Hepatitis C virus proteins interfere with the activation of chemokine gene promoters and downregulate chemokine gene expression

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Hepatitis C virus proteins interfere with theactivation of chemokine gene promoters anddownregulate chemokine gene expression

Maarit Sillanpaa, Pasi Kaukinen, Krister Melen and Ilkka Julkunen

Correspondence

Maarit Sillanpaa

[email protected]

Department of Viral Diseases and Immunology, National Public Health Institute, FIN-00300Helsinki, Finland

Received 13 July 2007

Accepted 16 October 2007

The hepatitis C virus (HCV) non-structural (NS) 3/4A protein complex inhibits the retinoic acid

inducible gene I (RIG-I) pathway by proteolytically cleaving mitochondria-associated

CARD-containing adaptor protein Cardif, and this leads to reduced production of beta interferon

(IFN-b). This study examined the expression of CCL5 (regulated upon activation, normal T-cell

expressed and secreted, or RANTES), CXCL8 (interleukin 8) and CXCL10 (IFN-c-activated

protein 10, or IP-10) chemokine genes in osteosarcoma cell lines that inducibly expressed

NS3/4A, NS4B, core-E1-E2-p7 and the entire HCV polyprotein. Sendai virus (SeV)-induced

production of IFN-b, CCL5, CXCL8 and CXCL10 was downregulated by the NS3/4A protein

complex and by the full-length HCV polyprotein. Expression of NS3/4A and the HCV polyprotein

reduced the binding of interferon regulatory factors (IRFs) 1 and 3 and, to a lesser extent, nuclear

factor (NF)-kB (p65/p50) to their respective binding elements on the CXCL10 promoter

during SeV infection. Furthermore, binding of IRF1 and IRF3 to the interferon-stimulated response

element-like element, and of c-Jun and phosphorylated c-Jun to the activator protein 1 element of

the CXCL8 promoter, was reduced when NS3/4A and the HCV polyprotein were expressed.

In cell lines expressing NS3/4A and the HCV polyprotein, the subcellular localization of

mitochondria was changed, and this was kinetically associated with the partial degradation of

endogenous Cardif. These results indicate that NS3/4A alone or as part of the HCV polyprotein

disturbs the expression of IRF1- and IRF3-regulated genes, as well as affecting mitogen-activated

protein kinase kinase- and NF-kB-regulated genes.

INTRODUCTION

Hepatitis C virus (HCV) belongs to the family Flaviviridae.Its single-stranded RNA genome is 9.6 kb and encodes alarge polyprotein that is cleaved into 11 structural andnon-structural (NS) proteins by cellular and viral-encodedproteases. The structural region of the HCV genomecontains the core, E1, E2 and p7 proteins and an alternativereading frame protein (Moradpour et al., 2002). The coreprotein forms the viral nucleocapsid, E1 and E2 are viralenvelope glycoproteins, and p7 functions as a cationchannel (Griffin et al., 2003). The HCV genome alsoencodes six NS proteins. NS2 is a cysteine protease andNS3 a serine protease, and they are involved in processingof the HCV polyprotein. NS3 also functions as an RNAhelicase. NS4A is an essential co-factor for NS3 proteaseactivity. NS4B is a membrane-associated protein with noassigned functions, NS5A is a phosphoprotein and NS5B isan RNA-dependent RNA polymerase (Lindenbach & Rice,2005; Moradpour et al., 2002).

In the early stages of virus infection, the production of theantiviral cytokines alpha and beta interferon (IFN-a/b) and

several proinflammatory chemokines such as CCL5 [regu-lated upon activation, normal T-cell expressed and secreted(RANTES)], CXCL8 [interleukin (IL) 8], and CXCL10 [IFN-c-activated protein 10 (IP-10)] are activated. These chemo-kines are chemoattractive for T cells, neutrophils, and naturalkiller and Th1-type T cells, respectively. Until now, it hasbeen reported that several HCV proteins may interfere withhost cell signalling pathways. The core protein inhibits IFNand tumour necrosis factor-a signalling, whilst E2 and NS5Ainterfere with the activation of protein kinase R (PKR) andthe antiviral response (reviewed by Gale & Foy, 2005). Recentstudies have also shown that the NS3/4A protein complexcan block type I IFN production (Cheng et al., 2006; Foyet al., 2003; Li et al., 2005b).

Host-cell IFN gene expression is triggered by dsRNAproduced during RNA virus infection. dsRNA is recog-nized by Toll-like receptor 3 (TLR3) (Alexopoulou et al.,2001), retinoic acid inducible gene I (RIG-I) andmelanoma differentiation-associated gene 5 (MDAs)(Yoneyama et al., 2004). These receptors interact andactivate their downstream adaptor molecules, Toll/IL-1receptor domain-containing adaptor inducing IFN-b

