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Department of Viral Diseases and Immunology, National Public Health Institute, FIN-00300 Helsinki, Finland
Correspondence
Maarit Sillanpää
maarit.sillanpaa{at}ktl.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-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-β, 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)-
B (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-B-regulated genes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The core protein forms the viral nucleocapsid, E1 and E2 are viral envelope glycoproteins, and p7 functions as a cation channel (Griffin et al., 2003). The HCV genome also encodes six NS proteins. NS2 is a cysteine protease and NS3 a serine protease, and they are involved in processing of the HCV polyprotein. NS3 also functions as an RNA helicase. NS4A is an essential co-factor for NS3 protease activity. NS4B is a membrane-associated protein with no assigned functions, NS5A is a phosphoprotein and NS5B is an RNA-dependent RNA polymerase (Lindenbach & Rice, 2005; Moradpour et al., 2002).
In the early stages of virus infection, the production of the antiviral cytokines alpha and beta interferon (IFN-
/β) and several proinflammatory chemokines such as CCL5 [regulated upon activation, normal T-cell expressed and secreted (RANTES)], CXCL8 [interleukin (IL) 8], and CXCL10 [IFN--activated protein 10 (IP-10)] are activated. These chemokines are chemoattractive for T cells, neutrophils, and natural killer and Th1-type T cells, respectively. Until now, it has been reported that several HCV proteins may interfere with host cell signalling pathways. The core protein inhibits IFN and tumour necrosis factor- signalling, whilst E2 and NS5A interfere with the activation of protein kinase R (PKR) and the antiviral response (reviewed by Gale & Foy, 2005). Recent studies have also shown that the NS3/4A protein complex can block type I IFN production (Cheng et al., 2006; Foy et al., 2003; Li et al., 2005b).
Host-cell IFN gene expression is triggered by dsRNA produced during RNA virus infection. dsRNA is recognized by Toll-like receptor 3 (TLR3) (Alexopoulou et al., 2001), retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDAs) (Yoneyama et al., 2004). These receptors interact and activate their downstream adaptor molecules, Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) (Yamamoto et al., 2002) and Cardif (also called MAVS/IPS-1/VISA), respectively (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). Recent reports have shown that RIG-I-mediated activation is further regulated by tripartite motif protein family member 25, which ubiquitinates RIG-I (Gack et al., 2007), and NEMO, which is part of the IB kinase (IKK-/β/) complex (Zhao et al., 2007). Eventually, the IKK-related kinases IKK
and Tank-binding kinase 1 (TBK1) are activated and they in turn activate nuclear factor (NF)-B and IFN regulatory factor 3 (IRF3) transcription factors, which translocate into the nucleus and initiate the transcription of type I IFN genes (Wathelet et al., 1998). The NS3/4A protein complex may target both TRIF (Ferreon et al., 2005; Li et al., 2005a) and Cardif (Kaukinen et al., 2006; Li et al., 2005b; Lin et al., 2006a; Loo et al., 2006; Meylan et al., 2005), causing their proteolytic degradation. In addition, there is evidence that NS3 can interact directly with TBK1 and IKK (Otsuka et al., 2005). Interference of the TLR3 and RIG-I pathways by NS3/4A leads to inhibition of IRF3 phosphorylation and reduced IFN-β gene expression (Cheng et al., 2006; Foy et al., 2003; Meylan et al., 2005).
In the present study, we analysed the effect of HCV protein expression on host-cell chemokine gene expression in an osteosarcoma model cell system expressing HCV genes in a tetracycline-regulated fashion (Hugle et al., 2001; Moradpour et al., 1998; Wolk et al., 2000). We observed that, in the cell lines expressing NS3/4A or the full-length HCV polyprotein, Sendai virus (SeV)-induced chemokine gene expression was clearly reduced. Reduced binding of IRF1, IRF3 and NF-B transcription factors to CXCL10 gene promoter regulatory elements was observed. Inhibition of host-cell chemokine gene expression in NS3/4A- and HCV polyprotein-expressing cells correlated with partial cleavage of the mitochondrion-associated Cardif adaptor. More importantly, the subcellular localization of mitochondria was dramatically altered by NS3/4A expression and this may lead to impaired RIG-I-mediated signalling.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; 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 ml–1, 50 µg streptomycin ml–1, 500 µg G418 ml–1, 1 µg puromycin ml–1 and 1 µg tetracycline ml–1 (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 with antibody pairs and standards purchased from BD Pharmingen. CCL5-specific antibody pairs and standard were obtained from R&D Systems.
