06) 58–68www.elsevier.com/locate/yviro

Virology 354 (20

Hepatitis C virus core protein induces expression of genes regulating immuneevasion and anti-apoptosis in hepatocytes

Hau Nguyen, Sumathi Sankaran, Satya Dandekar ⁎

Department of Medical Microbiology and Immunology, School of Medicine, Topper Hall, Room 3146, University of California, Davis, CA 95616, USA

Received 14 February 2006; returned to author for revision 17 March 2006; accepted 24 April 2006Available online 28 July 2006

Abstract

Hepatitis C virus (HCV) Core protein is implicated in the development of hepatocellular carcinoma (HCC). We utilized a HepG2 humanhepatocyte cell line with inducible expression of HCV Core protein (HCV-1b) to investigate the early effects of Core protein on hepatocyte geneexpression and to identify molecular processes modulated by the Core protein. A significant change was observed in the expression of 407 genes,which included genes regulating apoptosis, immune response, and cell cycle. Some of these genes were previously known to be tumor markers.The decreased expression of chemo-attractants such as TNFSF10, CCL20, and osteopontin was observed, which suggested that HCV Coreexpression could lead to suppression of inflammatory response as well as trafficking of macrophages and neutrophils to the site of HCV infection.An increased expression of anti-apoptosis factors including PAK2, API5, BH1, Tax1BP1, DAXX, and TNFAIP3/A20 was observed. Some ofthese genes were also linked to the regulation of NFKB activation and that the alteration of their expression levels, by HCV Core, might lead to thesuppression NFKB activation of inflammatory responses. Our data suggested that Core expression may contribute to the viral persistence byprotecting infected hepatocytes from cell death by the suppressing apoptosis and inflammatory reaction to HCV viral infection.© 2006 Elsevier Inc. All rights reserved.

Keywords: HCV Core; Immune regulation; Apoptosis; Microarray

Introduction

Hepatitis C virus (HCV) is the etiologic agent of acute andchronic hepatitis affecting more than 100 million peopleworldwide (Uchida, 1994). Chronic hepatitis is one of theleading causes of liver cirrhosis and hepatocellular carcinoma(HCC) (Saito et al., 1990), yet the mechanisms of viralpersistence and HCV associated tumorigenesis have not beenfully elucidated. HCV, a member of the flavivirus family, has a9.5-kb positive single-stranded RNA genome, which encodes apolyprotein that is processed into at least 10 different structuraland nonstructural proteins (Choo et al., 1989; Ray and Ray,2001). The HCV Core protein is derived from the N-terminus ofthe polypeptide and has a highly basic N-terminal region and ahighly hydrophobic C-terminus (Kunkel and Watowich, 2002).In addition to being the major component of the viral

⁎ Corresponding author. Fax: +1 530 752 8692.E-mail address: sdandekar@ucdavis.edu (S. Dandekar).

0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2006.04.028

nucleocapsid, this multifunctional protein has also beenimplicated in hepatocyte proliferation and cellular transforma-tion, although molecular mechanisms mediating these processesare not fully determined.

Several human and murine studies have investigatedmechanisms of HCV Core protein in the development ofHCC which included the effects on the cell cycle and growth.We reported that the expression of HCV Core proteinmodulated cell cycle of hepatocytes as indicated by an increasedlevel of the cell-cycle-dependent kinase inhibitor (CdkI) p21, atarget of the p53 tumor suppressor, and found that the increasedlevel of p21 corresponded to a decreased level of cdk2 kinaseactivity and arrested HepG2 hepatocytes in the G0/G1 phase ofthe cell cycle (Nguyen et al., 2003). The CdkI p21 has adominant role in arresting cells in the G0/G1 phase of the cellcycle by inhibiting the kinase activity of Cdk2-cyclin Ecomplex. Other studies have also demonstrated that HCVCore protein modulates the expression of p21 (Oka et al., 2003;Yamanaka et al., 2002). It has been reported that the p53 tumor

Fig. 1. (A) Expression of HCV Core protein in hepatocytes. Western blotanalysis demonstrates the inducible expression of HCV Core protein in HepG2-Core cells. Induced (I) and uninduced (UI) in triplicates. The numbers in theparentheses indicate the individual experiment subsets of the triplicateexperiment. (B) Expression of HCV Core protein in HepG2-Core cells reducedcell proliferation. The Core expression in HepG2-Core cells was induced withPonA for 48 h and compared to uninduced HepG2-Core cells. Live cells werecounted with Trypan blue dye. Nontransfected HepG2 cells with and withoutPonA induction served as negative control for cell proliferation assay. Each cellgrowth assay was performed in triplicates.

59H. Nguyen et al. / Virology 354 (2006) 58–68

suppressor is activated in the presence of HCV Core proteinexpression (Lu et al., 1999) which could lead to an increasedexpression of p21. Cyclin E, a protein instrumental in thetransversal of the G1/S checkpoint, was shown to be elevated incells expressing HCV Core protein (Cho et al., 2001). Inaddition, Core protein activates the c-myc promoter butsuppresses the fos promoter, indicating that HCV Core is apotential regulator of oncogenes (Ray et al., 1995). In ourprevious studies, we observed that HepG2 cells with HCV Coreexpression accumulated in the G0/G1 phase of the cell cycle(Nguyen et al., 2003). Other studies of HCV Core proteinexpression using the Huh-7 cell line reported either enhanced orno change in cell proliferation (Fukutomi et al., 2005; Li et al.,2002). The Huh7 cell line contains a point mutation of the p53gene, while the HepG2 cell line has the wt p53 gene copies,which could lead to different effects of Core protein on cellproliferation. However, these studies together suggest that Coreprotein may have an impact on HCV pathogenesis throughmodulation of cell cycle regulatory mechanisms.