Journal of General Virology (2008), 89, 432–443 DOI 10.1099/vir.0.83316-0

432 0008-3316 G 2008 SGM Printed in Great Britain


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(TRIF) (Yamamoto et al., 2002) and Cardif (also calledMAVS/IPS-1/VISA), respectively (Kawai et al., 2005; Meylanet al., 2005; Seth et al., 2005; Xu et al., 2005). Recent reportshave shown that RIG-I-mediated activation is furtherregulated by tripartite motif protein family member 25,which ubiquitinates RIG-I (Gack et al., 2007), and NEMO,which is part of the IkB kinase (IKK-a/b/c) complex (Zhaoet al., 2007). Eventually, the IKK-related kinases IKKe andTank-binding kinase 1 (TBK1) are activated and they in turnactivate nuclear factor (NF)-kB and IFN regulatory factor 3(IRF3) transcription factors, which translocate into thenucleus and initiate the transcription of type I IFN genes(Wathelet et al., 1998). The NS3/4A protein complex maytarget both TRIF (Ferreon et al., 2005; Li et al., 2005a) andCardif (Kaukinen et al., 2006; Li et al., 2005b; Lin et al.,2006a; Loo et al., 2006; Meylan et al., 2005), causing theirproteolytic degradation. In addition, there is evidence thatNS3 can interact directly with TBK1 and IKKe (Otsuka et al.,2005). Interference of the TLR3 and RIG-I pathways by NS3/4A leads to inhibition of IRF3 phosphorylation and reducedIFN-b gene expression (Cheng et al., 2006; Foy et al., 2003;Meylan et al., 2005).

In the present study, we analysed the effect of HCV proteinexpression on host-cell chemokine gene expression in anosteosarcoma model cell system expressing HCV genes ina tetracycline-regulated fashion (Hugle et al., 2001;Moradpour et al., 1998; Wolk et al., 2000). We observedthat, in the cell lines expressing NS3/4A or the full-lengthHCV polyprotein, Sendai virus (SeV)-induced chemokinegene expression was clearly reduced. Reduced binding ofIRF1, IRF3 and NF-kB transcription factors to CXCL10 genepromoter regulatory elements was observed. Inhibition ofhost-cell chemokine gene expression in NS3/4A- and HCVpolyprotein-expressing cells correlated with partial cleavageof the mitochondrion-associated Cardif adaptor. Moreimportantly, the subcellular localization of mitochondriawas dramatically altered by NS3/4A expression and this maylead to impaired RIG-I-mediated signalling.

METHODS

Cell culture and virus infection. The U-2 OS human osteosarcoma-

derived cell lines (kindly provided by Dr Moradpour, University of

Lausanne, Switzerland) UNS3-4A-24 expressing the NS3/4A complex,

UNS4Bcon-4 expressing NS4B and UHCV-11 expressing the entire

HCV polyprotein have been described previously (Hugle et al., 2001;

Moradpour et al., 1998; Wolk et al., 2000). The UCp7con-9 cell line

expressing the core, E1, E2 and p7 proteins will be described in detail

elsewhere. These cell lines were maintained in Dulbecco’s modified

Eagle’s medium supplemented with 10 % fetal calf serum (Integro),

50 U penicillin G ml21, 50 mg streptomycin ml21, 500 mg G418 ml21,

1 mg puromycin ml21 and 1 mg tetracycline ml21 (Sigma). Cell lines

were grown for 24 h in tetracycline-free medium to reach steady-state

expression of HCV proteins before they were infected with SeV (strain

Cantell; National Public Health Institute, Helsinki, Finland) at an m.o.i.

of 5.

Cytokine determination. Cytokine levels in cell culture supernatants

were determined by ELISA as described previously (Miettinen et al.,

1998). The amounts of CXCL8 and CXCL10 were determined withantibody pairs and standards purchased from BD Pharmingen. CCL5-specific antibody pairs and standard were obtained from R&DSystems.

Real-time PCR analysis. Total cellular RNA was isolated fromguanidium isothiocyanate-lysed cells (Chirgwin et al., 1979) bycentrifugation through a caesium chloride cushion (Glisin et al.,1974). Samples containing equal amounts (2 mg) of total cellular RNAwere treated with RNase-free DNase I according to the manufacturer’sinstructions (Roche Diagnostics). cDNA synthesis was performed inTaqMan RT buffer with 5.5 mM MgCl2, 500 mM dNTPs, 2.5 mMrandom hexamers, 0.4 U RNase inhibitor ml21 and 1.25 U MultiScribereverse transcriptase ml21 (Applied Biosystems). The cDNA sampleswere then amplified in Master Mix buffer (Applied Biosystems) withassay-on-demand oligonucleotides (Applied Biosystems) to analysemRNA levels for IFN-b (Hs00277188_s1), CCL5 (Hs00174575_m1),CXCL8 (Hs00174103_m1) and CXCL10 (Hs00171042_m1). EachcDNA sample was amplified in triplicate for a studied cytokine andalso with 18S TaqMan rRNA Control Reagents (Applied Biosystems)from diluted (1 : 1000) cDNA sample with an ABI PRISM 7500Sequence Detector. The amounts of cytokine mRNA and 18S rRNAwere calculated from a standard curve and the relative mRNA levelswere normalized against 18S rRNA.

Statistical analysis. The significance of differences in results wasanalysed using Student’s t-test and values of P,0.05 and P,0.01 wereconsidered to be significant and highly significant, respectively.

