasing amount of a plasmid that encodes FLAG-tagged IKKß (lanes 3 to 5). The total amount of transfected DNA was normalized as described in Materials and Methods. Transfected cells were further incubated without serum overnight and treated with or without LPS (1 µg/ml) for 4 h. Expression of COX-2 was measured by Western blotting with 20 µg of total cell lysate.

To assess the specificity of this response, we examined whethersuperrepressor IkB ${alpha}$ ^S32,36A suppresses COX-2 expression inducedby IKKß. First, RAW 264.7 cells were transfected withan NF- ${kappa}$ B luciferase reporter construct along with the IKKß-expressingplasmid in the absence or presence of the superrepressor-expressingplasmid. As shown in Fig. 8A, transfection of the superrepressorI ${kappa}$ B ${alpha}$ ^S32,36A markedly inhibits NF- ${kappa}$ B-mediated luciferase activityinduced by IKKß. Next, to test whether COX-2 proteinproduction induced by IKKß is suppressed by I ${kappa}$ B ${alpha}$ ^S32,36A,RAW 264.7 cells were infected with replication-deficient adenovirusesencoding kinase-active IKKß, I ${kappa}$ B ${alpha}$ ^S32,36A, or ß-Gal.Western blot analysis in Fig. 8B shows that infection with IKKß-expressingadenovirus induced COX-2 expression, while infection with ß-Gal-expressingadenovirus failed to do so. Coinfection with two adenovirusesencoding IKKß and I ${kappa}$ B ${alpha}$ ^S32,36A blunted the productionof COX-2. Taken together, these data demonstrate that selectiveNF- ${kappa}$ B activation results in COX-2 expression in macrophage celllines.

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FIG. 8. COX-2 induction by NF- ${kappa}$ B is suppressed by the superrepressor I ${kappa}$ B ${alpha}$ ^S32,36A. (A) I ${kappa}$ B ${alpha}$ ^S32,36A inhibits NF- ${kappa}$ B activated by IKKß. RAW 264.7 cells were transfected with 0.2 µg of a tk-Renilla luciferase construct (tk-Renilla-Luc) and 0.5 µg of a luciferase reporter construct containing NF- ${kappa}$ B-binding sites (NF- ${kappa}$ B-Luc) along with an empty vector plasmid (lanes 1 and 2) or the indicated amounts of the IKKß-expressing plasmid (lanes 3 to 5). To examine the inhibitory effect of I ${kappa}$ B ${alpha}$ ^S32,36A on NF- ${kappa}$ B activated by IKKß, an increasing amount of a plasmid encoding I ${kappa}$ B ${alpha}$ ^S32,36A was cotransfected (lanes 4 and 5). At 48 h posttransfection, the cell extract was prepared and enzymatic activity was measured. The result shown here is a representative of three independent experiments. (B) COX-2 expression elicited by activated NF- ${kappa}$ B is inhibited by I ${kappa}$ B ${alpha}$ ^S32,36A. RAW 264.7 cells were infected with adenovirus either encoding ß-Gal (lanes 1 and 2) or IKKß (lanes 3 and 4) along with I ${kappa}$ B ${alpha}$ ^S32,36A (lane 4). Each infection was normalized with adenovirus encoding ß-Gal to a multiplicity of infection of 0.2. At 24 h postinfection, the total cell lysate was prepared and 20 µg of total protein was used for Western blotting of COX-2. Infected cells were serum starved overnight before LPS treatment for 4 h (lane 2). COX-2 was measured by Western blotting with 20 µg of total cell lysate.

HCV core suppresses COX-2 expression induced by LPS. Finally, we examined whether the inhibitory effect of HCV coreon NF- ${kappa}$ B activity leads to down regulation of COX-2 expression.RAW 264.7 cells were transfected with either an empty host plasmidor a plasmid expressing the HCV core protein. Subsequently,transfected cells were treated with LPS to induce COX-2. Asindicated in Fig. 9, HCV core protein, by itself, did not induceCOX-2 expression (lanes 3 and 4). In contrast, HCV core proteinstrongly suppressed COX-2 expression induced by LPS treatment(lanes 5 and 6).

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FIG. 9. The HCV core protein suppresses COX-2 expression. RAW 264.7 cells were transfected with either empty vector (lanes 1 and 2) or the HCV core-expressing plasmid (lanes 3 to 6). Transfected cells were treated with LPS (1 µg/ml) for 4 h to induce COX-2 (lanes 2, 5, and 6). Expressed COX-2 was measured by Western blotting with 20 µg of total protein.

