Hepatitis C virus inhibits intracellular interferon alpha expression in human hepatic cell lines

Authors

  • Ting Zhang,

    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Rong-Tuan Lin,

    1. Lady Davis Institute for Medical Research, McGill University, Montreal, QC, Canada
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  • Yuan Li,

    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Steven D. Douglas,

    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Catherine Maxcey,

    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Chun Ho,

    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Jian-Ping Lai,

    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Yan-Jian Wang,

    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Qi Wan,

    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Wen-Zhe Ho

    Corresponding author
    1. Division of Allergy & Immunology, Joseph Stokes Jr. Research Institute at The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA
    • Division of Allergy & Immunology, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, 34th Street & Civic Center Boulevard, Philadelphia, PA 19104
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    • fax: 215-590-2025


  • Potential conflict of interest: Nothing to report.

Abstract

The chronicity of hepatitis C virus (HCV) infection raises the question of how HCV is able to persist in hepatic cells. We show that human primary hepatocytes and human hepatic cell lines (Huh7 and HepG2) spontaneously produce interferon (IFN)-α that is inhibited in the HCV replicon cells (Huh.8 and FCA-1). Silencing IFN-α gene expression by IFN-α small interfering RNA (siRNA) in the HCV replicon cells resulted in increased HCV replicon expression. The activation of IFN-α expression by interferon regulatory factor (IRF-7) led to the inhibition of HCV replicon expression, whereas the anti–IFN-α receptor antibody could partially block IRF-7–mediated HCV replicon inhibition. In addition, the blockade of IFN-α receptor by anti–IFN-α receptor antibody on the replicon cells increased HCV replicon expression. Among the HCV nonstructural (NS) proteins tested, NS5A is the most potent inhibitor of IFN-α expression by the hepatic cells. Investigation of the mechanism of HCV action on IFN-α showed that IRF-7–induced IFN-α promoter activation was inhibited in the HCV replicon cells. Furthermore, IRF-7 expression was restricted in the HCV replicon cells. In conclusion, we provide direct evidence that HCV undermines the intracellular innate immunity of the target cells, which may account for HCV persistence in hepatic cells. (HEPATOLOGY 2005;42:00–00.) (HEPATOLOGY 2005;42:819–827.)

Hepatitis C virus (HCV) is estimated to chronically infect more than 170 million people worldwide, causing a spectrum of liver disease ranging from an asymptomatic carrier state to end-stage liver disease. The clinical outcome of HCV infection and the degree of liver damage is the result of complicated interactions between the virus and host immune responses. Although cellular and humoral immune responses are present during acute and chronic HCV infection,1 the immune response is rarely effective in eradicating the virus. Most HCV-infected subjects develop chronic infection, suggesting that HCV may have evolved strategies to overcome or evade efficient innate immune responses of the host.2–6

The innate immune response is the earliest phase of immune defense and also regulates the adaptive immune response.7 Because the innate immune response of the immune cells or virus-infected cells plays a crucial role in controlling viral infection, damage to the innate immune response may generate a favorable microenvironment for HCV infection and replication. Interferons (IFNs) are well-characterized components of the innate host defense against viral infections. Type 1 IFNs (IFN-α/β) are produced by virus-infected host cells and constitute the primary defense mechanism against viral infection and replication. Rapid induction of type I IFNs is a central event in initiating the innate antiviral response.8, 9 The IFN system and the generation of the antiviral state are the earliest immunological defense mechanisms that have evolved to combat virus infection. Molecular regulation of IFN gene expression is tightly regulated by extracellular and intracellular signals generated during primary infection, culminating in the activation of nuclear factor-kappaB (NF-κB) and IFN regulatory factor (IRF)-3 transcription factors, which trigger an immediate early IFN response characterized by the release of IFN-β and IFN-α1. Secreted IFN acts through an autocrine and paracrine loop, which requires intact IFN receptor and Janus kinase (JAK) signal transducer and activation of transcription (STAT) pathways. IFN activation of the IFN-stimulated gene factor (ISGF) 3 complex results in the transcriptional upregulation of IRF-7.10–14 Virus infection activates IRF-7 through inducible phosphorylation, and phosphorylated IRF-7 participates together with IRF-3 in the transcriptional induction of immediate-early and delayed-type IFN genes.1, 2 Activation of the ISGF3 complex also binds to interferon-stimulated response elements (ISRE) found in hundreds of IFN-stimulated genes (ISGs), including 2′-5′ oligoadenylate synthase (2′-5′ OAS), myxovirus resistance protein (Mx), double-stranded RNA (dsRNA) activated kinase (PKR) and major histocompatibility complex class I, resulting in the induction of proteins that impair viral gene expression and replication.8, 14, 15 Clinically, IFN-α–based treatment combined with ribavirin is the major therapeutic choice available for chronic HCV infection. Because IFNs are a critical component of the innate defense against viral infection, HCV may have a strategy to counteract IFN-mediated host defense mechanism(s). Many lines of evidence suggest that HCV interferes with IFN signaling pathway and expression of IFN-stimulated genes.4 However, little information is available about reciprocal interaction between HCV and intracellular IFN-α in the hepatic cells, the primary target for HCV infection.