Oligonucleotide DNA precipitation and Western blot analysis.Samples from osteosarcoma cell lines were collected at different timesafter stimulation and nuclear extracts were prepared as describedpreviously (Osterlund et al., 2005; Siren et al., 2005). Nuclei werelysed and protein concentrations of nuclear extracts were determinedusing the Bradford method (Bradford, 1976). Equal amounts ofprotein (150–200 mg, depending on experiment) from these extractswere incubated with streptavidin–agarose beads (Pierce) coupled to59-biotinylated oligonucleotides containing sequences for theCXCL10 interferon-stimulated response element (ISRE) (59-GGATCCTGTTTTGGAAAGTGAAACCTAATTCAC), CXCL10 NF-kB (59-GGATCCGCAGAGGGAAATTCCGTAACTTGG), CXCL8AP-1 (59-GGATCCGTGATGACTCAGGTT), CXCL8 NF-kB (59-GGATCCAAATCGTGGAATTTCCTCTGACATAAT) and CXCL8ISRE (59-GGATCCGATAAGGAACAAATAGGAAGTGTGAT) (DNATechnology A/S). Oligonucleotide-bound proteins were released inSDS-PAGE sample buffer by boiling. The proteins were separated bySDS-PAGE and transferred to Immobilon-P membranes (Millipore).To confirm that equal amounts of protein were used as the startingmaterial in these oligonucleotide DNA precipitation assays, 10 mgnuclear protein and 12 mg whole-cell proteins were separated by SDS-PAGE and stained for nucleolin, an abundant nuclear protein.Antibodies against p50, p65, c-Jun, C23/nucleolin and actin werepurchased from Santa-Cruz Biotechnology. Phospho-c-Jun antibodywas obtained from Cell Signalling. Antibodies against HCV coreprotein (Melen et al., 2004), RIG-I (Matikainen et al., 2006), Cardif(Kaukinen et al., 2006), IKKe and IRF3 (Veckman et al., 2006) andIRF1 (Matikainen et al., 1996) have been described previously.Horseradish peroxidase-conjugated goat anti-rabbit, goat anti-guineapig and goat anti-mouse (DakoCytomation) IgG were used forsecondary staining of antibodies. Binding of antibodies was visualizedon Amersham HyperMax films using an enhanced chemilumin-escence system (Amersham Biosciences). The intensity of bands wasdetermined using Kodak Digital Science 1D (version 3.0.2) software.

Indirect immunofluorescence and laser-scanning confocal

microscopy. The osteosarcoma cell lines UNS3-4A-24, UNS4Bcon-4, UCp7con-9 and UHCV-11 were grown on glass coverslips in the

HCV interferes with chemokine gene expression

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presence or absence of tetracycline. To stain mitochondria with

MitoTracker Red 580 (Molecular Probes), cells were incubated for

30 min in growth medium supplemented with 500 nM MitoTracker.

Cells were washed with growth medium and then fixed for 15 min

with 3 % paraformaldehyde diluted in growth medium. Cells were

washed with PBS and permeabilized with ice-cold methanol for 5 min

at 220 uC. Localization of Cardif was studied by staining the cells

with guinea pig anti-Cardif and FITC-labelled goat anti-guinea pig

IgG F(ab9)2 fragment (Chemicon).

Possible co-localization of Cardif with different HCV proteins was

studied by double staining using guinea pig or rabbit anti-Cardif, and

mouse anti-NS3 (US Biological) or rabbit anti-core or human patient

serum from an HCV-positive individual to recognize NS4B.

Secondary antibodies were FITC-labelled goat anti-mouse (Jackson

ImmunoResearch Laboratories), FITC-labelled goat anti-rabbit

F(ab9)2 fragment (Jackson ImmunoResearch Laboratories) or FITC-

labelled anti-human IgG (H+L) (Vector Laboratories) and

Rhodamine Red-X-labelled goat anti-guinea pig or Rhodamine

Red-X-labelled goat anti-rabbit (Jackson ImmunoResearch

Laboratories). Cells were visualized under a Leica TCS NT confocal

microscope.

RESULTS

Expression of HCV proteins interferes with SeV-induced chemokine production

It has previously been shown that transient expression ofNS3/4A interferes with the RIG-I pathway leading toreduced expression of the IFN-b gene (Li et al., 2005b;Meylan et al., 2005). To study further the mechanisms ofHCV-mediated inhibition of innate immune responses, weused cell lines expressing different HCV proteins in atetracycline-regulated manner (Hugle et al., 2001;Moradpour et al., 1998; Wolk et al., 2000). First, weanalysed SeV-induced chemokine production in relation toHCV protein expression, which was detected whentetracycline levels were decreased to less than 10 ng ml21

(Fig. 1b). There was clear inhibition of SeV-induced CCL5,CXCL8 and CXCL10 production in UNS3-4A-24 andUHCV-11 cell lines expressing NS3/4A protein and thefull-length HCV polyprotein, and the differences werehighly significant (P,0.01) when analysed using Student’st-test. Expression of NS4B had no effect on chemokineproduction, whereas expression of the HCV structuralregion, core-E1-E2-p7, led to increased production ofCXCL8 and slightly reduced production of CCL5 andCXCL10 (Fig. 1a) at a lower significance level of P,0.05.

