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1 Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan
2 Department for Hepatitis Control, Tokyo Medical and Dental University, Tokyo, Japan
3 First Department of Internal Medicine, University of Yamanashi, Yamanashi, Japan
4 Laboratory of Molecular Genetics, Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kyoto, Japan
5 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
Correspondence
Naoya Sakamoto
nsakamoto.gast{at}tmd.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and IRF-3. This study analysed the mechanisms of dsRNA-induced IFN responses in various cell lines that supported subgenomic hepatitis C virus (HCV) replication. Transfection of dsRNA into Huh7, HeLa and HEK293 cells induced an IFN expression response as shown by IRF-3 dimerization, whilst these responses were abolished in corresponding cell lines that expressed HCV replicons. Similarly, RIG-I-dependent activation of the IFN-stimulated response element (ISRE) was significantly suppressed by cells expressing the HCV replicon and restored in replicon-eliminated cells. Overexpression analyses of individual HCV non-structural proteins revealed that NS4B, as well as NS34A, significantly inhibited RIG-I-triggered ISRE activation. Taken together, HCV replication and protein expression substantially blocked the dsRNA-triggered, RIG-I-mediated IFN expression response and this blockade was partly mediated by HCV NS4B, as well as NS34A. These mechanisms may contribute to the clinical persistence of HCV infection and could constitute a novel antiviral therapeutic target.
These authors contributed equally to this work. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Taniguchi & Takaoka, 2002). Hepatitis C virus (HCV) infection is characterized by persistence and replication of the virus in the liver, despite an intact host immune system (Alter, 1997). Indeed, even after administration of the currently most potent IFN reagents, as many as half of the patients are refractory to the treatment and fail to eradicate the virus (Fried et al., 2002). These features have led to speculation that HCV escapes from or attenuates the host antiviral response (Katze et al., 2002).
Cellular antiviral responses are primarily mediated by IFN and IFN-stimulated genes (ISGs), including 2,5-oligoadenylate synthetase, dsRNA-dependent protein kinase R (PKR) and MxA proteins, as well as by as yet uncharacterized genes (Itsui et al., 2006; Stark et al., 1998). A study of experimental chimpanzee HCV infection has shown that various cytokines and chemokines are induced in the liver during the course of acute HCV infection and its clearance, and that a considerable proportion of the genes is induced by type I IFN (Bigger et al., 2001).
Control of expression of ISGs is mediated by binding of type I IFNs to their receptors. Following receptor binding, STAT1 and STAT2 are phosphorylated to form ISGF-3, which translocates to the nucleus and binds the IFN-stimulated response element (ISRE), located in the promoter/enhancer region of ISGs, and activates transcription of ISGs (Samuel, 2001; Taniguchi et al., 2001; Taniguchi & Takaoka, 2002). ISRE-dependent gene expression is also mediated by binding of the ISRE by molecules such as IRF-1, IRF-3 and IRF-7 (Kanazawa et al., 2004). IRF-3 is a transducer of virus-mediated signalling and plays a critical role in the induction of cellular antiviral responses (Lin et al., 1998; Sato et al., 2000; Taniguchi et al., 2001; Yoneyama et al., 1998). Transcriptional activation and suppression of IRF-3 are inversely correlated with the level of HCV replication in vitro (Yamashiro et al., 2006). Following virus infection, IRF-3 is phosphorylated by two cytoplasmic kinases, TBK1 and IKK (Fitzgerald et al., 2003; Sharma et al., 2003). The phosphorylated IRF-3 forms a homodimer, translocates to the nucleus and predominantly activates expression of the IFN-
gene and certain ISGs (Doyle et al., 2002; Nakaya et al., 2001; Taniguchi & Takaoka, 2002).
RIG-I is a recently identified cytoplasmic DExD/H box RNA helicase that participates in recognition of virus-related dsRNA as a pathogen-related molecular pattern (Yoneyama et al., 2005). RIG-I contains two caspase-recruitment domains (CARDs) in the N terminus and a DExD/H box RNA helicase in the C terminus. MDA5 has been identified as another CARD-containing DExD/H box RNA helicase (Andrejeva et al., 2004). More recently, an adaptor molecule of RIG-I and MDA5, Cardif (also known as IPS-I, MAVS and VISA), has been identified by four independent groups (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). On association with dsRNA, RIG-I or MDA5 causes conformational changes and homo-oligomerization, and binds the CARD of Cardif (Saito et al., 2007). Cardif subsequently recruits the kinases TBK1 and IKK, which catalyse phosphorylation and activation of IRF-3 (Yoneyama et al., 1998).