One of the major obstacles for the investigation of innate host defense against HCV replication is the lack of cell models. The establishment of the HCV replicon system16, 17 has greatly facilitated the analysis of the dynamics of HCV replication and virus-host interaction. The studies using HCV replicon system are beginning to shed light on HCV interaction with the host cell and the IFN system.15 A major question that needs to be addressed in the immunopathogenesis of HCV infection is how HCV can persist in the hepatic cells. To address this issue, we investigated the interplay between HCV and intracellular IFN-α in the HCV replicon system.

Abbreviations

HCV, hepatitis C virus; IFN, interferon; IRF, interferon regulatory factor; JAK-STAT: Janus kinase (JAK) signal transducer and activation of transcription (STAT); ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-stimulated response element; ISGs, interferon stimulatory genes; 2′-5′ OAS, 2′-5′ oligoadenylate synthetase; PKR, the double-stranded RNA (dsRNA)-dependent protein kinase; NS, nonstructural; DMEM, Dulbecco's modified Eagle medium; HRP, horseradish peroxidase; RT-PCR, reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; LPS, lipopolysaccharide; FCS, fetal calf serum; HHV-8, human herpesvirus 8; NDV, Newcastle disease virus.

Materials and Methods

Cell Culture.

Human primary hepatocytes were purchased from Cambrex Bio Science Rockland, Inc. (Rockland, ME) and cultured according to the protocol provided by the manufacturer. HCV replicon cell line (Huh.8) was obtained from Dr. Charles Rice (The Rockefeller University and Apath, L.L.C., St. Louis, MO).17 The FCA-1 cell line was obtained from Dr. Christoph Seeger (Fox Chase Cancer Center, Philadelphia, PA).18 Huh7 is the parental cell line of the HCV replicon cell lines. Huh7.5 is a Huh.8 cell clone in which HCV self-replicating subgenomic RNA has been eliminated by prolonged treatment with IFN-α.19 HepG2, a human hepatocellular carcinoma cell line, was obtained from American Type Culture Collection (ATCC, Manassas, VA). These cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco-BRL, Grand Island, NY) as previously described.20 For all experiments, functionally endotoxin-free media and reagents were used. All cell cultures were checked for mycoplasma contamination by MycoAlert Mycoplasma Detection Kit (Cambrex).

Antibodies.

Rabbit polyclonal antibody against actin was purchased from Sigma-Aldrich Co. (St. Louis, MO). Mouse monoclonal antibody against HCV NS5A was purchased from Virostat (Portland, ME). Mouse monoclonal antibodies against HCV NS4A and 4B were purchased from Virogen (Watertown, MA). Rabbit polyclonal antibodies against HCV NS3, NS5B were gifts obtained from Dr. Bill Sun (Thomas Jefferson University, Philadelphia, PA). Mouse monoclonal antibody against IFN-α/β receptor was purchased from Chemicon International, Inc. (Catalog no. MAB1155, Temecula, CA).21 Rabbit polyclonal antibodies against IRF-3 and IRF-7 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody and goat anti-rabbit IgG were purchased from Jackson Immune Research Labs (West Grove, PA).

Plasmids.

The plasmids (pIFN-A4-luc, pIFN-A1-luc, pIFN-A14-luc, and wild-type IRF-7) and control plasmids (pFLAG-CMV-2 and pGL3) have been previously described.9 The HCV NS3, NS3-4A, NS4AB, NS4B, NS5A, NS5B plasmids, and control plasmid (pcDNA 3.1) were obtained from Dr. Michael Gale (University of Texas Southwestern Medical Center, Dallas, TX).3

Reverse Transcription Polymerase Chain Reaction and Real Time Reverse Transcription Polymerase Chain Reaction Analysis.