HCV Core protein is also linked to the formation of steatosisboth in in vitro cell cultures and in vivo. Core protein expressionincreased the formation of cellular lipid droplets, and Core co-localized with apolipoprotein AII on the surface of cytoplasmiclipid droplets (Barba et al., 1997; Sabile et al., 1999). Expressionof Core protein in the liver of transgenic mice induced hepaticsteatosis (Moriya et al., 1997) and promoted the development ofhepatocellular carcinoma (Moriya et al., 1998). The ability ofCore protein to mimic HCV disease in the mousemodel suggeststhat theexpressionof thisprotein is integral toHCVpathogenesis.

To investigate the molecular mechanisms of the HCV Coreprotein induced changes in hepatocytes, we examined the geneexpression profiles of HepG2 hepatocytes in the presence ofHCV Core protein by DNA microarray analysis. Our datashowed that HCV Core protein expression led to modulation ofgenes regulating immune defense response, anti-apoptosis, andgenes previously known as markers of hepatic tumorigenesis.The analysis of the interconnecting network of gene functionssuggests that HCV Core may interfere with the immune defenseresponse primarily through regulation and quenching of NFKBactivation. The alteration of NFKB function could lead to thesuppression of inflammatory responses and ensure the survivalof HCV infected hepatocytes from immune surveillance. Inaddition, Core expression also altered the gene expression thatcould confer protection of cells from apoptosis. Our datasuggest that HCV Core may play an important role in themechanism of HCV viral persistence in addition to acting as thebuilding blocks of the viral capsid.

Results

HCV Core protein expression led to decreased hepatocyte cellproliferation

We investigated the effect of HCV Core protein onhepatocyte proliferation following the induction of Coreexpression in HepG2-Core hepatocyte cells. We used anecdysone-inducible expression system (Materials and methods

section) to achieve inducible expression of HCV Core protein inHepG2 hepatocytes. This system allowed us to examinealterations in hepatocyte proliferation and gene expressionimmediately following the induction of Core protein expres-sion. The advantage of the inducible expression systems is thatit prevents any modification of the normal cell phenotype that isassociated with exposure to high constitutive expression of Coreprotein in the cells. We also appended to the N-terminus of HCVCore protein with a sensitive 3XFLAG peptide tag to enhancethe sensitivity of detection of HCV Core expression. As shownby Western blot analysis using anti-FLAG antibodies (Fig. 1A),HCV Core protein was expressed in hepatocytes induced byponasterone A (PonA), a synthetic analog of insect ecdysone,but not in uninduced HepG2 cells. The numbers in theparentheses indicate the subsets of the triplicate experiment.The exaggerated intensity of Core protein expression shown inthe figure is due to the sensitivity of the 3XFLAG tag. We havepreviously shown that the level of Core protein in ourexpression system was significantly less pronounced when weused anti-Core antibody to directly detect Core proteinexpression (Nguyen et al., 2003). There was no visualindication of significant cytotoxicity or cell death in HepG2cells following the induction of Core protein expression, asindicated by the normal morphology of the cells in the culturesand the lack of cellular uptake of the Trypan Blue dye, an

Fig. 2. Graphical hierarchical clustering pattern demonstrated gene expressioninduced by HCV Core protein in HepG2 cells. The 3 columns on the leftrepresent the mRNA expression profiles of HepG2-Core-expressing cells, andthe 3 right columns represent expression profiles of HepG2 control cells(blue = low expression, red = high expression). The graphical cluster wasgenerated using the dChip array analysis program.

60 H. Nguyen et al. / Virology 354 (2006) 58–68

indicator for cell death. There was also no visual indication ofcytotoxicity in mock-induced control HepG2 cells following theaddition of PonA. The expression of Core protein for 48 h ininduced HepG2-Core cells resulted in a lower number of cellcounts as compared to uninduced HepG2-Core cell cultures(Fig. 1B). The counted cell number was 25% less for cellsexpressing Core protein compared to cells without Core proteinexpression. There was no significant difference in the number ofcells counted in untreated and PonA treated HepG2 controls(NT I vs. NT U), indicating that the lower cell number wasdependent on Core protein expression and was independent ofPonA treatment. This observation is consistent with ourprevious studies, which suggested that HCV Core blockedcell proliferation at the G1-S phase of the cell cycle (Nguyen etal., 2003).

HCV Core protein induced changes in hepatocyte geneexpression

In order to identify genes and molecular processes associatedwith Core induced changes in hepatocyte cell proliferation, weevaluated gene expression profiles of Core-expressing HepG2-Core cells in comparison to PonA induced control nontrans-fected HepG2 cells. The Core expression led to changes (1.3-fold or more and P value of less than or equal to 0.05) in mRNAexpression of 534 genes; 453 upregulated genes, 81 down-regulated genes. The initial screenings were conducted withmore stringent fold change standards to screen for mostprominent changes. However, the high rigid fold changethresholds produced spotty data that omitted many smallerchanges of genes that have significant statistical confidential Pvalues. We included in this report genes that have small butconsistent fold changes that also accompanied with significantconfident P values. Hierarchical clustering graph of thesignificant gene list showed a distinct expression patternassociated with HCV Core protein expression (Fig. 2). GOOntological classification of this list of genes showed that Coreprotein expression led to a modulation of genes involved in theregulation of immune response as well as apoptosis (Tables 1,2). These genes and their respective biological functions andprocesses were further analyzed to determine the possibleeffects from their altered expression.

