Inhibition of RIG-I-Dependent Signaling to the Int
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病菌学杂志 2005年第6期
Unité Hépacivirus, Institut Pasteur, Paris, France
Lady Davis Institute for Medical Research and Departments of Microbiology & Immunology and Medicine, McGill University, Montreal, Canada
Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka
Department of Tumor Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Bunkyo-ku, Japan
ABSTRACT
Interferon (IFN) is one important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor (TLR)-dependent and -independent mechanisms. As part of its pathogenic strategy, hepatitis C virus (HCV) interferes with the innate immune response and induction of IFN-? via the HCV NS3/4A protease activity which inhibits phosphorylation of IRF-3, a key transcriptional regulator of the IFN response. In the present study, we demonstrate that inhibition by the protease occurs upstream of the noncanonical IKK-related kinases IKK and TBK-1, which phosphorylate IRF-3, through partial inhibition of the TLR adapter protein TRIF/TICAM1-dependent pathway. Use of TRIF–/– mouse embryo fibroblasts however revealed the presence of a TRIF-independent pathway involved in IFN induction that was also inhibited by NS3/4A. Importantly, we show that NS3/4A can strongly inhibit the ability of the recently described RIG-I protein to activate IFN, suggesting that RIG-I is a key factor in the TRIF-independent, NS3/4A-sensitive pathway. Expression of IFN signaling components including IKK, TBK-1, TRIF, and wild type or constitutively active forms of RIG-I in the HCV replicon cells resulted in IFN-? promoter transactivation, with IKK displaying the highest efficiency. Subsequently, overexpression of IKK resulted in 80% inhibition of both the positive and negative replicative strands of the HCV replicon. The partial restoration of the capacity of the host cell to transcribe IFN-? indicates that IKK expression is able to bypass the HCV-mediated inhibition and restore the innate antiviral response.
INTRODUCTION
The cellular innate immune response is triggered in response to a variety of pathogens, such as bacteria or viruses, and is essential to limit the initial spread of these pathogens. Microbial agents are recognized through specific motifs or pathogen-associated molecular patterns by different members of the Toll-like receptor (TLR) family. After pathogen-associated molecular pattern recognition, the cytosolic Toll/interleukin (IL)-1 receptor domain (TIR) of the TLRs interacts with adaptor proteins, including Myd88, TIRAP/Mal, and TRIF, and lead sto activation of mitogen-activated protein kinases, NF-B, and IRF-3 transcription factors by several distinct or overlapping signaling pathways (6).
Alpha-beta interferon (IFN-/?) is one of the important elements of the innate immune response. The initial signaling events in IFN activation involve at least three distinct TLR components: TLR3, TLR4, and TLR7, which are activated in response to viral double-stranded RNA (2), lipopolysaccharide (28), and single-stranded viral RNA (16), respectively. In response to double-stranded RNA, the cytoplasmic TIR domain of TLR3 recruits the adapter molecule TICAM-1/TRIF (65), while with lipopolysaccharide, the TLR4 receptor complex recruits the adapter molecule TICAM2/TRAM, which may serve as a bridging adapter to recruit TICAM-1/TRIF (17, 44). Whereas TLR3 andTLR4 act through MyD88-independent pathways, the recently described TLR7 response to single-stranded RNA appears to use the MyD88 and Mal adapter proteins.
IFN induction requires the phosphorylation of the transcription factors IRF-3 and IRF-7 through an amplification process in which IRF-3 is required in an early phase and IRF7 in a late phase (35, 41). Two kinases, TBK-1 (TANK-binding kinase1), also known as T2K or NAK (10, 50, 62), and IKK (46), initially isolated as IKK-i (58), were recently shown to be responsible for IRF-3 and IRF7 phosphorylation (18, 57). TBK-1 associates with the N terminal domain of TRIF, suggesting a relatively direct pathway to IRF-3/IRF7 activation (54). On the other hand, TRIF also associates with TRAF6 to mediate activation of NF-B and mitogen-activated protein kinases (54). Ultimately, the TLR-dependent pathways converge at the level of the classical IKK/IKK? complex to activate NF-B and at the level of TBK-1 and IKK to activate IRF-3.
Several human pathogenic viruses have evolved strategies to inhibit the early signaling events leading to IFN activation at distinct steps in the pathway, illustrating the importance of IFN production in the early host response to viral infection. The NS1 protein of influenza virus, the E3L protein of vaccinia virus, and the Ebola virus VP35 protein all inhibit IRF-3 activation (7, 59, 61); the human papillomavirus 16 E6 oncoprotein interacts directly with IRF-3 and facilitates degradation (51), and the human herpesvirus 8 IRF homologue blocks IRF-3 recruitment of the CBP and p300 coactivators (34). Recently, the viral protease NS3/4A encoded by hepatitis C virus (HCV) has been added to the list of viral inhibitors of the IRF-3 phosphorylation and IFN induction pathway (20). The NS3/4A protein is responsible for cleavage of the majority of the HCV nonstructural proteins after translation of the single polyprotein precursor from the 9.6-kb positive-strand RNA genome (4).