Real-time PCR analysis.
Total cellular RNA was isolated from guanidium isothiocyanate-lysed cells (Chirgwin et al., 1979) by centrifugation through a caesium chloride cushion (Glisin et al., 1974). Samples containing equal amounts (2 µg) of total cellular RNA were treated with RNase-free DNase I according to the manufacturer's instructions (Roche Diagnostics). cDNA synthesis was performed in TaqMan RT buffer with 5.5 mM MgCl2, 500 µM dNTPs, 2.5 µM random hexamers, 0.4 U RNase inhibitor µl–1 and 1.25 U MultiScribe reverse transcriptase µl–1 (Applied Biosystems). The cDNA samples were then amplified in Master Mix buffer (Applied Biosystems) with assay-on-demand oligonucleotides (Applied Biosystems) to analyse mRNA levels for IFN-β (Hs00277188_s1), CCL5 (Hs00174575_m1), CXCL8 (Hs00174103_m1) and CXCL10 (Hs00171042_m1). Each cDNA sample was amplified in triplicate for a studied cytokine and also with 18S TaqMan rRNA Control Reagents (Applied Biosystems) from diluted (1 : 1000) cDNA sample with an ABI PRISM 7500 Sequence Detector. The amounts of cytokine mRNA and 18S rRNA were calculated from a standard curve and the relative mRNA levels were normalized against 18S rRNA.
Statistical analysis.
The significance of differences in results was analysed using Student's t-test and values of P<0.05 and P<0.01 were considered to be significant and highly significant, respectively.
Oligonucleotide DNA precipitation and Western blot analysis.
Samples from osteosarcoma cell lines were collected at different times after stimulation and nuclear extracts were prepared as described previously (Osterlund et al., 2005; Siren et al., 2005). Nuclei were lysed and protein concentrations of nuclear extracts were determined using the Bradford method (Bradford, 1976). Equal amounts of protein (150–200 µg, depending on experiment) from these extracts were incubated with streptavidin–agarose beads (Pierce) coupled to 5'-biotinylated oligonucleotides containing sequences for the CXCL10 interferon-stimulated response element (ISRE) (5'-GGATCCTGTTTTGGAAAGTGAAACCTAATTCAC), CXCL10 NF-B (5'-GGATCCGCAGAGGGAAATTCCGTAACTTGG), CXCL8 AP-1 (5'-GGATCCGTGATGACTCAGGTT), CXCL8 NF-B (5'-GGATCCAAATCGTGGAATTTCCTCTGACATAAT) and CXCL8 ISRE (5'-GGATCCGATAAGGAACAAATAGGAAGTGTGAT) (DNA Technology A/S). Oligonucleotide-bound proteins were released in SDS-PAGE sample buffer by boiling. The proteins were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore). To confirm that equal amounts of protein were used as the starting material in these oligonucleotide DNA precipitation assays, 10 µg nuclear protein and 12 µg 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 were purchased from Santa-Cruz Biotechnology. Phospho-c-Jun antibody was obtained from Cell Signalling. Antibodies against HCV core protein (Melen et al., 2004), RIG-I (Matikainen et al., 2006), Cardif (Kaukinen et al., 2006), IKK and IRF3 (Veckman et al., 2006) and IRF1 (Matikainen et al., 1996) have been described previously. Horseradish peroxidase-conjugated goat anti-rabbit, goat anti-guinea pig and goat anti-mouse (DakoCytomation) IgG were used for secondary staining of antibodies. Binding of antibodies was visualized on Amersham HyperMax films using an enhanced chemiluminescence system (Amersham Biosciences). The intensity of bands was determined 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 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 –20 °C. Localization of Cardif was studied by staining the cells with guinea pig anti-Cardif and FITC-labelled goat anti-guinea pig IgG F(ab')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(ab')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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Meylan et al., 2005). To study further the mechanisms of HCV-mediated inhibition of innate immune responses, we used cell lines expressing