To study whether the altered chemokine production inHCV protein-expressing cells was due to altered transcrip-tion, we analysed the kinetics of SeV-induced CCL5,CXCL8 and CXCL10 mRNA expression. The relative levelof IFN-b mRNA was included as an internal control andwas clearly decreased at the 8 h time point in UNS3-4A-24cells and at 24 h in UHCV-11 cells (Fig. 2). In both of thesecell lines, there was also a consistent reduction in therelative expression of CCL5, CXCL8 and CXCL10 mRNAs(Fig. 2). These differences were highly significant (P,0.01)except for that of CXCL8 mRNA in UHCV-11 cells. In

NS4B protein-expressing cells, there was an increase inrelative IFN-b and CXCL8 mRNA levels at the 24 h timepoint (Fig. 2). The expression of HCV structural proteinsincreased the relative mRNA levels of IFN-b and CXCL8 at24 h but decreased that of CCL5 mRNA (Fig. 2). Overall,the alterations in virus-induced chemokine mRNA andprotein expression patterns (see Fig. 1) in HCV protein-expressing cells were consistent with each other. As theELISA analysis was based on three independent samples,the variation was larger than in RT-PCR analysis, which isbased on one sample run in triplicate.

Binding of SeV-activated transcription factors topromoter elements of the CXCL8 and CXCL10genes in HCV protein-expressing cells

HCV NS3/4A and the full-length polyprotein decreasedSeV-induced expression of CXCL8 and CXCL10 mRNAs(Fig. 2). To study this inhibition further, we carried outDNA affinity binding experiments, which showed tran-scription factor binding to their specific response elements(REs) on the CXCL8 and CXCL10 promoters. The CXCL10promoter contains ISRE and NF-kB REs (Ohmori &Hamilton, 1993), which are able to bind virus-activatedtranscription factors IRF1, IRF3 and NF-kB (Genin et al.,2000; Veckman et al., 2006). The binding of activated IRF1and IRF3 to the ISRE of CXCL10 (Fig. 3a) and of the p65/p50 heterodimer to the NF-kB RE of CXCL10 (Fig. 3b) wasclearly decreased in the UNS3-4A-24 cell line by 6 h afterSeV infection, whereas reduced binding was detected laterat the 18 h time point in the UHCV-11 cell line. Noinhibition of SeV-induced IRF1, IRF3 or p65/p50 bindingto CXCL10 promoter elements was seen in NS4B or core-E1-E2-p7 protein-expressing cell lines.

The CXCL8 promoter contains NF-kB, AP-1 and C/EBPREs (Mukaida et al., 1994) as well as an ISRE/HNF-3element (Casola et al., 2000). The NF-kB RE is indispens-able for activation of the CXCL8 gene, whereas AP-1 andC/EBP REs have a less important role in CXCL8 geneexpression, depending on the cell type (Hoffmann et al.,2002). The ISRE-like element participates in activation ofthe CXCL8 promoter during respiratory syncytial virusinfection (Casola et al., 2000) or when dsRNA signallingpathways are activated (Wagoner et al., 2007). In NS3/4Aprotein-expressing cells, there was less c-Jun and phos-phorylated c-Jun bound to the CXCL8 promoter (Fig. 4b)and binding of IRF1 and IRF3 to the ISRE of CXCL8 wasalso reduced (Fig. 4c). There was only a modest reductionin binding of the p65/p50 heterodimer to the NF-kB RE ofCXCL8 in UNS3-4A-24 cells (Fig. 4a). In the UHCV-11 cellline, binding of p65/p50 was slightly reduced, whereasbinding of the IRF1, IRF3 and phosphorylated c-Juntranscription factors to their respective elements on theCXCL8 promoter was reduced more strongly (Fig. 4). Inthe cell line expressing NS4B protein, there were nochanges in the binding of transcription factors to their REson the CXCL8 promoter (Fig. 4). In the UCp7con-9 cell

M. Sillanpaa and others

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line, binding of IRF1 and IRF3 to the ISRE of CXCL8 wasreduced at 18 h after infection (Fig. 4c).

Expression of components of the RIG-I signallingpathway

Based on transfection experiments, it has been shown thatthe HCV NS3/4A serine protease complex directly targetsCardif (MAVS/IPS-1/VISA), causing its degradation anddissociation from the mitochondrial membrane (Kaukinenet al., 2006; Li et al., 2005b; Meylan et al., 2005). Recently,degradation of endogenous Cardif was reported in HCVreplicon (HCV 1b subgenomic replicon) cell lines Huh7-K2040 (Li et al., 2005b), Huh7-HP, Huh7-JFHR (JFH-1HCV 2a subgenomic replicon RNA) (Loo et al., 2006) andHuh8 (Lin et al., 2006a). To confirm that Cardif is a

proteolytic target for NS3/4A in our experimental system,we analysed Cardif protein expression in UHCV cell lines(Fig. 5). Increased HCV protein expression starting at 24 hafter HCV protein induction correlated with a decrease inthe full-length form and the appearance of a C-terminallytruncated form of Cardif in the UNS3-4A-24 and UHCV-11 cell lines. No degradation of Cardif was seen in cell linesexpressing the NS4B or the structural region proteins.