DISCUSSION

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Discussion

Primary HCV infection evades protective immunity and establishesa chronic infection (4, 16). Chronic HCV-infected patients havereduced Th1-type CD4⁺ T-cell activity and increased Th2-typeCD4⁺ T-cell activity (16, 23). Some chronic hepatitis C patientstreated with alpha interferon (IFN- ${alpha}$ ) (14) and ribavirin showenhanced Th1 CD4⁺ T-cell effector function with improvementof the medical condition (32, 65, 69). An HCV murine model alsoshowed the compromised production of Th1 cytokines, such asinterleukin-2 (IL-2) and IFN- ${gamma}$ , by HCV core (39). Similarly,there is a decreased Th1 cytokine production in transgenic micethat express HCV core (62). These data, together, suggest thatHCV evades the host immune response through a mechanism thatis mediated by HCV core. Recent experimental evidence showsthat HCV infection occurs with a broad cellular tropism encompassingnot only hepatocyte cells but also immune cells, such as B andT lymphocytes and monocytes (1). However, the impact of HCVon infected immune cells is not understood. In this study, weaddressed this issue by examining an inhibitory effect of HCVcore on NF- ${kappa}$ B, a key transcription factor in innate and adaptiveimmunity. Our data showed that, through inhibition of IKK kinaseactivity, HCV core hinders NF- ${kappa}$ B activity, which results in bluntedexpression of an important inflammatory protein, COX-2, in macrophages.

Along with constitutively expressed COX-1, COX-2 metabolizesarachidonic acid to prostaglandin H₂, which is further metabolizedto generate various prostanoids (24). Prostanoids comprise threegroups: prostaglandins, such as D2 (PGD₂), E2 (PGE₂), and F2(PGF₂); prostacylin, such as PGI₂; and thromboxane A₂. In general,prostanoids are considered to promote inflammation by inducingvasodilation and increasing vascular permeability and cellularmigration to the site of inflammation (22, 73, 74). Notably,prostanoids are also important modulators of adaptive immunity(28, 30); however, effects of prostanoids on immunity are complex,and the net result depends on the surrounding milieu. For example,PGE₂ promotes dendritic cell maturation and migration to lymphoidorgans in peripheral tissue, while it suppresses T-cell activationby dendritic cells in lymphoid organs (28). In the case of PGD₂,while enhancing the Th2-type immune response, PGD₂ and its derivativecyclopentenone prostaglandins are known to have anti-inflammatoryactivities by inhibiting NF- ${kappa}$ B, inducing apoptosis of macrophagesand neutrophils in inflammatory lesions, and activating anti-inflammatorytranscription factor PPAR- ${gamma}$ (64). Other prostanoids, such asPGI₂ and thromboxane A₂, also increase T-cell functions, andPGI₂ enhances natural killer (NK) cell functions (19, 35, 68,70). Nevertheless, these results clearly illustrate that prostanoidsare implicated in innate and adaptive immunity.

It is not well documented that there is a relationship betweenHCV infection and COX-2 expression that might influence HCVpathogenesis. As yet, combined treatment with IFN- ${alpha}$ and ribavirinis the popular measure to control hepatitis C, albeit with limitedsuccess. A report that IFN- ${alpha}$ increased PGE₂ production suggestedthat elevated PGE₂ might be attributed to unresponsiveness toIFN- ${alpha}$ treatment, given the immunosuppressive nature of PGE₂ (6).Accordingly, IFN- ${alpha}$ signaling can be enhanced by indomethacin,one of the nonsteroidal anti-inflammatory drugs (NSAIDs) thatblocks prostanoids production by inhibiting COX-1 and COX-2activities (25). However, combined treatment of HCV patientswith NSAIDs and IFN- ${alpha}$ has shown limited success and is less effectivethan treatment with ribavirin (5). This result might be dueto complex effects of prostanoids on host immunity. Interestingly,recent studies have shown that arachidonic acid is able to suppressHCV genomic replication and has a beneficial effect on HCV-infectedpatients (40, 51). Although it is not understood how arachidonicacid affects HCV, it is conceivable that impaired arachidonicacid metabolism might be favorable for HCV survival.

Our data implicate HCV in COX-2 expression. Although additionalstudies are apparently required to understand the biologicalsignificance of deregulated COX-2 expression in HCV-infectedcells, possible roles of COX-2 in HCV pathogenesis can be hypothesized.It is possible that HCV avoids unwanted stimulation of adaptiveimmune responses against itself by down regulating COX-2 expressionin HCV-infected antigen-presenting cells, such as macrophagesand dendritic cells. Another possibility is that, by down regulatingCOX-2 expression, HCV-infected immune effector cells avoid lysisby NK cells and/or apoptosis caused by cyclopentenone 15d-PGJ₂,which is derived from PGD₂ during inflammatory responses. Lastly,HCV avoids evoking antiviral activities of prostanoids by suppressingCOX-2 expression. Although a precise mechanism is not known,PGA and PGJ series prostaglandins have been reported to haveantiviral activities against various DNA and RNA viruses (64).Recently, PGI₂ was reported to have antiviral activity againstrespiratory syncytial virus in a respiratory syncytial virus-induceddisease mouse model (31). Similarly, in a vaccinia virus-induceddisease mouse model, PGI₂-treated mice showed enhanced survival,whereas NSAIDs-treated mice succumbed to virus-induced mortality(80). Therefore, impaired COX-2 expression could be attributableto host immune evasion by HCV, favoring persistent HCV infection.