different HCV proteins in a tetracycline-regulated manner (Hugle et al., 2001; Moradpour et al., 1998; Wolk et al., 2000). First, we analysed SeV-induced chemokine production in relation to HCV protein expression, which was detected when tetracycline levels were decreased to less than 10 ng ml–1 (Fig. 1b). There was clear inhibition of SeV-induced CCL5, CXCL8 and CXCL10 production in UNS3-4A-24 and UHCV-11 cell lines expressing NS3/4A protein and the full-length HCV polyprotein, and the differences were highly significant (P<0.01) when analysed using Student's t-test. Expression of NS4B had no effect on chemokine production, whereas expression of the HCV structural region, core-E1-E2-p7, led to increased production of CXCL8 and slightly reduced production of CCL5 and CXCL10 (Fig. 1a) at a lower significance level of P<0.05.
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). In both of these cell lines, there was also a consistent reduction in the relative 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 in relative IFN-β and CXCL8 mRNA levels at the 24 h time point (Fig. 2). The expression of HCV structural proteins increased the relative mRNA levels of IFN-β and CXCL8 at 24 h but decreased that of CCL5 mRNA (Fig. 2). Overall, the alterations in virus-induced chemokine mRNA and protein expression patterns (see Fig. 1
) in HCV protein-expressing cells were consistent with each other. As the ELISA analysis was based on three independent samples, the variation was larger than in RT-PCR analysis, which is based on one sample run in triplicate.
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). To study this inhibition further, we carried out DNA affinity binding experiments, which showed transcription factor binding to their specific response elements (REs) on the CXCL8 and CXCL10 promoters. The CXCL10 promoter contains ISRE and NF-B REs (Ohmori & Hamilton, 1993), which are able to bind virus-activated transcription factors IRF1, IRF3 and NF-B (Genin et al., 2000; Veckman et al., 2006). The binding of activated IRF1 and IRF3 to the ISRE of CXCL10 (Fig. 3a) and of the p65/p50 heterodimer to the NF-B RE of CXCL10 (Fig. 3b) was clearly decreased in the UNS3-4A-24 cell line by 6 h after SeV infection, whereas reduced binding was detected later at the 18 h time point in the UHCV-11 cell line. No inhibition of SeV-induced IRF1, IRF3 or p65/p50 binding to CXCL10 promoter elements was seen in NS4B or core-E1-E2-p7 protein-expressing cell lines.
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B, AP-1 and C/EBP REs (Mukaida et al., 1994) as well as an ISRE/HNF-3 element (Casola et al., 2000). The NF-B RE is indispensable for activation of the CXCL8 gene, whereas AP-1 and C/EBP REs have a less important role in CXCL8 gene expression, depending on the cell type (Hoffmann et al., 2002). The ISRE-like element participates in activation of the CXCL8 promoter during respiratory syncytial virus infection (Casola et al., 2000) or when dsRNA signalling pathways are activated (Wagoner et al., 2007). In NS3/4A protein-expressing cells, there was less c-Jun and phosphorylated c-Jun bound to the CXCL8 promoter (Fig. 4b) and binding of IRF1 and IRF3 to the ISRE of CXCL8 was also reduced (Fig. 4c). There was only a modest reduction in binding of the p65/p50 heterodimer to the NF-B RE of CXCL8 in UNS3-4A-24 cells (Fig. 4a). In the UHCV-11 cell line, binding of p65/p50 was slightly reduced, whereas binding of the IRF1, IRF3 and phosphorylated c-Jun transcription factors to their respective elements on the CXCL8 promoter was reduced more strongly (Fig. 4). In the cell line expressing NS4B protein, there were no changes in the binding of transcription factors to their REs on the CXCL8 promoter (Fig. 4). In the UCp7con-9 cell line, binding of IRF1 and IRF3 to the ISRE of CXCL8 was reduced at 18 h after infection (Fig. 4c).