Intracellular localization of Cardif and HCVproteins

Cardif localizes to mitochondria (Li et al., 2005b; Lin et al.,2006a; Seth et al., 2005). In order to verify this observation,we carried out co-localization experiments withMitoTracker and anti-Cardif antibody staining in different

Fig. 1. SeV-induced chemokine production in U-2 OS human osteosarcoma-derived cell lines expressing different HCVproteins. (a) UNS3-4A-24 cells expressing the NS3/4A complex, UNS4Bcon-4 cells expressing NS4B, UCp7con-9 cellsexpressing core-E1-E2-p7 and UHCV-11 cells expressing the entire HCV polyprotein were induced to express HCV proteinsby incubating them in decreasing concentrations of tetracycline (from 1000 to 0 ng ml”1) for 24 h. Thereafter, the cells wereinfected with SeV (m.o.i. of 5) for 24 h or left uninfected as controls, and the cell culture supernatants were collected andCCL5, CXCL8 and CXCL10 chemokine levels were determined by ELISA. Chemokine levels represent the means±SD oftriplicate culture specimens. Different scales are used in the CCL5 and CXCL8 panels for results from the UHCV-11 cell line.The significance of differences between cells grown with tetracycline at 1000 ng ml”1 and at 100, 10, 3, 1 or 0 ng ml”1 wereanalysed using Student’s t-test, and are indicated as significant (*, P,0.05) or highly significant (**, P,0.01). (b) Theexpression of HCV proteins was determined by Western blotting (10 mg cellular protein per lane) by staining with patient serarecognizing different HCV proteins. Equal loading of samples was verified by staining with anti-actin antibodies. The experimentwas performed twice with similar results.




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HCV protein-expressing cell lines. Cardif protein stronglyco-localized with Mitotracker, indicating that the intrinsicCardif protein is primarily located in the mitochondria(Fig. 6). Surprisingly, the subcellular localization of themitochondria changed when the NS3/4A protein wasexpressed for 2 days in the UNS3-4A-24 cell line, and themitochondria accumulated as clusters near the nucleus.There was a similar change in the distribution ofmitochondria, although to a lesser extent compared withUNS3-4A-24 cells, in the full-length HCV polyprotein-expressing cell line (UHCV-11). In the UNS4Bcon-4 andUCp7con-9 cell lines, HCV protein expression did notaffect the subcellular localization of mitochondria (Fig. 6).

Next, we studied whether Cardif could co-localize withdifferent HCV proteins in these cell lines (Fig. 7). Generally,the NS3/4A protein was tightly co-localized with Cardif inthe endoplasmic reticulum (ER)/mitochondria-like struc-tures, although some NS3/4A protein was also seen in theperinuclear space, apparently associated with the nuclear

membrane (at 24 h after induction of NS3/4A proteinexpression). It has been shown previously that the NS3/4Aprotein complex in the UNS3-4A-24 cell line is located inboth the ER and the mitochondria (Wolk et al., 2000). Twodays after the induction of expression of NS3/4A protein, aclear change in the distribution of mitochondria was seen inUNS3-4A-24 cells. Cardif and NS3 proteins also fully co-localized (Fig. 7). In the UNS4Bcon-4 cell line, NS4B proteinwas expressed evenly throughout the cytoplasm, as reportedpreviously (Melen et al., 2004), and no co-localization withCardif was detected (Fig. 7). The core protein showed apartial co-localization with Cardif in mitochondria in theUCp7con-9 cell line. Previous studies have shown that thecore protein is located in lipid storage vesicles (Barba et al.,1997) and also in the outer membrane of mitochondria(Schwer et al., 2004). In the UHCV-11 cell line, the stainingfor NS3 was weaker compared with that seen in UNS3-4A-24 cells. However, in UHCV-11 cells, the distribution ofmitochondria also changed and co-localization of NS3 withCardif was clearly seen (Fig. 7).

Fig. 2. Relative mRNA levels of IFN-b, CCL5, CXCL8 and CXCL10 after SeV infection in UNS3-4A-24, UNS4Bcon-4,UCp7con-9 and UHCV-11 cell lines. Cells were grown in the presence (1 mg ml”1) or absence of tetracycline for 24 h,followed by infection with SeV (m.o.i. of 5); control cells were not infected. Cells were collected at 4, 8 and 24 h after infection,total cellular RNA was isolated and quantitative PCR analysis was carried out. Results are based on one cDNA sample run intriplicate using a Taqman assay-on-demand system where chemokine mRNA level was normalized against 18S rRNA. Relativeexpression levels of IFN-b and chemokine mRNAs are shown in the presence of HCV proteins (open columns) and in theabsence of HCV proteins (filled columns). The significance of differences between HCV protein-expressing and non-expressingcells was studied using Student’s t-test and highly significant differences are indicated (*, P,0.01).