COX-2 expression is regulated by diverse transcription factorsthat include NF- ${kappa}$ B, CREB, and C/EBP-ß. However, therelative importance of these pathways is not clear and, moreimportantly, the impact of NF- ${kappa}$ B on COX-2 expression in macrophagesis controversial. Our results show that COX-2 is induced byselective activation of NF- ${kappa}$ B alone and is suppressed by expressionof superrepressor I ${kappa}$ B ${alpha}$ , suggesting that NF- ${kappa}$ B activation is sufficientfor COX-2 expression.

Our data in Fig. 8 show that COX-2 induction by LPS is substantiallyinhibited by HCV core. Given the complexity of cox-2 transcriptionalregulation and the diverse transcriptional modulating functionof HCV core, the possibility that HCV core also influences functionof other transcription factors that are involved in COX-2 expressioncannot be excluded. It is also possible that the significantdecrease of COX-2 is due to a direct interruption of TNF- ${alpha}$ orother cytokine production, via NF- ${kappa}$ B suppression by HCV core,which otherwise contributes to COX-2 expression in an autocrinefashion, as LPS treatment induces TNF- ${alpha}$ and other cytokines that,along with TLR4 signaling, enhance COX-2 expression (11). Finally,it is conceivable that HCV core could alter the half-life orincrease the turnover rate of COX-2 protein, although transcriptionof the cox-2 gene is closely related to the level of COX-2 proteinexpression (M. D. Bryer, personal communication), and the half-lifeof COX-2 mRNA is significantly prolonged by LPS treatment (10).

NF- ${kappa}$ B is either activated or suppressed by HCV core, dependingon subtype. For example, while the core protein of HCV type1b activates NF- ${kappa}$ B activity, that of HCV type 1a (Hutchinsonstrain) suppresses NF- ${kappa}$ B activity (54). A mutagenesis study showedthat a single amino acid substitution in the core protein ofHCV type 1a converting either from Lys at 9 (K9) to Arg or fromAsn at 11 (N11) to Thr is sufficient to relieve its inhibitoryfunction (54). However, it is not understood how these aminoacid residues affect modulation of NF- ${kappa}$ B activity.

Previously, it was proposed that activation of NF- ${kappa}$ B by type1b HCV core is related to IKKß, although a directbiochemical interaction between the two proteins and directfunctional interference by the HCV core protein were not presented(79). In the present study, we report evidence that type 1aHCV core protein biochemically interacts with IKKßand inhibits IKK kinase activity (Fig. 6A and 7). Although wedo not fully understand the mechanism that, unlike type 1a HCVcore, type 1b HCV core activates IKK signalsome and NF- ${kappa}$ B activity,our data raise the possibility that a capability of HCV coreto physically interact with IKKß might determine differentialregulation of NF- ${kappa}$ B activity. It is possible that the type 1aHCV core protein sequesters IKKß and prevents formationof a fully functional IKK signalsome, which impairs NF- ${kappa}$ B signaling.Alternatively, it is possible that the HCV core protein interfereswith stoichiometric interactions between IKKß and,subsequently, other subunits of IKK signalsome. More importantly,given that a single amino acid change at either K9 or N11 relievesinhibitory functions of the HCV core protein type 1a, it isconceivable that microenvironmental changes such as electriccharge or physical hindrance affect interactions between IKKßand its substrates.

Another observation in our study that might lead to understandingof the mechanism by which HCV core suppresses IKK activity isthe disruption of cytoplasmic-nuclear partitioning of IKKß.As shown in Fig. 4, either transfected or endogenous IKK-ßis located in both cytoplasmic and nuclear fractions; however,the HCV core protein selectively disturbs the nuclear localizationof IKK-ß. In similar experiments to measure cellulardistributions of IKK ${alpha}$ and IKK ${gamma}$ , HCV core did not affect the distributionsof these two subunits of the IKK signalsome (data not shown).The functional significance of nuclear IKKß and thebiological significance of blocked translocation of IKKßby the HCV core protein are areas of intense investigation.

In summary, we studied the inhibitory function of HCV core onNF- ${kappa}$ B activity in macrophages. Our data indicate that inhibitionof NF- ${kappa}$ B activity by the HCV core protein might be related toits physical interaction with and interrupted nuclear localizationof IKKß. Furthermore, our data show that COX-2 expressionis induced by NF- ${kappa}$ B activation and suppressed by the HCV coreprotein. These results suggest the possibility that HCV-infectedmacrophages have impaired immune responses at least in partdue to impaired COX-2 expression.

ACKNOWLEDGMENTS

This work was supported by the Department of Veterans Affairsand National Institutes of Health grants AI057591 (Y.S.H.),HL075557 (J.W.C.), and HL061419 (T.S.B.) and program projectHL066196.

We thank Tamara Lasakow for editorial assistance with the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2650. Phone: (615) 343-2365. Fax: (615) 343-7748. E-mail: myungsoo.joo{at}vanderbilt.edu.

REFERENCES

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