HCV Core expression modulates immune response genes

The expression of Core protein in HepG2 cells resulted in themodulation of genes regulating host immune responses topathogens (Table 1). The altered expression pattern of immuneresponse genes indicated that the Core protein may dampen theeffectiveness of the immune response to HCV infection. HCVCore led to an increased expression of some genes involved ininnate immunity. Genes with increased expression includedMHC class I polypeptide-related sequence B (MICB), NK celltranscript 4 (NK4/IL-32), defensin beta 1 (DEFB1), beta-2-microglobulin (B2M) and nuclear factor of kappa inhibitor lightalpha (NFKBIA/IKBα). MICB is a ligand of NKG2D type IIreceptor that can activate the cytolytic response of natural killer

(NK) cells, CD8 αβ T cells, and γδ T cells (Groh et al., 1998).NK4 was initially discovered as a product of IL-2 activated NKcells (Dahl et al., 1992), and it is recently suggested to induceinflammatory factors, and it is also an antagonist to HGFinduced cell growth and an inhibitor of angiogenesis (Matsu-moto and Nakamura, 2003). Defensin B1 is a member of themicrobactericidal peptide family that plays an important role ininnate immune response at mucosal surfaces. Defensin B1 hasbeen shown to be expressed and readily detectable inhepatocytes, and the presence of Defensin B1 in hepatic tissueswas proposed to act as defense mechanism against pathogenicinfection of the hepatic biliary system (Harada et al., 2004).B2M has a close homology to the immunoglobulins, and itsexpression is essential for the expression and assembly of HLAclass I (Arce-Gomez et al., 1978; D'Urso et al., 1991). TheNFKBIA (IKBα) protein has a crucial role in regulatingimmune response, inflammation and apoptosis since it is themain regulator of NFKB activation (Viatour et al., 2005). Theincreased expression of IKBα could result in a widespreaddysregulation of NFKB transactivated genes. The pro-inflam-matory genes downstream of NFKB activation could besuppressed due to the enhanced expression of IKBα by HCVCore.

The expression of HCV Core expression also resulted in thedownregulation of tumor necrosis factor (ligand) superfamily,member 10 (TNFSF10/TRAIL), liver activation regulatedchemokine (LARC/CCL20/MIP3A), secreted phosphoprotein

Table 1Immune response related genes were modulated by HCV Core expression

Gene name and probe set Foldchange

P value

LARC/MIP3A/CCL20 (liver activation regulatedchemokine, 205476_AT)

−3.15 0.00108

SSP1/Osteopontin (secreted phosphoprotein 1,209875_S_AT)

−3.04 0.00374

TNFSF10 (tumor necrosis factor (ligand) superfamily,member 10, 202688_AT)

−1.89 0.01131

IL1RL1LG/ST2L (interleukin 1 receptor-like 1 ligand,203679_AT)

−1.54 0.03166

FN1 (fibronectin 1, 212464_S_AT) −1.45 0.01966MAP2K3 (mitogen-activated protein kinase kinase 3,

215499_AT)1.36 0.03018

B2M (Beta-2-microglobulin, 201891_S_AT) 1.42 0.01596CTSC (cathepsin C, 201487_AT) 1.42 0.00750MICB (MHC class I polypeptide-related sequence B,

206247_AT)1.56 0.01557

NK4 (NK cell transcript 4, 203828_S_AT) 1.63 0.01613PLA2G2A (phospholipase A2, group IIA, 203649_s_at) 1.76 0.00007Nuclear factor of kappa inhibitor light alpha (NFKBIA,

201502_s_at)1.90 0.00040

DEFB1 (defensin, beta 1, 210397_AT) 3.04 0.00449

Table 2Genes regulating apoptosis were modulated by HCV core expression

Gene name and probe set Foldchange

Pvalue

Tax1 (human T cell leukemia virus type I) binding protein 1(TAX1BP1, 200976_s_at)

1.34 0.032

BAX inhibitor 1/Testis enhanced gene transcript (BI1,200803_s_at)

1.36 0.0016

Apoptosis inhibitor 5 (API5, 201687_s_at) 1.49 0.0358Death-associated protein 6 (DAXX, 201763_s_at) 1.55 0.0387Tumor necrosis factor, alpha-induced protein 3 (A20,202644_s_at)

1.65 0.0115

p21 (CDKN1A)-activated kinase 2 (PAK2, 208877_at) 1.79 0.0119

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1 (SPP1/Osteopontin) and phospholipase A2 group 2A(PLA2G2A). TNFSF10 is a member of the TNF family ofproteins that function as strong mediators of immune regulationand the inflammatory response. The presence of TNFSF10 wasdemonstrated in liver tissues of HCV infected patients andcultured hepatocytes (Mundt et al., 2003). TNFSF10 was shownto act through the TRAIL pathway to induce apoptosis ondiverse tumor cell lines (Wiley et al., 1995). PLA2G2A is bestknown as the inflammatory factor that is highly expressed insynovial fluid of arthritic joints, and its downregulation in thepresent of HCV Core expression is interesting from theperspective of its role in the pro-inflammatory process. LARCand osteopontin function as chemo-attractants for recruitment ofCD4+, CD8+, and CD44+ T lymphocytes (Homey et al., 2000;Nelson et al., 2001; Weber et al., 1996). As the functions of thegene products were briefly outlined above, the data suggest thatthe expression of HCV Core could hamper the recruitment ofimmune cells by inhibiting the expression pattern of immuneand chemokine factors.