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DISCUSSION

Several different HCV proteins are able to interfere with thehost-cell antiviral response by inhibiting IFN production andsignalling, as well as by inhibiting the functions of IFN-induced antiviral proteins (Gale & Foy, 2005). The coreprotein interferes with IFN signalling and the antiviral

response (Gale & Foy, 2005; Melen et al., 2004), for exampleby binding to the SH2 domain of signal transducers andactivators of transcription (STAT) 1 (Lin et al., 2006b).Furthermore, the core protein can downregulate thetranscription of IRF1 (Ciccaglione et al., 2007). In addition,the E1 and NS5A proteins have been shown to block theactivation of PKR, leading to a reduced IFN-induced

Fig. 3. HCV protein expression interferes with transcription factor binding to ISRE and NF-kB REs on the CXCL10 promoter.HCV protein-expressing cell lines were grown in the absence of tetracycline (”) to induce HCV protein expression or in mediumcontaining tetracycline (+) to maintain repression of HCV protein expression. At 24 h after the removal of tetracycline, the cellswere infected with SeV (m.o.i. of 5). Cells were collected at the indicated times after infection and equal amounts of nuclearprotein were precipitated with Sepharose-immobilized oligonucleotides containing sequences of the ISRE and NF-kB REs ofthe CXCL10 promoter. Bound proteins were analysed by Western blotting using anti-IRF1- and anti-IRF3-specific (a) or anti-p65- and anti-p50-specific antibodies (b). Control samples C1 and C2 contained whole-cell extracts. The intensities of thebands were quantified using Kodak Digital Science 1D software and are shown below each band as a percentage in relation tothe strongest band whose intensity was assigned a value of 100 %. The use of equal amounts of protein in the oligonucleotide-binding assays was confirmed by Western blotting; 10 mg samples of nuclear proteins and 12 mg samples of whole-cellproteins were stained with anti-nucleolin antibody (c). The experiment was performed in duplicate with similar results.




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antiviral response (Gale & Foy, 2005). Several recent studieshave shown that the NS3/4A complex targets the RIG-Ipathway and proteolytically cleaves the mitochondrion-associated Cardif/MAVS/IPS-1/VISA adaptor molecule,which leads to reduced expression of the IFN-b gene(Kaukinen et al., 2006; Lin et al., 2006a; Loo et al., 2006;Meylan et al., 2005; Seth et al., 2005). These studiesconcentrated mainly on revealing the molecular mechanismof action of the NS3/4A protein complex on IFN-b geneexpression and less attention was paid to analysing the effectsof HCV protein expression on the regulation of chemokinegene expression.

The production of cytokines during virus infection requiresa coordinated action between different transcription factor

families; for example, IRFs, NF-kB and AP-1 are requiredfor the transcription of IFN-b (Wathelet et al., 1998), IRFsand NF-kB for the transcription of CCL5 (Genin et al.,2000) and CXCL10 (Ohmori & Hamilton, 1993), and NF-kB, IRFs, AP-1 and C/EBP for the transcription of CXCL8(Casola et al., 2000; Mukaida et al., 1994). Inhibition ofIFN-b gene expression was apparently due to impairedphosphorylation of IRF3 (Foy et al., 2003) and impairedbinding of activated IRF3 and NF-kB to the respective IFN-b promoter elements (Foy et al., 2005). Our results supportthese observations, as NS3/4A and full-length HCVpolyprotein expression, but not that of core-E1-E2-p7 orNS4B protein, clearly reduced endogenous IFN-b mRNAexpression (Fig. 2) in our stable cell line systems. Thisinhibitory effect was not restricted to the IFN-b gene, as

Fig. 4. Effect of HCV protein expression ontranscription factor binding to ISRE, NF-kBand AP-1 REs on the CXCL8 promoter. HCVprotein-expressing cell lines were grown in theabsence of tetracycline (”) to induce HCVprotein expression or in medium containingtetracycline (+) to prevent HCV proteinexpression. At 24 h after the removal oftetracycline, cells were infected with SeV(m.o.i. of 5). Cells were collected at theindicated times and equal amounts of nuclearprotein were precipitated with oligonucleotidescontaining sequences of the NF-kB, AP-1 andISRE elements of the CXCL8 promoter. Boundproteins were analysed by Western blottingusing anti-p65- and anti-p50-specific (a), anti-phospho c-Jun- and anti-c-Jun-specific (b) oranti-IRF1- and anti-IRF3-specific (c) antibod-ies. Control samples C1 and C2 containedwhole-cell extracts. The intensities of bandswere quantified using Kodak Digital Science1D software and are shown below each blot asa percentage in relation to the strongest band,which was assigned a value of 100 %. The useof equal amounts of protein in the oligonucleo-tide-binding assays was confirmed by Westernblotting; 10 mg samples of nuclear proteinsand 12 mg samples of whole-cell proteinswere stained with anti-nucleolin antibody (d).The experiment was performed in duplicatewith similar results.