Total RNA was extracted from human primary hepatocytes, Huh7, HepG2, and HCV replicon cells using Tri-Reagent (Molecular Research Center, Cincinnati, OH) as described.22 Total cellular RNA (1 μg) was subjected to reverse transcription using the reverse transcription system from Promega (Madison, WI), with specific primers (antisense) for IFN-α, HCV, and IRF-7 gene for 1 hour at 42°C. The reaction was terminated by incubating the reaction mixture at 99°C for 5 minutes and then kept at 4°C. The cDNA served as a template for polymerase chain reaction (PCR) amplification. IRF-7 gene primers: 5′-CTG TGG TGG TGG GAC AGC TGC-3′ (sense) and 5′-CCC CAC GCG TGC TGT TCG GAG-3′ (anti-sense). β-actin gene primers: 5′-ATG TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3′ (sense) and 5′-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3′ (anti-sense). The thermal cycling conditions were designed as follows: 95°C 8 minutes, followed by 40 cycles of 95°C 30 seconds, 60°C 30 seconds, 72°C 30 seconds, and elongation at 72°C for 7 minutes. After PCR amplification, the samples were electrophoresed on ethidium bromide-stained 3% NuSieve 3:1 agarose gel (FMC BioProducts, Rockland, ME).

The HCV real time reverse transcription (RT)-PCR assay that we have developed22 was used for the quantification of HCV RNA. HCV genome primers: 5′-CGG GAG AGC CAT AGT GGT CTG CG-3′ (S130) and 5′-CTC GCA AGC ACC CTA TCA GGC AGT A-3′ (AS311). The sequence probe (molecular beacon, MB) of HCV is: 5′-FAM-GCG AGC CAC CGG AAT TGC CAG GAC GAC CGC TCG C-DABCYL-3′. A standard curve of HCV was generated with 10-fold dilutions of HCV 5′-NCR RNA control that had been pre-quantified by a spectrophotometer (Eppendorf Scientific, Inc., Westbury, NY). Thermal cycling conditions were designed as follows: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

The real time RT-PCR for the quantification of IFN-α mRNA was performed with the iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). IFN-α gene primers: 5′-TTT CTC CTG CCT GAA GGA CAG-3′ (sense) and 5′-GCT CAT GAT TTC TGC TCT GAC A-3′ (antisense). Real-time PCR reaction mixtures contained 12.5 μL of 2X SYBR Green Supermix, 0.5 μL of each primer (final concentration is 400 nmol/L), and 2 μL template cDNA; water was added to a final volume of 25 μL. Samples were subjected to the following thermal cycling conditions: 95°C 3 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 1 minute. The final step consisted of a decrease of 0.5°C every 10 seconds (80 times). This additional step allowed us to check the melting temperature of the formed amplicons and thus the specificity of the primers. The amplification results were visualized and analyzed using the software MyiQ provided with the thermocycler (iCycler iQ real time PCR detection system; Bio-Rad Laboratories). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels of the samples in the same plate were analyzed by the real time RT-PCR to normalize the mRNA contents among the samples tested. GAPDH gene primers were 5′-GGT GGT CTC CTC TGA CTT CAA CA-3′ (sense) and 5′-GTT GCT GTA GCC AAA TTC GTT GT-3′ (anti-sense).

Transfection, Luciferase, and IFN-α Small Interfering RNA Silencing Assays.

Transfection of plasmid DNA was carried out with FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) as recommended by the manufacturer. Luciferase activities in cell lysates were quantified using a Luciferase Assay System (Promega) and a TD-20/20 Luminometer (Turner Designs, Inc., Sunnyvale, CA).20 The results were presented as relative light units (RLU) normalized by the total proteins in the cell lysates. For IFN-α promoter-luciferase reporter experiments, the transfection was performed in 24-well plates, in which each well contained 105 cells in 1 mL DMEM. The cells were transfected with the plasmids (pIFN-A4-luc, pIFN-A1-luc, pIFN-A14-luc, wild-type IRF-7, HCV NS3, NS3-4A, NS4AB, NS4B, NS5A, and NS5B) using FuGENE 6 at a ratio of FuGENE 6: plasmid 6:1 (μL:μg) in a total volume of 50 μL serum-free cell culture media. The empty vector for HCV NS genes containing constructs were used as the control and included in parallel for each transfection experiment. IFN-α small interfering RNA (siRNA), and scramble siRNA were designed as follows: IFN siRNA, CUC CUG CCU GAA GGA CAG AUU (sense), UCU GUC CUU CAG GCA GGA GAA (antisense). Scramble siRNA (catalog no. 1022076, QIAGEN Inc., Valencia, CA), UUC UCC GAA CGU GUC ACG U dTdT (sense), ACG UGA CAC GUU CGG AGA A dTdT (anti-sense).