HCV Core induced anti-apoptosis factors

Expression of HCV Core protein in hepatocytes led toenhanced expression of genes with anti-apoptotic functions.This gene category included p21-activated protein kinase(PAK)-2, apoptosis inhibitor 5 (API5), BAX inhibitor 1(BH1), Tax1 (human T-cell leukemia virus type I) bindingprotein 1 (Tax1BP1), death-associated protein 6 (DAXX), andtumor necrosis factor-alpha-induced protein 3 (TNFAIP3/A20)(Table 2).

PAK-2 is a gamma protein member of the PAK family that isphosphorylated by the p21 CdKI. PAK2 participates in multiplecellular processes that include acting as a substrate for caspase 3cleavage during the apoptotic process (Chan et al., 1998).

However, the expression of full-length PAK-2 was shown toprotect NIH-3T3 mouse fibroblast cell line against apoptosisstimulated by TNF-α or growth factor removal or UV light bypreventing the pro-apoptosis Bad protein from dimerizationwith Bcl2 (Jakobi et al., 2001). The API5 factor is another anti-apoptosis factor whose expression was increased in the presenceof HCV Core protein. The API5 was shown to protect NIH-3T3and human cervical cell lines from undergoing apoptosisfollowing serum starvation (Kim et al., 2000; Tewari et al.,1997). The expression of BI1 was also increased with theexpression of Core. The BI1 is a known anti-apoptosis factorthat can block Bax induced apoptosis (Xu and Reed, 1998). Ourdata showed that the expression of Tax1BP1/T6BP andTNFAIP3/A20 genes was also increased in the presence ofHCV Core. Together, Tax1BP1 and A20 form a proteincomplex that effectively blocks TNF-α induced apoptosis of3T3 cells (De Valck et al., 1999). An increased expression ofDAXX was also observed in Core-expressing HepG2 cells. Theeffect of DAXX expression on apoptosis has not been welldefined. DAXX was shown to enhance Fas mediated apoptosis(Pluta et al., 1998; Yang et al., 1997), however, RNAi silencingof DAXX was shown to sensitize cells to apoptosis (Chen andChen, 2003; Michaelson and Leder, 2003).

The data of showed that HCV Core expression correlatedwith the increased expression of factors known to protect cellsagainst apoptosis. Interestingly, both Tax1BP1/T6BP and A20were shown to inhibit activation of NFKB and downstreamtransactivation inflammatory responses (Heyninck et al., 1999;Iha et al., 2000). DAXX was also reported to repress NFKBactivation and expression (Michaelson and Leder, 2003).

Expression of known tumor markers

The HCV Core protein induced expression of genespreviously known to be the markers of various human tumors.Increased levels of Ep-CAM, S100A6, IGF-2, Kras2, andCyclin D1 were found in Core-expressing hepatocytes (Table3). The transmembrane glycoprotein Ep-CAM is expressed innormal epithelium of different tissues. Overexpression of thisgene has been documented in a variety of human carcinomas(Went et al., 2004) and is considered as a marker associated withthe development of HCC (Ruck et al., 2000). DNA microarrayanalysis showed that the expression of Ep-CAM increased intissues of individuals identified to be at high risk to develop

Table 4RT-PCR validation of DNA microarray data

Gene name Fold change (HepG2-Core induced vs. HepG2 induced)

GAPDH normalized 18S normalized Microarray value

CCL20/MIP3A −2.56 −1.61 −3.15IGF2 1.62 2.58 3.49KRAS2 2.93 4.66 7.50PCNA 2.17 3.46 1.89

Table 3Tumor marker genes were modulated by HCV Core expression

Gene name and probe set Foldchange

P value

IGF2 (insulin-like growth factor 2/Somatomedin A,202410_X_AT)

3.49 0.00006

CCND1 (cyclin D1, 208711_S_AT) 2.71 0.00483Ep-CAM/TACSTD1 (tumor-associated calcium signaltransducer 1, 201839_S_AT)

1.47 0.04533

S100A6 (S100 calcium binding protein A6, 217728_AT) 1.46 0.00848KRAS2 (v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogenehomolog, 214352_S_AT)

7.5 0.00414

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HCC (Kim et al., 2004). The S100A6 is a member of the S100calcium-binding protein family that has been linked with cancertumorigenesis and metastasis. S100A6 was highly expressed inthe metastasizing fronts of colorectal adenocarcinomas and livermetastases (Cross et al., 2005; Komatsu et al., 2002). IGF2 is asecreted factor that appears to be an autocrine regulator of cellproliferation (Lund et al., 2004). Increased expression of IGF2was reported in HCC samples compared to normal liver samples(Xu et al., 2001). IGF2 expression levels were also found to beelevated in Wilms tumor tissues (Scott et al., 1985). The loss ofpaternal imprinting of IGF2 is a potential correlate of colorectalcancer development (Cui et al., 2003). The loss of imprint ofIGF-2 has also been suggested to contribute to the progressionof HCC (Iizuka et al., 2004; Tannapfel et al., 2003). KRAS2 is aGTPase that transduces signal from cell surface receptors. Theactivation of Kras2 either due to mutations or by increasedexpression has been shown to be a corollary of various forms ofcancer. Cyclin D1 has been implicated in cell transformation,and its expression was altered in cirrhotic and HCC tissues(Smith et al., 2003; Xu et al., 2001). Cyclin D1 expressioncontributed to rat kidney cell transformation by complementinga defective adenovirus E1A oncogene (Hinds et al., 1994). Thedata indicated that HCV Core expression resulted in theenhanced expression of genes that are markers of tumorsincluding hepatic tumor.