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expression of SeV-induced CCL5, CXCL8 and CXCL10protein (Fig. 1) and mRNA (Fig. 2) was reduced when NS3/4A or the HCV polyprotein was expressed. At least in thecase of the CXCL10 gene, this inhibition is likely to occur atthe transcriptional level, as oligonucleotide-binding experi-ments revealed that NS3/4A expression decreased IRF1,IRF3 and p65/p50 heterodimer binding to their respectiveelements on the CXCL10 promoter (Fig. 3a, b). Thus, NS3/4A-mediated inhibition of the CXCL10 gene expressionoccurs by a mechanism similar to that of the IFN-b gene. Inthe UHCV-11 cell line, the inhibition of IRF1 and IRF3binding to the ISRE and p65/p50 binding to the NF-kBelement on the CXCL10 promoter was reduced at a laterstage compared with that seen in the UNS3A-4-24 cell line(Fig. 3a, b). This is consistent with the reduction seen inmRNA levels, which was also detected earlier in UNS3-4A-24 cells than in the UHCV-11 cell line (Fig. 2).

The regulation of CXCL8 production involves bothactivation of transcription and stabilization of mRNA.Activation of the CXCL8 gene occurs mainly via NF-kB,whilst AP-1 and C/EBP contribute to maximal CXCL8 geneexpression depending on the cell type (Hoffmann et al.,2002). In addition, an ISRE-like element of the CXCL8promoter participates in activation of the CXCL8 geneduring virus infection (Casola et al., 2000; Wagoner et al.,2007). Transcription of CXCL8 is induced rapidly by virusinfection (Hoffmann et al., 2002), as also seen in ourexperiment with SeV (Fig. 2). CXCL8 mRNA containsseveral AU-rich elements (Winzen et al., 1999, 2004),which control the stability of CXCL8 mRNA. Severalproteins are involved in the stabilization of CXCL8 mRNA:mitogen-activated protein kinase (MAP) kinase-activatedp38 and its substrate, MAP kinase-activated protein kinase2 (Hoffmann et al., 2002; Winzen et al., 1999), theconstitutively active form of RIG-I (Wagoner et al., 2007),and also HCV proteins in some HCV replicon cell lines(Green et al., 2006). Our experiments with UHCV cell linesshowed that the expression of NS3/4A and the HCVpolyprotein clearly inhibited the expression of CXCL8

mRNA and the production of CXCL8 protein (Figs 1 and2). These data are consistent with the observed decreasedbinding of phosphorylated c-Jun, IRF1 and IRF3 to theirREs on the CXCL8 promoter (Fig. 4). In several HCVreplicon cell lines, CXCL8 production is increased duringHCV replication (Koo et al., 2006). The promoter ofCXCL8 was activated by JFH-1 strain HCV RNA and virus(Wagoner et al., 2007). The difference from the currentstudy is that we used SeV to induce chemokine productionin UHCV cell lines that express HCV proteins, whereasWagoner et al. (2007) used HCV RNA to activate RIG-Isignalling. Although expression of the HCV structuralproteins, core-E1-E2-p7, increased the expression ofCXCL8 mRNA and protein (Figs 1 and 2) in ourexperiments, we could not detect enhanced transcriptionfactor binding to the regulatory elements on the CXCL8promoter (Fig. 4). It has been shown that the core proteincan activate NF-kB-regulated pathways leading toenhanced CXCL8 promoter activation (Kato et al., 2000),but this effect has been suggested to be dependent on theHCV genotype (Ray et al., 2002). Thus, there may be somepost-transcriptional regulatory steps involved in theproduction of CXCL8 in the UCp7con-9 cell line thatneed to be studied further.

Although the dsRNA-activated TLR3 and RIG-I pathwaysare not always operational in the same cells (Melchjorsen etal., 2005), they are apparently functional in UHCV celllines (Li et al., 2005a). In the present study, weconcentrated on the RIG-I pathway, as the crucial adaptorprotein of the RIG-I pathway, Cardif/MAVS/IPS-1/VISA,has been identified as the proteolytic target for the NS3/4Aprotein complex (Li et al., 2005b; Lin et al., 2006a; Loo etal., 2006; Meylan et al., 2005). Similarly, in the UNS3-4A-24 and the UHCV-11 cell lines, degradation of endogenousCardif was observed, starting 24 h after induction of HCVprotein expression (Fig. 5). It is noteworthy that weobserved only a partial degradation of Cardif in NS3/4A-and HCV polyprotein-expressing cells. Another studycarried out with the UNS3-4A-24 cell line showed almost

Fig. 5. Partial cleavage of endogenous Cardif in HCV protein-expressing cells. UHCV cell lines were grown in the absence oftetracycline to induce HCV protein expression and samples were collected at the indicated times. Cellular proteins (15 mg perlane) were separated by SDS-PAGE, and Western blot analyses were carried out with anti-Cardif or anti-actin antibodies orwith patient sera obtained from a person chronically infected with HCV. Cardif-DTM represents the truncated Cardif proteinform. The experiment was performed in triplicate with similar results.