The IRF-3-mediated IFN- induction pathway could be a target for viruses to counteract antiviral responses and promote their replication in host cells. Ebola virus, bovine viral diarrhea virus (BVDV) and influenza A virus interfere with the activation of IRF-3 through interactions of their virus-encoded proteins (Basler et al., 2003; Schweizer & Peterhans, 2001; Talon et al., 2000). There are several reports that HCV proteins interact with IFN-mediated antiviral systems. The NS5A and E2 proteins have been reported to interfere with the action of IFN by inhibiting the activity of PKR (He & Katze, 2002). It was reported recently that the HCV NS34A protease blocks virus-induced activation of IRF-3, possibly by proteolytic cleavage of Cardif (Foy et al., 2003; Meylan et al., 2005).
The HCV subgenomic replicon is an in vitro model that simulates autonomous cellular replication of HCV genomic RNA (Lohmann et al., 1999). Expression of the HCV replicon can be abolished by treatment with small amounts of type I and type II IFNs (Blight et al., 2000; Frese et al., 2002; Guo et al., 2001), suggesting intact IFN receptor-mediated cellular responses. In contrast, viral expression persists in the absence of the exogenous IFN. Baseline expression levels of ISG were substantially decreased in cells expressing the HCV replicon compared with parental Huh7 cells (Kanazawa et al., 2004). These findings led us to speculate that intracellular virus-induced antiviral responses are attenuated or caused to malfunction by the expression of viral proteins.
In this study, we investigated cell lines that support subgenomic HCV replication and HCV cell culture for the dsRNA-induced cellular IFN expression pathway. Here, we report that RIG-I- and Cardif-mediated IFN gene activation is uniformly attenuated in several replicon-expressing cell lines of different lineages and, more importantly, that the HCV NS4B protein is involved in the suppression of antiviral IFN responses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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RIG-I expressed full-length and C-terminally truncated RIG-I protein, respectively (Yoneyama et al., 2004). The plasmid pER-flagRIG-IKA (RIG-IKA) has a point mutation in the putative ATP-binding site of the RIG-I helicase domain and was used as a negative control for RIG-I and RIG-I full transfection assays. Expression plasmids for full-length Cardif (Cardif), Cardif CARD (CARD) and CARD-truncated Cardif (CARD) were provided by Dr J. Tschopp (University of Lausanne, Switzerland) (Meylan et al., 2005). Expression plasmids for toll-like receptor 3 (TLR3) and TIR domain-containing adaptor inducing IFN- (TRIF), the transmembrane receptor of dsRNA and the adaptor molecule of TLR3, respectively, were provided by Dr S. Akira (Osaka University, Japan). Plasmids expressing HCV NS345, NS3, NS34A, NS4A, NS4B, NS5A and NS5B were amplified from HCV pCV-J4-L4S (Yanagi et al., 1997) by PCR and subcloned. The DNA fragments were inserted into the vector pcDNA4/TO/myc-His (Invitrogen). Nucleotide sequences were confirmed by sequencing. Plasmids TOPO-NS34A (HCV N), TOPO-NS4B (HCV N) and pcDNA-NS4B (HCV JFH1) expressed Myc-tagged NS34A and NS4B proteins derived from the HCV N (Beard et al., 1999) and HCV JFH1 (Wakita et al., 2005) strains, as indicated. Plasmid pISRE-TA-Luc (Invitrogen) contained five copies of consensus ISRE motifs upstream of the firefly luciferase gene. Plasmid pIFN-Fluc was constructed by cloning the human IFN- promoter region, spanning nt –110 to –36, upstream of the firefly luciferase gene of pGL3 Basic (Promega). Plasmid pcDNA3.1 (Invitrogen) was used as an empty vector for mock transfection. pRL-CMV (Promega), which expressed the Renilla luciferase protein, was used for correction of transfection efficiency.
Cell culture.
HCV strain JFH1-infected Huh7.5.1, Huh7, Huh7.5.1 (kindly provided by Dr F. Chisari, The Scripps Institute, CA, USA; Zhong et al., 2005), HeLa and HEK293 cells were maintained in Dulbecco's modified minimal essential medium (Sigma) supplemented with 2 mM L-glutamine and 10 % fetal calf serum at 37 °C with 5 % CO2. Cells expressing the HCV replicon were cultured in medium containing 100 µg G418 (Wako) ml–1.
HCV replicon constructs and transfected cell lines.