Validation of DNA microarray data by real-time RT-PCR andWestern blotting

We utilized Real time RT-PCR to measure the transcriptlevels of selected genes to validate the changes in geneexpression observed by DNA microarray analysis. Expressionlevels of LARC/CCL20, IGF2, Kras2, and PCNA werequantitated using 18S ribosomal and GAPDH genes as theinternal controls. These genes were selected for validation asthey represented a diverse magnitude of changes and intensityof expression as seen from the DNA microarray data. Theincrease or decrease in expression of these genes by DNAmicroarray analysis was in general agreement with the real-timeRT-PCR validation data (Table 4). Table 4 shows the data forinduced HepG2-Core and induced nontransfected HepG2 celllines to validate the numbers obtained from our DNAmicroarray data. We also determined the changes in the geneexpression between the PonA induced and noninduced cell

cultures to compensate for differences due to the presence ofPonA. A minimal change in gene expression levels between thePonA induced and noninduced cells was observed. This slightlyaffected the numerical fold change values between inducedHepG2-Core and induced nontransfected HepG2 samples, butthe compensated values did not alter the direction of change(data not shown).

The DNA microarray data showed that proliferating cellnuclear antigen (PCNA) mRNA was upregulated in HepG2cells with HCV Core expression. PCNA is an importantcomponent of the DNA polymerase complex that is essential forthe DNA synthesis process that occurs during cell division andproliferation (Bowman et al., 2004). PCNA is also known tointeract with p21 and act as a component of the RAD6-dependent DNA repair mechanism (Hoege et al., 2002). It ispossible that the arrest of cells at the G0/G1 and the increase ofp21 levels could cause the initiation of PCNA transcription toactivate the DNA damage and repair surveillance mechanisms.Other studies utilizing DNA microarrays also reported anincreased expression of PCNA in HCV infected liver samples(Smith et al., 2003; Zindy et al., 2005). Western blot analysis ofPCNA expression was performed to correlate changes observedat the mRNA level to the changes at the protein level. The levelsof PCNA protein in induced (I) cells expressing HCV Coreprotein (Fig. 3, lanes 3 and 6) were found to be higher than innontransfected (NT, lanes 1 and 4) and uninduced (UI, lanes 2and 5) cells without HCV Core expression. The addition ofPonA to HepG2 cells did not alter PCNA protein levels (lanes 1vs. 2 and 4 vs. 5). Each lane was loaded with equal amounts ofproteins as shown by the GAPDH levels. The change of PCNAprotein level is small but consistent with the change of PCNAmRNA expression in HCV Core-expressing cells. AdditionalWestern blots were conducted to verify the protein levels forIKBα and PAK2. The direction of changes of protein levels ofIKBα and PAK2 also reflected the direction of changes of theirmRNA.

Discussion

In this study, the DNA microarray analysis of HCV Coreprotein expressing HepG2 cells provided additional informationto changes that have been detected in the HepG2 cell line geneexpression profile induced by HCV-1b Core protein. Theexpression data suggested that HCV Core expression led to thedecrease of cell proliferation, repression of pro-inflammatorysignals, and enhanced expression of anti-apoptosis factors. Thiscould lead to prevention of host cell death from apoptosis and

Fig. 3. Western blot analysis of PCNA, IKBα and PAK2 protein levels in HCVCore-expressing hepatocytes. PCNA, IKBα and PAK2 protein levels weredetected in HepG2-Core induced (I) cells (lanes 3 and 6) and HepG2-Coreuninduced (UI) cells (lanes 2 and 5) and nontransfected (NT) HepG2 inducedcontrol cells (lane 1 and 3). The detection of the Flag-tagged HCV Core proteinis shown on the top panel. The levels of GAPDH are shown to demonstrate theamount of protein loaded in each lane. The same membrane blot was used toprobe for each protein, the membrane was sectioned and probed (or re-probed)with different antibodies to ensure consistency.

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suppression of the immune surveillance system. The analysis ofgene functions and regulators suggested that the regulation ofNFKB might be a central target for HCV Core in its attempts tomodulate apoptosis and immune suppression in hepatocytes. Inaddition, HCV Core affected the expression of other cellularfactors that may result in the alteration of cell growthcharacteristics. It is unlikely that inhibitions in downstreamtranslational processes would nullify the interpretation of thetranscriptional data since, in the HepG2 cell expression system,it was shown that HCV Core expression did not affect cap-dependent translation (Li et al., 2003; Shimoike et al., 2006).Furthermore, cap-dependent translation was not affected byvarying the amount of HCV Core in the in vitro translationsystem (Boni et al., 2005).

The gene expression data from this study provide support tothe hypothesis that HCV Core expression decreases cellproliferation. We observed an increased of Geminin andKHDRBS1 (1.5- and 1.57-fold increase). These genes were ofspecial interest due to their anti-proliferative functions thatcould potentially contribute to the observed slower proliferationof Core-expressing cells. The increased expression of Gemininhas been shown to inhibit DNA replication during S, G2, and Mphases by preventing complete assembly of the DNA pre-replication complex (McGarry and Kirschner, 1998).KHDRBS1 is a tyrosine-phosphorylated, Src-associated proteinin mitotic cells. KHDRBS1 expression was demonstrated toinhibit serum-induced DNA synthesis, suggesting that it mayplay a role in controlling the G1/S cell cycle transition (Barlat etal., 1997). In addition, the decreased expression of LARC,hepatocyte growth factor receptor (HGRF/met, 1.34-fold

decrease), and alpha fetal protein (AFP, 2.64-fold decrease)was observed. LARC functions primarily as a chemotaxisfactor, it can act as an autocrine/paracrine factor that enhanceshepatic cell growth (Fujii et al., 2004). The suppression ofLARC expression correlated with decreased proliferation ofHCV Core-expressing cells. The decreased expression of twohepatic pro-growth factors, HGFR and AFP in HepG2 cellsexpressing Core protein also are supportive of the observationthat HCV Core protein expression suppresses cell proliferation.Decreased expression of HGFR could reduce the number ofreceptors that are available to receive and transmit growthsignals. It is thought that an increased serum AFP level isprognostic of active liver regeneration (Karvountzis andRedeker, 1974), and conversely a decrease in AFP transcriptioncould be interpreted as an indicator of lower hepatocyteregeneration and proliferation.