Both IFN-α siRNA and scramble siRNA were prepared by QIAGEN Inc. For IFN-α siRNA or scramble siRNA transfection experiments, the cells were transfected with IFN-α siRNA or scramble siRNA with a ratio of RNAiFect Reagent (QIAGEN Inc.): siRNA 15 μL: 2.5 μg in a total volume of 100 μL of serum-free cell culture media according to the protocol provided by the manufacturer.

Immunoassays.

For assessment of actin, IRF-3, IRF-7, HCV NS 3, NS4A, NS 4B, NS5A, and NS5B protein expression, the hepatic cells were harvested 48 hours after transfection or after subculture. The total proteins (∼50 μg) were extracted from the cells, quantitated, and subjected to Western blot assay as described.20 For immunofluorescent evaluation of HCV NS5A protein, Huh7 cells transfected with the plasmid containing HCV NS5A gene were cultured on glass coverslips (12 mm) in a 24-well plate for 48 hours. The cells were fixed with 75% ice-cold acetone and then pretreated with a blocking solution for 30 minutes. The coverslips were then incubated with a mouse monoclonal anti-HCV NS5A Ab (1:100) in blocking solution at room temperature for 60 minutes, and subsequently incubated with HRP-conjugated goat–anti-mouse IgG antibody (1:200) for 30 minutes and followed by Fluorophore Tyramide (NEN Life Science Products Inc., Boston, MA) working solution for 15 minutes (in dark). The coverslips were washed 3 times with 1× phosphate-buffered saline (PBS), mounted in vectorshield (Vector Labs, Burlingame, CA), and viewed with a fluorescence microscope (Zeiss, Germany). ELISA kits for IFN-α were purchased from PBL Biomedical Laboratories (Piscataway, NJ), and the assay was performed according to the protocol provided by the manufacturer.23

Results

HCV Suppresses IFN-α.

In an initial set of experiments, we examined whether human primary hepatocytes and the hepatic cell lines (Huh7 and HepG2) express intracellular IFN-α. As shown in Fig. 1, these cells spontaneously expressed IFN-α mRNA and released IFN-α protein. We then examined whether differential expression of IFN-α occurs between the HCV replicon cells and their parental cells (Huh7). Spontaneous IFN-α production was significantly suppressed in the HCV replicon cells at both mRNA (Fig. 1A) and protein (Fig. 1B) levels, as demonstrated by the real time RT-PCR and ELISA. To rule out the possibility that lipopolysaccharide (LPS) contamination in fetal calf serum (FCS) contributed to the observed intracellular IFN-α expression in the hepatic cells, we maintained the cells in the media with or without FCS. There was no significant difference in IFN-α expression between the hepatic cells cultured with FCS and the cells cultured without FCS (data not shown). In addition, treating the cells with polymyxin B did not affect the levels of intracellular IFN-α in the hepatic cells (data not shown).

Figure 1.

Constitutive IFN-α expression in the human hepatic cells. (A) IFN-α mRNA expression in primary human hepatocytes and the hepatic cell lines. Total cellular RNA extracted from the cell cultures was subjected to IFN-α real time RT-PCR (SYBR Green). The data shown are the mean ± SD of triplicate cultures, representative of 3 independent experiments. (B) IFN-α protein levels in culture supernatants of primary human hepatocytes and the hepatic cell lines. Huh7, HepG2, human primary hepatocytes, and the HCV replicon cells (Huh.8 and FCA-1) were cultured for 48 hours, and the supernatants were harvested for IFN-α ELISA. The results shown are the mean ± SD of triplicate cultures. IFN, interferon; RT-PCR, reverse transcription polymerase chain reaction.

Intracellular IFN-α Is a Powerful HCV Inhibitor.

Although exogenous IFN-α inhibits HCV replicon expression,17, 18 little information is available about the role of hepatocyte-released IFN-α in HCV suppression. We hypothesized that intracellular IFN-α produced by the hepatic cells has the ability to suppress HCV replicon expression. Three experimental strategies were used to address this hypothesis. First, we used IFN-α siRNA to purge intracellular IFN-α gene expression in the hepatic cells. The hepatic cells transfected with IFN-α siRNA showed decreased expression of IFN-α protein in comparison with the cells transfected with scramble siRNA (Fig. 2A). This decreased IFN-α expression by IFN-α siRNA corresponded to increased HCV RNA expression in the replicon cells (Fig. 2B). Second, we examined whether the activation of intracellular IFN-α expression by IRF-7 inhibits HCV RNA expression. IRF-7 has a critical role in rapid induction of type I IFN expression, a central event in establishing the innate antiviral response.8, 24, 25 As shown in Fig. 3A, IRF-7 induced intracellular IFN-α protein expression in a dose-dependent fashion, which corresponded to the decrease of HCV RNA in the replicon cells (Fig. 3B). The antibodies to IFN-α receptors partially blocked IRF-7–mediated reduction of HCV RNA expression (Fig. 3B). Third, we used the antibodies to IFN-α receptor to block the autocrine pathway of intracellular IFN-α, which interferes with the biological function of intracellular IFN-α. The replicon cells (Huh.8 and FCA-1) incubated with the antibody against IFN-α receptor expressed 3- to 4-fold higher levels of HCV RNA in comparison with the control cells (Fig. 3B). The effectiveness of the antibody to IFN-α receptor was validated by its blocking effect on the exogenous IFN-α (100 IU/mL, R&D Systems Inc., Minneapolis, MN) that significantly inhibited HCV RNA expression (data not shown).