An HCV subgenomic replicon plasmid, pHCVIbneo-delS (designated pRep-N), was derived from an HCV clone of strain N, genotype 1b, and pSGR-JFH1 was derived from HCV JFH1, genotype 2a (Guo et al., 2001; Wakita et al., 2005). These replicons were reconstructed by substituting the neomycin phosphotransferase gene with a fusion gene comprising Renilla luciferase and neomycin phosphotransferase to construct pRep-Reo-1b and pRep-Reo-2a, respectively (Tanabe et al., 2004; Yokota et al., 2003). RNA was synthesized from the replicons using T7 polymerase (Promega) and transfected into Huh7, HeLa and HEK293 cells. After culture in the presence of G418, cell lines stably expressing the replicon were established (Huh7/1bReo, Huh7/2aReo, HeLa/2aReo and 293/2aReo). We have previously reported that firefly luciferase activities of Feo-replicon-expressing cells correlate well with HCV NS3, NS4A and NS5A protein expression levels and with the levels of replicon RNA (Yokota et al., 2003).
Transient transfection.
Transient DNA transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. ISRE reporter assays were carried out as previously described (Nakagawa et al., 2004). To analyse IFN expression in HCV JFH1 cell cultures, a total of 1x105 Huh7.5.1, JFH-1 infected Huh7.5.1 and IFN-treated Huh7.5.1 cells were seeded into 24-well plates the day before transfection. Plasmids pISRE-TA-Luc and RIG-I (200 ng each) were transfected using 1 µl Lipofectamine 2000. RIG-IKA was used as a control. Luciferase assays were performed on day 3 post-transfection.
For further study, 400 ng of each non-structural protein was added to 1x104 Huh7 or HEK293 cells that had been seeded into 96-well plates the day before transfection. pISRE-TA-Luc and RIG-I (40 ng each) were transfected using 0.5 µl Lipofectamine 2000. RIG-IKA was used as a control.
Western blotting.
Preparation of the cytoplasmic and nuclear fractions of cell lysates was carried out as described previously (Tanabe et al., 2004). Protein (20 µg) was separated using NuPAGE 4–12 % Bis/Tris gels (Invitrogen) and blotted onto an Immobilon PVDF membrane (Roche). The membrane was immunoblotted with anti-IRF-3 (Santa Cruz) and detected by chemiluminescence (BM Chemiluminescence Blotting Substrate; Roche).
RT-PCR.
Interleukin (IL)-8 mRNA was detected by RT-PCR as described previously (Itsui et al., 2006). The primers used were IL8-S (5'-GCACAAACTTTCAGAGACAGCAGAGCACAC-3') and IL8-AS (5'-CAGAGCTGCAGAAATCAGGAAGGCTGCCAA-3').
Indirect immunofluorescence assay.
Cells seeded onto tissue culture chamber slides were fixed with cold acetone. The cells were incubated with anti-protein disulphide isomerase (PDI) or anti-Myc antibodies and subsequently with Alexa 488- or Alexa 568-labelled secondary antibodies. Cells were mounted with VECTA SHIELD Mounting Medium and DAPI (Vector Laboratories) and visualized by fluorescence microscopy (BZ-8000; Keyence).
Luciferase reporter assays.
Luciferase activity was measured using a 1420 Multilabel Counter (ARVO MX; PerkinElmer) using a Bright-Glo Luciferase Assay System (Promega) or a Dual Luciferase Assay System (Promega). Assays were carried out in triplicate and the results expressed as means±SD.
MTS assay.
To evaluate cell viabilities, dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assays were performed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega) according to manufacturer's instructions.
Statistical analyses.
Statistical analyses were performed using an unpaired, two-tailed Student's t-test. P values of less than 0.05 were considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Activation of RIG-I or MDA5 induces phosphorylation and homodimerization of IRF-3. Following transfection of poly(I : C) into Huh7, HeLa or HEK293 cells, IRF-3 dimers were detected (Fig. 1). However, in cells supporting HCV replicons, IRF-3 dimer formation was almost completely abolished. These findings showed that expression of HCV proteins blocked activation of dsRNA-mediated IFN expression and that these effects were consistently found in several cell lines of different origin.