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; Li et al., 2005b; Meylan et al., 2005). Recently, degradation of endogenous Cardif was reported in HCV replicon (HCV 1b subgenomic replicon) cell lines Huh7-K2040 (Li et al., 2005b), Huh7-HP, Huh7-JFHR (JFH-1 HCV 2a subgenomic replicon RNA) (Loo et al., 2006) and Huh8 (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 h after HCV protein induction correlated with a decrease in the full-length form and the appearance of a C-terminally truncated form of Cardif in the UNS3-4A-24 and UHCV-11 cell lines. No degradation of Cardif was seen in cell lines expressing the NS4B or the structural region proteins.
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; Lin et al., 2006a; Seth et al., 2005). In order to verify this observation, we carried out co-localization experiments with MitoTracker and anti-Cardif antibody staining in different HCV protein-expressing cell lines. Cardif protein strongly co-localized with Mitotracker, indicating that the intrinsic Cardif protein is primarily located in the mitochondria (Fig. 6). Surprisingly, the subcellular localization of the mitochondria changed when the NS3/4A protein was expressed for 2 days in the UNS3-4A-24 cell line, and the mitochondria accumulated as clusters near the nucleus. There was a similar change in the distribution of mitochondria, although to a lesser extent compared with UNS3-4A-24 cells, in the full-length HCV polyprotein-expressing cell line (UHCV-11). In the UNS4Bcon-4 and UCp7con-9 cell lines, HCV protein expression did not affect the subcellular localization of mitochondria (Fig. 6).
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). Generally, the NS3/4A protein was tightly co-localized with Cardif in the endoplasmic reticulum (ER)/mitochondria-like structures, although some NS3/4A protein was also seen in the perinuclear space, apparently associated with the nuclear membrane (at 24 h after induction of NS3/4A protein expression). It has been shown previously that the NS3/4A protein complex in the UNS3-4A-24 cell line is located in both the ER and the mitochondria (Wolk et al., 2000). Two days after the induction of expression of NS3/4A protein, a clear change in the distribution of mitochondria was seen in UNS3-4A-24 cells. Cardif and NS3 proteins also fully co-localized (Fig. 7). In the UNS4Bcon-4 cell line, NS4B protein was expressed evenly throughout the cytoplasm, as reported previously (Melen et al., 2004), and no co-localization with Cardif was detected (Fig. 7). The core protein showed a partial co-localization with Cardif in mitochondria in the UCp7con-9 cell line. Previous studies have shown that the core 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 staining for NS3 was weaker compared with that seen in UNS3-4A-24 cells. However, in UHCV-11 cells, the distribution of mitochondria also changed and co-localization of NS3 with Cardif was clearly seen (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The core protein interferes with IFN signalling and the antiviral response (Gale & Foy, 2005; Melen et al., 2004), for example by binding to the SH2 domain of signal transducers and activators of transcription (STAT) 1 (Lin et al., 2006b). Furthermore, the core protein can downregulate the transcription of IRF1 (Ciccaglione et al., 2007). In addition, the E1 and NS5A proteins have been shown to block the activation of PKR, leading to a reduced IFN-induced antiviral response (Gale & Foy, 2005). Several recent studies have shown that the NS3/4A complex targets the RIG-I pathway and proteolytically cleaves the mitochondrion-associated Cardif/MAVS/IPS-1/VISA adaptor molecule, which leads to reduced expression of the IFN-β gene (Kaukinen et al., 2006; Lin et al., 2006a; Loo et al., 2006; Meylan et al., 2005; Seth et al., 2005). These studies concentrated mainly on revealing the molecular mechanism of action of the NS3/4A protein complex on IFN-β gene expression and less attention was paid to analysing the effects of HCV protein expression on the regulation of chemokine gene expression.