Figure 2.

Effect of IFN-α siRNA on IFN-α and HCV RNA expression. (A) Effect of IFN-α siRNA on IFN-α protein expression. Huh7 and the HCV replicon cells (Huh.8 and FCA-1) were transfected with IFN-α siRNA (2.5 μg) or scramble siRNA (2.5 μg) and then cultured for 48 hours. The supernatants were harvested from the cell cultures for IFN-α ELISA. The results shown are the mean ± SD of triplicate cultures, representative of 3 independent experiments. (B) Effect of IFN-α siRNA on HCV RNA expression in the replicon cells. The HCV replicon cells (Huh.8 and FCA-1) were transfected with IFN-α siRNA or scramble siRNA and cultured for 48 hours. Total cellular RNA extracted from the cells was then subjected to the real time RT-PCR assays for HCV RNA expression. The results shown are the mean ± SD of triplicate cultures, representative of 3 independent experiments. IFN, interferon; siRNA, small interfering RNA; HCV, hepatitis C virus; RT-PCR, reverse transcription polymerase chain reaction.

Figure 3.

Effect of IRF-7 or anti–IFN-α receptor antibody on expression of IFN-α and HCV in the replicon cells. (A) Effect of IRF-7 on IFN-α protein expression. Huh.8 and FCA-1 cells were transfected with the plasmid-containing IRF-7 gene (1-5 μg). The supernatants were harvested from the cell cultures 48 hours after transfection for IFN-α ELISA. (B) Effect of anti–IFN-α receptor antibody on IRF-7–mediated inhibition of HCV mRNA expression. Huh.8 and FCA-1 cells were pretreated with or without anti–IFN-α receptor antibody (50 μg/mL) for 1 hour, then transfected with or without 2 μg plasmid containing IRF-7 gene. Total RNA was extracted for the real-time RT-PCR 48 hours after transfection. The data are expressed as HCV RNA expression (Fold) relative to control cells transfected with the control plasmid. The results shown are the mean ± SD of triplicate cultures, representative of 3 independent experiments. IRF, interferon regulatory factor; IFN, interferon; HCV, hepatitis C virus; RT-PCR, reverse transcription polymerase chain reaction.

IRF-7–Induced IFN-α Promoter Activity Is Restricted in HCV Replicon Cells.

In the investigation of the mechanisms involved in HCV-mediated suppression of IRF-7 action on IFN-α, we examined whether IRF-7–induced IFN-α promoter activation is inhibited in the HCV replicon cells. We cotransfected the cells with the IRF-7 gene plasmid and the IFN-α promoter plasmid (IFNA4). IFN-α promoter was activated efficiently by the introduction of IRF-7 gene plasmid into Huh7 cells (Fig. 4). By contrast, IRF-7–induced IFN-α promoter activity was significantly suppressed in Huh.8 and FCA-1 cells (Fig. 4). This restriction, however, was restored in IFN-cured Huh7.5 cells (Fig. 4).

Figure 4.

Comparison of IRF-7–induced IFN-α promoter activation in different hepatic cells. The hepatic cells were co-transfected with or without the plasmid containing IRF-7 gene and the plasmid containing IFN-α promoter (IFNA4) linked to a luciferase gene for 48 hours. Luciferase activities were determined in the cell extracts. The results shown are the mean ± SD of triplicate cultures, representative of 3 independent experiments. IRF, interferon regulatory factor; IFN, interferon.

HCV Inhibits IRF-7 Expression.

To investigate the molecular mechanism(s) responsible for HCV-mediated suppression of endogenous IFN-α, we examined whether IRF-7 expression were restricted in the HCV replicon cells. The HCV replicon cells (Huh.8 and FCA-1) expressed lower basal levels of IRF-7 mRNA (Fig. 5A) and protein (Fig. 5B) than those in Huh7 cells. This suppression of IRF-7 mRNA and protein, however, was not observed in IFN-cured Huh7.5 cells (Fig. 5). We also examined IRF-3 protein expression in these cells. The levels of IRF-3 protein in the HCV replicon cells were compatible with those in Huh7 cells (Fig. 5B).