Overall, the change (increased or decreased) in theexpression of immune response-associated genes in the Core-expressing hepatocytes seemed to have the potential tonegatively impact the anti-viral immune response. The geneexpression data suggested that HCV Core may impairinflammatory responses and enhance cell survival of infectedcells by interfering with trafficking and infiltration of immunecells to the site of HCV infection and by preventing cell deathfrom necrosis and apoptosis. Downmodulation of the expres-sion of chemotactic factors such as LARC, and osteopontincould suppress immune cell trafficking. LARC is the knownchemotactic ligand for the CCR6 receptor on B cells, T cells,dendritic cells, and macrophages. The presence of LARCexpression is correlated to the increased necrosis levels ofinflamed liver tissue (Shimizu et al., 2001). Previous studiesshowed the levels of LARC increased in HCV patients thatresponded to INF treatment as compared to no change in thelevels of LARC in patients that were classified as nonresponders(Yamauchi et al., 2002). These findings suggested that LARCplays an important role of recruiting immune cells for theeffective mediation of inflammatory response and clearance ofviral infection. In this context, HCV Core could impair aneffective inflammatory response and prevent viral clearance byrepressing the expression of LARC. Our data also showed thatHCV Core suppressed osteopontin expression which isconsistent with a previous report (Ohkawa et al., 2003). Thesecretion of the extracellular matrix protein osteopontin by NKcells recruits and activates neutrophils and macrophages (Diaoet al., 2004; Weber et al., 2002). The correlation between highosteopontin levels and hepatic tissue injury due to the increaseof neutrophil infiltration was demonstrated in the rat model(Apte et al., 2005). The inverse relationship between LARC,osteopontin levels, and HCV Core expression suggests apossible mechanism of immune evasion employed by theHepatitis C virus.

HCV Core could contribute to viral persistence by regulatinganti-apoptosis factors that could prevents apoptosis and enhancethe survival of HCV infected host cells. The upregulation ofpredominantly anti-apoptosis genes such as PAK2, API5, BH1,Tax1BP1, TNFAIP3/A20, and DAXX demonstrated thepotential anti-apoptotic property of HCV Core. An enhanced

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expression of such anti-apoptosis genes has been shown toinhibit stress-induced apoptosis in various cell lines (De Valcket al., 1999; Kim et al., 2000; Tewari et al., 1997; Xu and Reed,1998). The functions of some of these anti-apoptosis genes areconserved across a wide spectrum of organisms. For example,the plant homologous of BI1 was shown to suppress Baxinduced apoptosis in yeast (Kawai et al., 1999; Sanchez et al.,2000; Xu and Reed, 1998). The normal expression of BI1 andDAXX is required for different cell lines to avoid spontaneousapoptosis (Grzmil et al., 2003; Kawai et al., 1999; Sanchez etal., 2000; Xu and Reed, 1998). The modulation of TNFSF10,API5, A20/TNFAIP3 by HCV Core was also recently shown by2-color DNA microarrays (Liu et al., 2005). It was proposedthat the TNFSF10 ligand/receptor system is a hepatic paracrinemechanism to eliminate virally infected hepatocytes (Mundt etal., 2003). Suppression of TNFSF10 expression by HCV Coremay minimize apoptosis of infected host cells and maintainHCV persistence which suggests an additional anti-apoptosismechanism employed by HCV Core to prevent viral clearance.These data suggest that HCV Core expression could contributeto the prevention of apoptosis by altering the expression ofimportant the anti-apoptosis factors.

The NFKB is a major transcription factor that is involved inthe activation of inflammatory and cell activation factors. Thesuppression of NFKB activation by HCV Core protein mayinclude multiple mechanisms. Our gene expression analysis hasidentified a number of genes that could contribute to themechanisms of inflammatory repression at the molecular level.Recent reports linked HCV Core protein to the inactivation ofNFKB (Joo et al., 2005; Ray et al., 2002). Anti-apoptosis genes(DAXX, Tax1BP1, A20) and immune response genes(PLA2G2A, NFKBIA/IkBa) have been previously demonstrat-ed to regulate the activity of NFKB. Using NFKB reporterconstructs, it was shown that increased expression of DAXXcaused the repression of the NFKB promoter (Michaelson andLeder, 2003). Similarly, overexpression of TaxBP1 led tosuppression of the NFKB promoter (Iha et al., 2000). The A20protein directly interacts with Tax1BP1, and exerts anti-apoptosis effects (De Valck et al., 1999). The A20 proteinwas a crucial anti-apoptotic factor in the mouse model and thatA20-deficient mice were susceptible to sub-lethal doses of TNF(Lee et al., 2000). The A20 protein was shown to block IL-1and TNF activation of NFKB via its interaction with TNFreceptor associated factor 2 (TRAF2) and TRAF6 (Heyninckand Beyaert, 1999; Heyninck et al., 1999) and consequentlyensure the cytoplasmic sequestration and inactivation of NFKBby the IkB complex. The function of PLA2G2A protein ininflammation is through its enzymatic activity to releasearachidonic acid from cellular phospholipids. Arachidonic acidis the precursor of prostaglandins and leukotrienes. Prosta-glandins are vasodilators and chemotaxis factors that promoteneutrophil migration and accumulation at the sites ofinflammation. PLA2G2A induced production of the monocytechemotactic protein-1 (MCP-1) chemoattractant (Fuentes et al.,2002) in macrophages. The injection of PLA2G2A has beenshown to exacerbate the inflammation of arthritic joints(Bomalaski et al., 1991). Following the stimulation of a