HCV is an enveloped positive-strand virus which belongs to the genus Hepacivirus in the Flaviviridae family (15, 52) and is a serious human pathogen that infects 3% of the world population. Approximately 20% of HCV-infected individuals recover from acute infection, and the cell-mediated immune response has been recognized as the major factor allowing this recovery. In contrast, a defective immune response is associated with persistent infection in 80% of infected individuals, leading to the development of chronic hepatitis infection and the occurrence of cirrhosis in 0.5 to 30% of cases and development of hepatocellular carcinoma at a rate of 1 to 3% per year (reviewed in reference 24). The current standard treatment is pegylated IFN and ribavirin, although positive results are obtained in only 40 to 60% cases, depending on the HCV genotype. A significant fraction of HCV-infected patients are therefore resistant to IFN treatment, and particular attention has focused on the ability of HCV to interfere with the IFN system, with the goal of improving therapeutic intervention.
In addition to its direct antiviral effect in cells through the JAK/STAT-mediated induction of several genes, IFN-/? is important for the maturation and activation of dendritic cells and thus links the innate immune pathway to the adaptive immune pathway (33). The ability of HCV to inhibit the early events of the IFN induction pathway may therefore represent one of the mechanisms by which virus infection compromises the host immune response and favors the establishment of chronic infection.
The objective of the present study was to investigate the molecular basis of NS3/4A interference with IRF-3 activation. We demonstrate that the IFN signaling pathway is inhibited in the presence of NS3/4A in an HCV replicon cell line and delineate that both TRIF-dependent and TRIF-independent pathways are inhibited by NS3/4A. Interestingly, the recently described RIG-I protein which has been implicated in TLR-independent, virus- and double-stranded RNA-mediated induction of IFN expression (66) is a major target of inhibition for NS3/4A. IKK expression partially restored the innate antiviral response in HCV-expressing cells. These results have important implications for virus-host interactions in HCV pathogenesis.
MATERIALS AND METHODS
Plasmids. The pcDNA3/AMP/zeo/flag (IKKwt), pcDNA3/AMP/zeo/flag (IKKK38A), pcDNA3/AMP/zeo/flag (IKKC), pcDNA3/AMP/zeo/flag (wild-type TBK-1), pcDNA3/AMP/zeo/flag (TBK-1 K38A), pCMV-flagIRF3, IFN-?-pGL3, pEF-flag-RIG-I, and pEF-flag-RIG-I plasmids were described previously (57,66). pcDNA1/AMP (NS5A) was a gift of G. Duverlie (Centre Hospitalier Universitaire-H?pital Sud, Amiens, France). The NS5A sequence was initially cloned by PCR from HCVJ (genotype 1b) serum. The pCMV-TRIF-flag, pRK5-TRAF6, pRK5 TRAF2, and pcDNA3.1-TANK expression plasmids (12) were a kind gift of K. Fitzgerald (University of Massachusetts Medical School, Worcester).
HR'TRIPU3(CMV), HR'TRIPU3 (CMV)-NS3/4A and HR'TRIPU3 (CMV)-NS3. The HR'TRIPU3 (CMV)-NS3/4A plasmid was constructed with the lentiviral vector HR'TRIPU3(CMV) GFP (67) in order to express NS3/4A, not only after transfection of cell lines but also eventually after transduction of primary hepatocytes. The HR'TRIPU3(CMV)GFP plasmid was first cut with BamHI and XhoI to remove green fluorescent protein (GFP). An adaptor sequence was then inserted with the sense oligonucleotide, 5'-GATCCCGGGAAGGGGTTCC, and the antisense oligonucleotide, 5'-TCGAGGAACCCCTTCCCGG, to recreate the BamHI and XhoI sites and create a SmaI site in addition.
The sequence coding for NS3 was amplified from a vaccine-derived construct expressing NS2 to 5B (a gift from C.Wychowsky; CNRS-UPR 2511, IBL, Lille, France) with the sense primer 5'-CGCGGATCCGCCACCATGGCGCCCATCACGGCGTACGCC-3', presenting a BamHI restriction site and an ATG start codon immediately upstream of the beginning of the NS3 sequence, and the antisense primer 5'-CGGGGTACCGATATCTTACGTGACGACCTCCAGGTC-3', containing the TAA stop codon at the end of the NS3 coding sequence and an additional KpnI site. The amplified product was subcloned into PCR2.1 TOPO, cut out with BamHI and KpnI (after verification of sequence) and inserted into HR'TRIPU3(CMV) previously digested by the same enzymes. NS4A was then amplified from the same vaccine construct with the sense primer 5'-ACTACAGTTAGGCTACGAGCG-3', starting at position 4603 in the HCV NS3 sequence, which allows us to include an adjacent SmaI site present in the NS3 sequence at position 4634 of NS3, and the antisense primer: 5'-CGGGGTACCGATATCTTAGCACTCTTCCATCTCATC-3', corresponding to the 3' end of NS4A with an additional TAA stop codon and a KpnI site. After subcloning into PCR 2.1 TOPO and sequence verification, the PCR product was cut out with SmaI and KpnI and inserted into HR'TRIPU3 (CMV-NS3) digested by the same enzymes.