Figure 5.

IRF-7 and IRF-3 expression in the hepatic cells. (A) IRF-7 mRNA expression in the hepatic cells. Total cellular RNA extracted from the indicated hepatic cells was subjected to RT-PCR for IRF-7 and β-actin RNA detection. β-Actin served as a control to ensure that RNA amounts used from the cells were equal. The data shown are representative of 3 experiments. (B) IRF-7 and IRF-3 protein expression in the hepatic cells. Equal amounts (50 μg) of total proteins extracted from the indicated hepatic cells were subjected to Western blot assay using the antibodies specific to IRF-7, or IRF-3, or actin. Arrows indicate the position of IRF-7, IRF-3, or actin. One representative result of 2 independent experiments is shown. IRF, interferon regulatory factor; RT-PCR, reverse transcription polymerase chain reaction.

HCV NS Proteins Suppress IFN-α Expression.

To determine which HCV protein(s) plays a major role in HCV-mediated downregulation of endogenous IFN-α in the hepatic cells, we transfected Huh7 cells with the plasmids containing the HCV genes that encode nonstructural proteins and with the plasmids containing either IFN-α promoter (A4) or IRF-7 gene. Huh7 cells, when transfected with HCV NS gene-containing plasmids, expressed HCV NS proteins as demonstrated by western blot assay (Fig. 6A). As expected, the vector plasmid without HCV NS genes had no effect on IRF-7–mediated IFN-α promoter activity (Fig. 6B). The expression of the HCV NS3, NS4AB, and NS4B proteins had little impact on IRF-7–induced IFN-α promoter activation (Fig. 6B); however, the expression of HCV NS3-4A, NS5A, and NS5B in Huh7 cells resulted in suppression of IRF-7–induced IFN-α promoter activation (Fig. 6B). Among these IFN-α suppressing proteins, NS5A was the strongest inhibitor of IFN-α promoter activation (Fig. 6B). In addition, HCV NS5A protein expression in Huh7 cells (Fig. 7A) inhibited endogenous IFN-α expression (Fig. 7B). Because IFN-α has multiple genotypes, we also examined the impact of NS5A on 3 IFN-α gene promoters (A1, A4, and A14). NS5A had the ability to suppress IRF-7–mediated activation of all 3 IFN-α promoters (Fig. 7C).

Figure 6.

Effect of HCV NS proteins on IRF-7–induced IFN-α promoter activity. (A) HCV NS protein expression in Huh7 cells. Equal amounts (30 μg) of total proteins extracted from Huh7 cells transfected with the plasmid containing HCV NS genes as indicated were subjected to Western blot assay using the antibodies specific to HCV NS3, NS 4A, NS4B, NS 5A, NS5B, and actin. Huh.8 cells were used as positive control (Pos). (B) Effect of HCV NS protein expression on IRF-7–induced IFN-α promoter activation. Huh7 cells co-transfected with the plasmids containing IFN-α promoter-luciferase gene and IRF-7 gene were also transfected with the plasmids containing HCV NS genes or control plasmid (vector) as indicated. The activation of IFN-α promoter was determined by luciferase activity at 48 hours after transfection. The results shown are the mean ± SD of triplicate cultures, representative of 3 independent experiments. HCV, hepatitis C virus; IFN, interferon.

Figure 7.

Effect of HCV NS5A protein on IFN-α expression and IRF-7–induced IFN-α promoter activity. (A) Immunoflurescence staining of HCV NS5A protein expression in Huh 7 cells. Huh7 cells were transfected with the plasmid containing NS5A gene and cultured for 48 hours. The cells then were stained with the antibody to NS5A protein and viewed with a fluorescence microscope (original magnification ×100). (B) Effect of NS5A on IFN-α mRNA expression in Huh7 cells. Huh7 cells were transfected with the plasmid containing NS5A gene or control plasmid (vector). IFN-α mRNA expression was analyzed by IFN-α real-time RT-PCR (SYBR Green) at 48 hours after tranfection with NS5A gene. (C) Effect of NS5A on IRF-7–induced IFN-α promoter activity in Huh7 cells. Huh7 cells co-transfected with the plasmids containing IRF-7 gene and IFN-α promoters (IFNA4, IFNA1, and IFNA14) were also transfected with the plasmid containing NS5A gene or control plasmid (vector). Luciferase activity was determined in the cell extracts at 48 hours after transfection. The results shown in (B) and (C) are the mean ± SD of triplicate cultures, representative of 3 independent experiments. HCV, hepatitis C virus; IFN, interferon; RT-PCR, reverse transcription polymerase chain reaction.