RAW264.7 macrophage cell line with PLA2G2A, an enhancedbinding of NFKB to its promoter sequences and degradation ofthe IkBa subunit was reported (Baek et al., 2001). Lastly, HCVCore expression led to increased expression of IkBa subunit ofthe IkB complex. The increased presence of IkB complexeswould enhance sequestration and inactivation of the NFKBtranscription complex. Our gene expression data providednovel findings that suggest that the expression of genesregulating NFKB was altered to suppress NFKB activation andsubsequently would decrease inflammation. Furthermore, thedirection of changes for anti-apoptosis, chemotaxis, andinflammation genes support the hypothesis that the Coreprotein could contribute to the immunosuppressive nature ofthe HCV.

HCV Core protein was suggested to have an immunosup-pressive effect through the downregulation of Cox-2 geneexpression (Jhaveri et al., 2005), which might be a result ofNFKB inactivation (Joo et al., 2005). Most recently, HCV Corewas shown to inhibit T cell function and chemokine productionthrough the interaction with complement receptor gC1qR (Yaoet al., 2005). The HCV Core protein expression also led to downmodulation of a number of immunity and inflammatoryresponse related genes in Huh-7 cell line (Fukutomi et al.,2005). The DNA microarray analysis in the chimpanzee modelof HCV infection showed that the control and clearance of HCVinfection was related to the intrahepatic infiltration of immunecells where as chronic infections correlated with impaired geneexpression for T cell recruitment (Su et al., 2002; Thimme et al.,2002). Expression of immune response related genes wasdownmodulated in liver during chronic HCV infection inhumans and chimpanzees (Bigger et al., 2004; Honda et al.,2001; Shao et al., 2005). The mechanisms could include theblock in the maturation process of T cells (Bigger et al., 2004)and an enhanced expression of anti-inflammatory genes (Hondaet al., 2001). HCV has also been shown to infect and replicate inlymph nodes, PBMC, monocyte/macrophages (Caussin-Schwemling et al., 2001; Laskus et al., 1998), which couldalso affect immune functions.

In summary, we investigated the effect of HCV Core onhepatocyte cell proliferation and gene expression. Our resultsshowed that the Core protein expression led to the suppressionof chemotaxis and inflammatory factors and enhanced theexpression of anti-apoptosis factors that could lead to theprotection of infected hepatocytes from immune clearance andapoptosis. Thus, HCV Core protein may play an important rolein the survival and persistence of Hepatitis C virus in infectedhepatocytes.

Materials and methods

Cell culture

HepG2 cells were obtained from American Type CultureCollection (Manassas, VA.). Cells were maintained in DMEMgrowth media supplemented with 10% fetal bovine serum,2 mM L-glutamine, and 100 U of penicillin-streptomycin/ml(GIBCO-BRL, Rockville, Md.).

65H. Nguyen et al. / Virology 354 (2006) 58–68

Transfection and establishment of a stable HepG2 cell line withinducible Core expression

The establishment of a HepG2 cell line with the ecdysoneinducible expression of HCV Core (HCV-1b) expression waspreviously described (Nguyen et al., 2003). Briefly, theecdysone inducible system consists of an inducible pINDexpression plasmid and an inducer pVgRxR. The inducibleplasmid pIND-Core was generated by cloning the sequence ofthe FLAG-tagged Core into ecdysone-inducible mammalianexpression vector pIND (Invitrogen, Carlsbad, CA) containinga neomycin (G418) selection marker. The expression of Core isunder the control of Drosophila melanogaster minimal heatshock promoter and the ecdysone and glucocorticoid responseelement (E/GRE) hybrid promoter. The E/GRE promoter isdriven the RxR and VgEcR ecdysone receptor subunits that areencoded and constitutively expressed by the pVgRxR plasmid(Invitrogen). The pVgRxR plasmid also contains a Zeocinselection marker for the generation of stable Zeocin-resistantcell clones. The binding of the RxR and VgEcR subunits to theE/GRE promoter is facilitated by the addition of ponasterone A(PonA).

HepG2 cells were co-transfected with linearized pIND-3XFLAG-Core and pVgRxR plasmids by electroporation of5 × 106 cells in 0.5 ml of growth media without serum at 250 Vand 950 μF using a Gene Pulser (Bio-Rad, Hercules, CA) andincubated overnight. The transfected cells were expanded inselection media containing 1000 μg/ml of G418 and 200 μg/mlof Zeocin for 2 weeks. The cell colonies were transferred into24-well plates and maintained in the G418/Zeocin selectionmedia for 2 to 3 weeks. Cell clones that survived the secondround of selection were expanded and evaluated for HCV Coreexpression by Western blots.

Hepatocyte growth conditions and collection

HepG2-Core cells and nontransfected (NT) control cell linewere grown in tissue culture plates for mRNA and proteinextraction. PonA was used to induce expression of HCV Coreprotein in the cells (10 μM). PonAwas also added to NT cells toserve as parallel controls. Four cell growth conditions were asfollowing: (i) HepG2-Core cells with induction using PonA, (ii)HepG2-Core cells without induction, (iii) untransfected controlHepG2 cells with PonA addition, (iv) untransfected controlHepG2 cells without PonA addition. Assay was performed intriplicates. Cells were collected and were immediately frozen.Total RNAwas isolated from HepG2-Core cells with inductionof Core expression (Core I), uninduced HepG2-Core cells (CoreU), induced NT control HepG2 cells (NT I), uninduced NTcontrol HepG2 (NT U) and used in the DNA microarrayanalysis and Real time RT-PCR (TaqMan) assay.