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complete degradation of transfected Cardif, whilst thestability of intrinsic Cardif was not analysed (Meylan et al.,2005). Our results may indicate that Cardif becomesinaccessible for degradation due to a change in itssubcellular localization in mitochondria (see below). Itmay well be that NS3/4A-induced change in subcellularlocalization of mitochondria, even in the absence of fullCardif degradation, may be sufficient for effective inhibi-tion of SeV-induced cytokine gene expression.

An interesting observation was that, in immunofluores-cence analysis (Figs 6 and 7), the expression of NS3/4A hada dramatic effect on the distribution of mitochondria in theUNS3-4A-24 and UHCV-11 cell lines. Clustering ofmitochondria was found after 2 days of HCV protein

Fig. 6. Intracellular localization of Cardif in HCV protein-expres-sing cells. UNS3-4A-24, UNS4Bcon-4, UCp7con-9 and UHCV-11 cell lines were grown in the absence of tetracycline (” Tet) toinduce HCV protein expression. Mitochondria were stained withMitoTracker in living cells that had been grown for 0, 24 or 48 h intetracycline-free growth medium. After MitoTracker staining, cellswere fixed, permeabilized and stained with guinea pig anti-Cardifantibodies, followed by staining with FITC-labelled secondaryantibodies. Stained cells were analysed by confocal laser-scanning microscopy to visualise the distribution of Cardif (green)and mitochondria (red); co-localization (yellow) is shown in themerged signal.

Fig. 7. Subcellular localization of Cardif and HCV proteins inUHCV cells lines. HCV protein expression was induced byremoving tetracycline (”Tet) from the growth medium. At theindicated times, the cells were fixed and double-stained withguinea pig anti-Cardif and monoclonal anti-NS3 antibody (forUNS3-4A-24 and UHCV-11 cells), or with patient serum forNS4B (for UNS4Bcon-4 cells) or rabbit anti-core antibody(for UCp7con-9 cells), followed by staining with FITC- orRhodamine Red-X-labelled secondary antibodies. Stained cellswere analysed by confocal laser-scanning microscopy to visualisethe distribution of Cardif (red) and HCV proteins (green); co-localization (yellow) is shown in the merged signal.




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expression and it was seen more clearly in the UNS3-4A-24cell line where the expression of NS3/4A is higher than inthe UHCV-11 cell line. Transient expression of NS3/4A inHuh7 cells has also been shown to change the intracellulardistribution of mitochondria (Nomura-Takigawa et al.,2006). A strong co-localization of NS3/4A was observedwith MitoTracker and Cardif when NS3/4A was expressedalone (UNS3-4A-24 cells) or in the context of the full-length HCV polyprotein (UHCV-11 cells). We did not findany marked cytoplasmic, non-mitochondrion-localizedCardif. Other studies have shown diffuse staining of thecytoplasm after partial dissociation of Cardif from themitochondrial membrane to the cytosol when NS3/4A wastransiently expressed in HEK293 cells (Li et al., 2005b),when Huh7 cells were infected with HCV (Loo et al., 2006)or when Huh8 cells expressed an HCV replicon (Lin et al.,2006a). Our results may indicate that further degradationof intrinsic Cardif may be relatively fast and therefore wefailed to observe the accumulation of truncated Cardif inthe cytoplasm. In addition, our data may imply that achange in the distribution of mitochondria and strong co-localization of NS3/4A with Cardif may as such besufficient to interfere with RIG-I signalling, rendering thecells incapable of producing antiviral and other pro-inflammatory cytokines. Mitochondria are also activeproducers of reactive oxygen species (ROS). HCV infectioncan induce oxidative stress, and NS3, core and NS5Aproteins have been shown to be able to induce ROSproduction (Koike & Miyoshi, 2006). Furthermore, anincrease in mitochondrial ROS production and disturbedmitochondrial function have been found in HCV poly-protein-expressing cells (Piccoli et al., 2006).Mitochondrial membrane may thus turn out to be anessential site for virus-induced signalling (Seth et al., 2005)and this deserves further investigation.

In the present study, we demonstrated that, in HCV NS3/4A protein-expressing cells, SeV-induced cytokine andchemokine gene expression was clearly reduced and thatNS3/4A-mediated inhibition also took place in the contextof the whole HCV polyprotein. The binding of transcrip-tion factors to their response elements on CXCL8 andCXCL10 promoters was disturbed by the expression ofNS3/4A and HCV polyprotein. A more novel finding wasthat the expression of NS3/4A protein changed thesubcellular localization of mitochondria. It is likely thatthis change in distribution of mitochondria renders themincapable of proper signalling and emphasizes an import-ant role for mitochondria in cellular resistance to HCVinfection.

ACKNOWLEDGEMENTS

This study was supported by grants from the European Commission(grant QLK2-CT-2002-00954) and the Medical Research Council ofthe Academy of Finland. We thank Dr Darious Moradpour forproviding us with the UHCV cell lines. The expert technical assistanceof Hanna Valtonen, Johanna Lahtinen, Mari Aaltonen, SinikkaSopanen, Anna Mantynen and Raija Tyni is acknowledged.

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Hepatitis C virus proteins interfere with the activation of chemokine gene promoters and downregulate chemokine gene expression