Discussion

The interaction of HCV and host innate immune response is an important determinant of the outcome of HCV infection. One of the critical elements in the innate host defense mechanism against viral infection is IFN-α.8, 26 IFN-α is the major clinical treatment for chronic HCV infection and inhibits HCV RNA expression in the HCV replicon cells.17, 18 IFN may act directly through its antiviral mechanisms or through its regulatory effect on innate and adaptive immune responses. Because IFN-α is a mediator of innate immune response against viral infection, HCV may have immune evasion strategies that circumvent or suppress the IFN response.15, 27, 28 HCV-encoded proteins, such as envelope protein E2, NS5A, and core proteins, interact with several steps of IFN signaling pathway.4, 29–34 Our observation that HCV has the ability to suppress intracellular hepatocyte-released IFN-α both quantitatively and functionally provides a potential mechanism by which HCV can persist in the host cells. These observations are also supported by the clinical findings that IFN-α levels are lower in the hepatic biopsy samples from HCV-infected subjects compared with the samples from non–HCV-infected subjects35, 36 and that intrahepatic IFN-α mRNA levels in HCV-infected subjects were significantly lower than those in nonalcoholic steatohepatitis.37

A weak IFN-α/β signal by the constitutively produced IFN-α/β is critical for the capacity to elicit strong innate immune responses against viruses.38 We demonstrated that, in the absence of stimuli, both primary hepatocytes and hepatoma cell lines (Huh7 and HepG2) constitutively produced IFN-α at a relative high level. This finding provides a possible explanation as to why the human hepatic cells are not permissive for HCV infection and replication in vitro. This spontaneous IFN-α expression in the hepatic cells is unlikely to be caused by LPS contamination, because all the media and reagents used for the cell cultures were endotoxin-free. Our data raise a major question: if our in vitro observation is applicable to the in vivo situation, how is HCV able to establish in vivo initial replication in the hepatic cells that spontaneously produce IFN-α? Thus, further studies to define in vivo role of the endogenous IFN-α in suppressing HCV replication in the hepatic cells are essential.

In an attempt to determine whether IFN-α produced by the hepatic cells has anti-HCV activity, we used IFN-α siRNA to purge IFN-α gene expression in the HCV replicon cells. Silencing the IFN-α gene in the HCV replicon cells resulted in a 4- to 5-fold increase of HCV RNA. The anti-HCV ability of endogenous IFN-α was demonstrated further by the observation that the activation of IFN-α gene expression by IRF-7 led to a decrease of HCV RNA expression. Moreover, the blockade of endogenous IFN-α binding to its receptors by the antibody against IFN-α receptors promoted HCV replicon expression. The anti-IFN-α receptor antibody partially blocked IRF-7–mediated HCV replicon inhibition (Fig. 3B), indicating additional effects of IRF-7 on HCV replicon expression. IRF-7 not only induces IFN-α, but also activates many ISGs, among which PKR, 2′-5′OAS, and the Mx protein have been well characterized for their antiviral activities.4 In addition, IRF may directly inhibit HCV replication. This speculation is supported by a recent report that IRF-7 can directly inhibit human herpesvirus 8 (HHV-8) replication.39 These data support the possibility that other anti-HCV factor(s) induced by IRF-7 also play a role in the observed HCV replicon inhibition. These findings indicated that endogenous IFN-α indeed has a critical role in controlling HCV replication in the hepatic cells. Although the levels of endogenous IFN-α are significantly suppressed in the HCV replicon cells, these low levels of endogenous IFN-α maintain viral suppression. Further suppression of endogenous IFN-α production and its function by IFN-α siRNA or by the blockage of IFN-α binding to its receptor resulted in a significant increase of HCV RNA in the HCV replicon cells. These data provide strong evidence that endogenous IFN-α is a critical self-defense mechanism against HCV in the hepatic cells.