The production of cytokines during virus infection requires a coordinated action between different transcription factor families; for example, IRFs, NF-B and AP-1 are required for the transcription of IFN-β (Wathelet et al., 1998), IRFs and NF-B for the transcription of CCL5 (Genin et al., 2000) and CXCL10 (Ohmori & Hamilton, 1993), and NF-B, IRFs, AP-1 and C/EBP for the transcription of CXCL8 (Casola et al., 2000; Mukaida et al., 1994). Inhibition of IFN-β gene expression was apparently due to impaired phosphorylation of IRF3 (Foy et al., 2003) and impaired binding of activated IRF3 and NF-B to the respective IFN-β promoter elements (Foy et al., 2005). Our results support these observations, as NS3/4A and full-length HCV polyprotein expression, but not that of core-E1-E2-p7 or NS4B protein, clearly reduced endogenous IFN-β mRNA expression (Fig. 2) in our stable cell line systems. This inhibitory effect was not restricted to the IFN-β gene, as expression of SeV-induced CCL5, CXCL8 and CXCL10 protein (Fig. 1) and mRNA (Fig. 2) was reduced when NS3/4A or the HCV polyprotein was expressed. At least in the case of the CXCL10 gene, this inhibition is likely to occur at the transcriptional level, as oligonucleotide-binding experiments revealed that NS3/4A expression decreased IRF1, IRF3 and p65/p50 heterodimer binding to their respective elements on the CXCL10 promoter (Fig. 3a, b). Thus, NS3/4A-mediated inhibition of the CXCL10 gene expression occurs by a mechanism similar to that of the IFN-β gene. In the UHCV-11 cell line, the inhibition of IRF1 and IRF3 binding to the ISRE and p65/p50 binding to the NF-B element on the CXCL10 promoter was reduced at a later stage compared with that seen in the UNS3A-4-24 cell line (Fig. 3a, b). This is consistent with the reduction seen in mRNA 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 both activation of transcription and stabilization of mRNA. Activation of the CXCL8 gene occurs mainly via NF-B, whilst AP-1 and C/EBP contribute to maximal CXCL8 gene expression depending on the cell type (Hoffmann et al., 2002). In addition, an ISRE-like element of the CXCL8 promoter participates in activation of the CXCL8 gene during virus infection (Casola et al., 2000; Wagoner et al., 2007). Transcription of CXCL8 is induced rapidly by virus infection (Hoffmann et al., 2002), as also seen in our experiment with SeV (Fig. 2). CXCL8 mRNA contains several AU-rich elements (Winzen et al., 1999, 2004), which control the stability of CXCL8 mRNA. Several proteins are involved in the stabilization of CXCL8 mRNA: mitogen-activated protein kinase (MAP) kinase-activated p38 and its substrate, MAP kinase-activated protein kinase 2 (Hoffmann et al., 2002; Winzen et al., 1999), the constitutively 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 lines showed that the expression of NS3/4A and the HCV polyprotein clearly inhibited the expression of CXCL8 mRNA and the production of CXCL8 protein (Figs 1 and 2). These data are consistent with the observed decreased binding of phosphorylated c-Jun, IRF1 and IRF3 to their REs on the CXCL8 promoter (Fig. 4). In several HCV replicon cell lines, CXCL8 production is increased during HCV replication (Koo et al., 2006). The promoter of CXCL8 was activated by JFH-1 strain HCV RNA and virus (Wagoner et al., 2007). The difference from the current study is that we used SeV to induce chemokine production in UHCV cell lines that express HCV proteins, whereas Wagoner et al. (2007) used HCV RNA to activate RIG-I signalling. Although expression of the HCV structural proteins, core-E1-E2-p7, increased the expression of CXCL8 mRNA and protein (Figs 1 and 2) in our experiments, we could not detect enhanced transcription factor binding to the regulatory elements on the CXCL8 promoter (Fig. 4). It has been shown that the core protein can activate NF-B-regulated pathways leading to enhanced CXCL8 promoter activation (Kato et al., 2000), but this effect has been suggested to be dependent on the HCV genotype (Ray et al., 2002). Thus, there may be some post-transcriptional regulatory steps involved in the production of CXCL8 in the UCp7con-9 cell line that need to be studied further.