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). Consistent results were obtained by overexpression of RIG-I, a constitutively active form. Transfection of RIG-I in Huh7 and HEK293 cells induced ISRE activation, whilst these responses were abolished or significantly suppressed in cell lines expressing HCV replicons and were recovered by eliminating the replicon by IFN treatment (data not shown). Similarly, ISRE activation by overexpression of Cardif, an adaptor molecule of RIG-I, was almost completely blocked in replicon-expressing cells and was recovered by eliminating the replicon from the cells (data not shown). The RIG-I-mediated IFN response was also suppressed in HCV JFH1 virus cell culture. In JFH1-infected Huh7.5.1 cells, RIG-I-induced ISRE reporter activation was significantly suppressed, but was recovered in IFN-treated, virus-eliminated cells (Fig. 2b and c). These results demonstrated that RIG-I- and Cardif-mediated antiviral responses were substantially suppressed by both subgenomic and genomic viral replication in both hepatocyte- and non-hepatocyte-derived host cells.
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). We transfected each expression plasmid with simultaneous activation of the RIG-I pathway by overexpression of RIG-I, Cardif, TBK1 and IKK (Fig. 3b–e). Expression of full-length non-structural (NS345) and NS34A proteins inhibited ISRE activation mediated by expression of RIG-I and Cardif but not that mediated by TBK1 and IKK. Interestingly, it was found that NS4B also inhibited ISRE activation mediated by expression of RIG-I and Cardif, but not by TBK1 and IKK. Consistent with Fig. 3(b), overexpression of NS4B significantly suppressed RIG-I-induced activation of the authentic IFN- promoter (Fig. 3f).
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). To investigate strain-specific differences in the characteristics of NS4B proteins, we performed co-transfection assays using NS4B expression constructs from HCV N (Beard et al., 1999) and JFH1 (Wakita et al., 2005) strains, as well as HCV strain J4 (Fig. 4a and b). All NS4B constructs suppressed RIG-I- or Cardif-mediated ISRE activation. These results suggested that the above-described effects of NS4B were independent of HCV strain.
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). The ER stress induces production of IL-8, which has been reported to interfere with the IFN system (Polyak et al., 2001). Therefore, we detected expression of IL-8 using RT-PCR in cells with and without overexpression of NS4B. As shown in Fig. 4(c)
, no significant difference was observed in IL-8 mRNA levels among mock-, NS34A- and NS4B-transfected cells. These results showed that NS4B overexpression in the present study did not induce expression of IL-8 and that the IFN-antagonizing effects of NS4B were independent of IL-8.
It has been reported that NS34A suppresses the TLR3-mediated IFN response (Breiman et al., 2005; Ferreon et al., 2005). However, overexpression of HCV non-structural proteins did not suppress ISRE activation that was induced by overexpression of TLR-3 or TRIF (Fig. 5a and b), nor did NS34A from two different HCV strains, J4 and N, show significant suppression of TRIF-mediated ISRE activation (Fig. 5c). Although strain-specific differences might be involved, these data suggest that neither NS34A nor NS4B affect the TLR3-triggered, TRIF-mediated IFN expression signalling pathway.
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) (Fig. 6a). Expression and subcellular localization of NS4B truncated proteins were visualized by indirect immunofluorescence assays (Fig. 7). Each of the NS4B truncated proteins was localized predominantly to the perinuclear rim as dense spots. Some of the spots were similar to the staining of the ER-resident host protein PDI, consistent with previous reports (Lindström et al., 2006; Lundin et al., 2006). These truncated expression plasmids were co-transfected with Cardif expression plasmids into Huh7 cells. As shown in Fig. 6(b), Cardif-mediated ISRE activation was significantly suppressed by co-transfection of NS4Bt1–156 and NS4Bt1–186, as well as full-length NS4B, whilst transfection of NS4Bt90–260 and NS4Bt110–260 did not significantly suppress Cardif-mediated ISRE activation. The shortest construct, NS4Bt131–260, partially retained the ability to reduce ISRE activity. These results suggested that the N-terminal domain of NS4B, which includes aa 1–110, might function directly to suppress RIG-I-mediated IFN expression responses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Several reports suggest that viruses target the IFN system to establish replication in the host cells (Kato et al., 2006). We have confirmed that the dsRNA-triggered, IRF-3-mediated IFN activation pathway was blocked in several replicon-supporting cell lines (Fig. 1). Similarly, the dsRNA responses were substantially suppressed in HCV JFH-1 cell culture compared with parental Huh7 cells (Fig. 2b and c). Overexpression analyses showed that RIG-I- and Cardif-mediated ISRE activation was significantly suppressed in HCV replicon-expressing cells, which recovered after elimination of the replicon by IFN treatment (Fig. 2a). In contrast, TBK1- or IKK-mediated ISRE activation was not suppressed in replicon-expressing cells. Overexpression of individual HCV non-structural proteins revealed that not only NS34A but also NS4B inhibited the ISRE activation signal (Figs 3, 4 and 5). These results suggested that HCV non-structural proteins suppress the IFN induction pathway and that the target host molecule could be Cardif or an unknown adaptor molecule acting between Cardif and TBK1/IKK.