Induction of IFN expression in response to viral infection requires posttranslational modification of transcription factors, including IRF-3 and IRF-7.10, 11, 40 These factors play a key role in initiating the cellular antiviral response. Recently, Foy et al.3 showed that HCV NS3/4A serine protease blocks the phosphorylation and effector action of IRF-3. In addition to IRF-3, IRF-7 is the master regulator of type I interferon–dependent immune responses,24 and overexpression of IRF-7 stimulates IFN gene expression in the absence of virus infection.9 IRF-7 amplifies the expression of other IFN genes that are not induced during the early stage of viral infection.10, 11, 41 Our investigation of the mechanism of HCV action on IFN-α showed that HCV inhibits basal level expression of IRF-7 at both mRNA and protein levels and that IRF-7–induced IFN-α promoter activation was restricted in the replicon cells. This HCV-mediated inhibition of IRF-7 expression and restriction of IRF-7–induced IFN-α promoter activation were not observed in IFN-α–cured Huh7.5 cells. Because Foy et al.3, 42 showed that HCV NS3/4A protease can inhibit virus- and retinoic acid–inducible gene I (RIG-I)–mediated IRF-3 phosphorylation and activation, we examined the basal levels of IRF-3 expression in the replicon cells. Our data showed that basal level of IRF-3 protein expression is not affected in the replicon cells in comparison with Huh7 cells (Fig. 5B). Thus, IRF-3 is unlikely to have a role in HCV-mediated endogenous IFN-α inhibition in the replicon cells. These findings provide a possible molecular mechanism whereby HCV inhibits IFN-α expression in hepatic cells.

HCV-encoded proteins, including envelope protein E2, NS5A, and core proteins, are immunosuppressive.4, 30, 33, 34, 43, 44 Among these HCV immunosuppressive proteins, NS5A has the ability to modulate a number of cell-cycle regulatory genes,45, 46 and has been implicated in the interference of IFN-mediated antiviral functions.6 NS5A inhibits IFN-induced, double-stranded RNA-activated protein kinase,31, 32 which mediates the antiviral effects of IFN. It also inhibits IFN-induced STAT/JAK signaling in human osteosarcoma cells, in epithelioid (Hela), and in murine fibroblasts (L929).47 In agreement with these findings, our data show that NS5A plays a major role in HCV-mediated suppression of endogenous IFN-α expression in hepatic cells. Our finding that NS5A suppressed IRF-7–induced IFN-α promoter activation is in disagreement with the observations of Ghosh et al., who showed that NS5A activates IFN-α promoter in HepG2 cells, with an associated increase of IRF-3.46 The discrepancy between our findings and the report by Ghosh et al. may be due to the differences in the reagents used for IFN-α promoter activation. In our study, we used IRF-7 as an inducer, whereas Ghosh et al. activated IFN-α promoter with Newcastle disease virus (NDV). It is possible that the interaction of NDV or NDV proteins with HCV NS5A may enhance IFN-α promoter activation. Nevertheless, HCV NS5A is likely to be a major contributor in HCV-mediated modulation of host innate defense mechanisms in the hepatic cells. In addition to NS5A, we showed that NS5B is also involved in suppression of IRF-7–mediated IFN-α promoter activation. NS5B has been identified as an RNA-dependent RNA polymerase that is responsible for the synthesis of negative-strand HCV RNA.48 Inhibition of NS5B has been long considered an attractive target for therapeutic intervention in HCV-infected patients.48, 49 Our data suggest that NS5B may have a role in interfering IFN signaling pathway. Future studies are needed to examine the mechanism involved in NS5B-mediated inhibition of the IFN signaling pathway. Finally, in support of the studies by Foy et al.42 and Breiman et al.,50 we showed that NS3/4A protease had the ability to inhibit IRF-7–induced IFN-α promoter activation (Fig. 6). This finding is not surprising and is even expected because NS3/4A can inhibit the signaling pathways leading to the activation of the IRF-3 and IRF-7.42, 50

In conclusion, our study demonstrating that HCV has the ability to undermine intracellular innate immunity of the host cells provides a plausible mechanism responsible for HCV persistence in the hepatic cells. Our data, in conjunction with the reports by Foy et al.3 and Wang et al.,39 suggest that HCV not only interferes with the IRF-3–mediated early protein pathway of type I IFNs, but also inhibits IRF-7–mediated IFN-α activation in the hepatic cells. Thus, HCV uses complex mechanisms to target multiple steps of the type I IFN signaling pathway in the hepatic cells, which results in significant suppression of endogenous IFN-α, a critical self-defense weapon used by the hepatic cells. To further elucidate the mechanism(s) implicated in HCV-mediated immune-modulating pathways—in particular, those related to the IFN pathway—we must develop an HCV permissive cell model as well as new targets for novel approaches toward preventive or therapeutic intervention of HCV disease.

Acknowledgements

The authors thank Dr. Charles Rice (The Rockefeller University, New York, NY) for providing Huh7, Huh7.5, and Huh.8 cell lines. We also thank Dr. Christoph Seeger (Fox Chase Cancer Center, Philadelphia, PA) for providing FCA-1 cell line. We thank Dr. Michael Gale (University of Texas Southwestern Medical Center, Dallas, TX) for providing the HCV NS3, NS3-4A, NS4AB, NS4B, NS5A, and NS5B plasmids.

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