Although the dsRNA-activated TLR3 and RIG-I pathways are not always operational in the same cells (Melchjorsen et al., 2005), they are apparently functional in UHCV cell lines (Li et al., 2005a). In the present study, we concentrated on the RIG-I pathway, as the crucial adaptor protein of the RIG-I pathway, Cardif/MAVS/IPS-1/VISA, has been identified as the proteolytic target for the NS3/4A protein complex (Li et al., 2005b; Lin et al., 2006a; Loo et al., 2006; Meylan et al., 2005). Similarly, in the UNS3-4A-24 and the UHCV-11 cell lines, degradation of endogenous Cardif was observed, starting 24 h after induction of HCV protein expression (Fig. 5). It is noteworthy that we observed only a partial degradation of Cardif in NS3/4A- and HCV polyprotein-expressing cells. Another study carried out with the UNS3-4A-24 cell line showed almost complete degradation of transfected Cardif, whilst the stability of intrinsic Cardif was not analysed (Meylan et al., 2005). Our results may indicate that Cardif becomes inaccessible for degradation due to a change in its subcellular localization in mitochondria (see below). It may well be that NS3/4A-induced change in subcellular localization of mitochondria, even in the absence of full Cardif degradation, may be sufficient for effective inhibition of SeV-induced cytokine gene expression.
An interesting observation was that, in immunofluorescence analysis (Figs 6 and 7), the expression of NS3/4A had a dramatic effect on the distribution of mitochondria in the UNS3-4A-24 and UHCV-11 cell lines. Clustering of mitochondria was found after 2 days of HCV protein expression and it was seen more clearly in the UNS3-4A-24 cell line where the expression of NS3/4A is higher than in the UHCV-11 cell line. Transient expression of NS3/4A in Huh7 cells has also been shown to change the intracellular distribution of mitochondria (Nomura-Takigawa et al., 2006). A strong co-localization of NS3/4A was observed with MitoTracker and Cardif when NS3/4A was expressed alone (UNS3-4A-24 cells) or in the context of the full-length HCV polyprotein (UHCV-11 cells). We did not find any marked cytoplasmic, non-mitochondrion-localized Cardif. Other studies have shown diffuse staining of the cytoplasm after partial dissociation of Cardif from the mitochondrial membrane to the cytosol when NS3/4A was transiently 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 degradation of intrinsic Cardif may be relatively fast and therefore we failed to observe the accumulation of truncated Cardif in the cytoplasm. In addition, our data may imply that a change in the distribution of mitochondria and strong co-localization of NS3/4A with Cardif may as such be sufficient to interfere with RIG-I signalling, rendering the cells incapable of producing antiviral and other pro-inflammatory cytokines. Mitochondria are also active producers of reactive oxygen species (ROS). HCV infection can induce oxidative stress, and NS3, core and NS5A proteins have been shown to be able to induce ROS production (Koike & Miyoshi, 2006). Furthermore, an increase in mitochondrial ROS production and disturbed mitochondrial function have been found in HCV polyprotein-expressing cells (Piccoli et al., 2006). Mitochondrial membrane may thus turn out to be an essential 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 and chemokine gene expression was clearly reduced and that NS3/4A-mediated inhibition also took place in the context of the whole HCV polyprotein. The binding of transcription factors to their response elements on CXCL8 and CXCL10 promoters was disturbed by the expression of NS3/4A and HCV polyprotein. A more novel finding was that the expression of NS3/4A protein changed the subcellular localization of mitochondria. It is likely that this change in distribution of mitochondria renders them incapable of proper signalling and emphasizes an important role for mitochondria in cellular resistance to HCV infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 July 2007;
accepted 16 October 2007.
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TM represents the truncated Cardif protein form. The experiment was performed in triplicate with similar results.