Cell viability and enumeration with Trypan blue

HepG2-Core (Core) and HepG2 control (NT) cells weretrypsinized to single cell suspensions and were counted toenumerate cell density. Equal number of cells (5 × 105) were

seeded in each well of the 6-well plates. Cells were grownovernight and subjected to PonA administration. Cells wereharvested 48 h following induction and suspended in 1 ml ofmedia after trypsinization with 0.5 ml of trypsin. Each cellsuspension was mixed with equal volume of Trypan Blue dye(25 μl/25 μl) to detect viable cells. The Trypan Bluesuspensions were spotted on a hemacytometer and live cellnumbers (without Trypan blue uptake) were counted per eachsample using a light microscope. For the measurement of cellgrowth for each set of experiments performed in triplicates, cellnumbers of Core uninduced and NT uninduced cell sampleswere considered to be 100% and were used as controls. Thenumbers of cells from each Core I and NT I cell samples werecompared with the controls and growth percentage values forCore I and NT I cells were derived.

DNA microarray analysis

The human HG-U133A GeneChips were used for the geneexpression profiling (Affymetrix, Santa Clara, CA, USA). RNAextraction, amplification, labeling, hybridization, staining, andscanning were performed as per recommendations (AffymetrixTechnical Manual). Fluorescence intensity values (.CEL files)generated from hybridized, stained GeneChips were subjectedto comparative and hierarchical clustering analyses using thedChip (Version 1.3) software program (Li and Wong, HarvardUniversity). The detection P value of 0.05 or better was used asa statistical criterion for including a given gene in subsequentcomparative or clustering analyses. A P value of 0.05 or lesswas used as a cutoff for identification of significant geneclusters. The initial hierarchical clustering was generated fromthe list of gene transcripts that were either upregulated ordownregulated at least 1.5-fold from comparisons betweenexperimental and control samples. We used D-chip's GeneOntology (GO) classification feature to group genes intofunctional categories. We also re-examined the data to includegenes with 1.3-fold change (P value < 0.05) that could provideadditional pertinent information.

Immunoblot analysis

HepG2 cells were lysed in NP-40 lysis buffer and 30 μg oftotal protein was separated on SDS-PAGE gels and transferredto BA-85 membrane (Schleicher and Schuell, Keene, NH). Themembrane was stained with Ponceau S to detect protein. It wasreacted with 5% nonfat dry milk in phosphate buffered saline(PBS) and 0.1% Tween for 1 hour before the addition of specificprimary antibodies to eliminate nonspecific binding. FLAG-tagged HCV Core, PCNA, GAPDH, IKBα, and PAK2 proteinswere detected using anti FLAG antibody (Stratagene. La Jolla,CA), anti-PCNA and anti-GAPDH antibodies (Santa CruzBiotech. Santa Cruz, CA), anti- IKBα and anti-PAK2 antibodies(Cell Signaling Technology. Beverly, MA). After washing andincubation with secondary mouse monoclonal or rabbitpolyclonal horseradish-peroxidase linked antibodies (Amer-sham Pharmacia, Piscataway, NJ), proteins were detected usingECL (Amersham Pharmacia). The same membrane blot was

66 H. Nguyen et al. / Virology 354 (2006) 58–68

used to probe for each protein, the membrane was sectioned intoparticular molecular weight ranges and probed (or re-probed)with different antibodies to ensure consistency.

Real-time PCR

Total RNA was used to synthesize cDNA by reversetranscription (Alluwaimi et al., 2003). Complementary DNAwas synthesized using random hexamer primers, and reactionswere carried out using PCR master mix (Applied Biosystems,Foster City, CA). The primer probe sets for chemokine (C-Cmotif) ligand 20 (CCL20), insulin-like growth factor 2 (IGF-2),Kirsten-ras2 (Kras2), proliferating cell nuclear antigen (PCNA),glyseraldehyde-3-phosphate dehydrogenase (GAPDH) and 18Sribosome were used for quantitation by real-time RT-PCR assay(Beckton Dickinson). The house keeping genes GAPDH and18S ribosome were used as internal controls for normalization.Each PCR reaction was set up according to manufacturerspecificity (TaqMan Universal PCR Mastermix; AppliedBiosystems). The reactions were placed in 96-well plates andamplified in an automated fluorometer (ABI PRISM 7700Sequence Detection System; Applied Biosystems). Amplifica-tion conditions included 2 min at 50 °C, 10 min at 95 °C, 40cycles of 15 s at 95 °C, and 60 s at 60 °C. For each gene, a cyclethreshold (Ct) value was obtained at the cycle that produced adetectable fluorescence signal. The relative Ct value of each ofthe candidate gene (in reference to the control gene, i.e., CCL20to GAPDH) was obtained by subtracting the Ct value of theGAPDH or 18S ribosome gene from the Ct value of eachcandidate gene. The average relative Ct value for each gene wascalculated from the triplicates of RNA samples. The fold changefor each gene was calculated from the difference between theaverage relative Ct values of the experimental and controlsamples.

Acknowledgments

We thank Michael George, Maria Mudryj, and DavidAsmuth for critical review of the manuscript. This work wassupported by NIH grant R01-DK61297.

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