NS4B protein is a 27 kDa hydrophobic integral membrane protein that is localized in the ER with other non-structural proteins. Studies on other flaviviruses such as Kunjin virus and BVDV support the notion that NS4B may indeed be an essential part of the replication mechanism (Grassmann et al., 2001; Khromykh et al., 2000; Li & McNally, 2001; Qu et al., 2001). These systems have demonstrated that intact NS4B is necessary in a cis configuration in the polyprotein for maintaining viral replication (Grassmann et al., 2001; Khromykh et al., 2000). Furthermore, single mutations in NS4B alter the cytopathic effects of BVDV and even mediate changes in the cellular tropism of Dengue virus (Hanley et al., 2003; Qu et al., 2001). In HCV, the search for cell-culture-adaptive mutations in HCV subgenomic replicons has led to the generation of mutations in the NS4B region that confer higher replication levels and resistance to IFNs, as well as broadening the tropism for different cell lines (Lohmann et al., 2003; Sumpter et al., 2004; Zhu et al., 2003). These pieces of evidence may imply that NS4B is not only part of the replication machinery but may also have other functions that enable establishment of viral replication.
NS4B truncation assays showed that RIG-I/Cardif-mediated ISRE activation was significantly suppressed by expression of N terminus-containing constructs (Fig. 6). These results imply that the N-terminal domain of NS4B, which is located between positions 1 and 110, may be essential for suppressing IFN expression responses in host cells. Lindström et al., (2006) investigated single point mutations in NS4B that negatively affected expression efficiency of the HCV replicon and reported that most of the active mutations were located around the N-terminal domain. A distinctive feature of NS4B is that it requires membrane rearrangement to form its native structure (Lindström et al., 2006; Lundin et al., 2006). The HCV polyprotein is translated from a single reading frame and subjected to proteolytic cleavage by the host signal peptidase and two viral proteases (Grakoui et al., 1993). The mature form of NS4B is localized in the ER and constitutes a subcellular structure called the membrane-associated focus (MAF) (Gretton et al., 2005). Once the NS4B is cleaved, the N-terminal peptide of NS4B is translocated from the cytoplasmic to the luminal side, giving it a fifth transmembrane region (Lundin et al., 2006). The N-terminal amphipathic helix (AH) 1 of NS4B is necessary for this translocation and for MAF formation; NS4B molecules that were truncated at the AH1 lacked the ability to create the MAF, to translocate and to replicate (Elazar et al., 2004; Lindström et al., 2006).
In our assay, NS4Bt131–260 regained the ability to reduce ISRE activity. As we confirmed that all mutants co-localized with the ER, there may be some effect of the N-terminal localization of this mutant. The precise mechanism of NS4B suppression is still not clear and further experiments are needed.
NS4B has been reported to induce an unfolded protein response or ER stress (Zheng et al., 2005). Accumulation of unfolded or misfolded proteins in the ER is detected by three ER sensor proteins, ATF6, IRE-1 and PKR-like ER kinase (PERK), and triggers the unfolded protein response as a stress response and induces expression of molecular chaperon proteins, global shut-off of protein translation and apoptotic cell death (Mori, 2000). Therefore, it may be possible that transgenic overexpression of NS4B induces ER stress and suppresses overall protein synthesis, including that of IFNs. In our experiments, however, NS4B suppressed the ISRE-mediated IFN gene activation but did not suppress non-specific protein expression, as demonstrated by Renilla luciferase activity in the control plasmid driven by the herpes simplex thymidine kinase promoter. In addition, the growth and viability of cells that overexpressed NS4B did not differ from untransfected cells or from those transfected with the other HCV proteins. IL-8 overproduction induced by ER stress was not observed in our NS4B-overexpressing cells (Fig. 4c). These findings indicated that the inhibitory effect of NS4B is specific to the IFN induction pathway and is not a non-specific effect through ER stress.
In conclusion, we have shown that dsRNA-induced IFN expression was suppressed by NS4B. These virus–host interactions probably contribute to HCV persistence and to the pathogenesis of HCV-associated liver disease.
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ACKNOWLEDGEMENTS |
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CARD and CARD, and to Dr Frank Chisari for providing the Huh7.5.1 cell line. This study was supported by grants from the Japan Society for the Promotion of Science, Miyakawa Memorial Research Foundation and Viral Hepatitis Research Foundation of Japan. |
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Received 4 April 2007;
accepted 27 July 2007.
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