HR'TRIPU3 CMV-NS5A. the NS5A coding sequence was amplified from pcDNA1/AMP-NS5A, with the sense primer 5'-TCCCCGGGCCAGCCATGGGTTCCGGCTCGTGG-3', designed to contain a SmaI restriction site, and the antisense primer 5'-CGGGGTACCTCAGCAGCAGACGACGTC-3', designed to contain a KpnI restriction site. After subcloning in the vector PCR 2.1 TOPO and verification of the sequence, the NS5A cDNA was cut out by SmaI and KpnI digestion and inserted into the HR'TRIPU3(CMV) vector previously digested by the same enzymes.
Cell culture. HEK 293T cells were cultured in Glutamax1-Dulbecco's modified Eagle's medium (with 110 mg of sodium pyruvate/liter and 4,500 mg of glucose/liter; Gibco BRL) containing 10% heat-inactivated fetal bovine serum, 50 U of penicillin G/ml, and 50 μg of streptomycin/ml. The Huh-7 cell line was cultured in the same medium with addition of nonessential amino acids (Gibco BRL).
For the preparation of the full-length HCV replicon cell line, in vitro transcription was first performed on the plasmid pFK-I398/neo/Core-3' containing the SfiI fragment from replicon 5.1 provided by R. Bartenschlager (48); 2 μg of the RNA was then introduced by electroporation into 2 x 106 Huh7 cells. After 24 h, the cells were cultured for 2 days in medium containing 100 μg of G418 (PAA Laboratories, Linz, Austria) per ml and then for 8 days in medium containing 400 μg of G418 per ml and subsequently grown in medium containing 100 μg of G418 per ml. Approximately 35 colonies were selected, and one of the clones (Huh-7Rep) was further isolated by limiting dilution, amplified, and used for subsequent experiments. Murine embryonic fibroblasts (MEFs) from wild-type mice (MEF TRIF+/+) and TRIF knockout mice (TRIF–/–) were described previously (64). MEF cells were cultured in minimal essential medium (Wisent) containing 10% heat-inactivated fetal bovine serum and antibiotics.
Virus. The Sendai virus (2,000 HAU/ml; Cantell strain, ATCC VR-907 Parainfluenza 1) was a gift of Veronique Kruys (Université Libre de Bruxelles, Brussels, Belgium) (45) (Fig. 1 and 4) or obtained from Charles River Laboratories (Fig. 3).
Immunofluorescence. Cells were incubated in eight-well chamber slides (Lab-Tek II; Nalge Nunc International, Naperville, Ill.). Fixation, incubation with different antibodies, and immunofluorescence analysis were as described (21). Observation was performed on a Zeiss Axioplan2 microscope with a x40 lens and digital pictures were collected with a Cool Snap HQ camera (Photometrics) and the Simple PCI software (Compix, Inc.).
Transfection and reporter assay. Eighteen to 24 h before transfection, the cells were seeded in 24-well plates (Falcon; Becton Dickinson) at different concentrations depending on the cell types, 100,000/well for HEK293T cells and Huh-7Rep cells, 80,000/well for Huh-7 cells. Three hours before transfection, the cells were washed and incubated in 300 μl of culture medium deprived of antibiotics and containing 10% serum. The required amount of Lipofectamine 2000 (from a 1 mg/ml stock solution; InVitrogen) was diluted with 75 μl of antibiotic-free and serum-free Dulbecco's modified Eagle's medium and mixed for 20 min at room temperature with 75 μl of the same medium containing DNA in the required combinations, to give a final ratio of 1.2 μl of Lipofectamine/μg of DNA.
For each sample, the DNA mix was adjusted to 1/10th of the total content with a plasmid expressing Rous sarcoma virus ?-galactosidase for normalization of the data; 150 μl of the medium covering the cells was removed and replaced with the 150 μl of the Lipofectamine-DNA mix. After 3 h of incubation with the mix, the cells were washed twice with 10% serum-antibiotic-free medium and further incubated in this medium in the presence of antibiotics. At 24 h after transfection, the culture medium was aspirated and the cells were washed once in phosphate-buffered saline. The cells in each well were then scraped and lysed in 200 μl of luciferase lysis buffer (25 mM Tris-phosphate [pH 7.6], 8 mM MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100, 15% glycerol) per well. For each sample, 50 μl was mixed with 50 μl of Bright-Glo luciferase assay reagent (Promega) and analyzed for luciferase activity with the Reporter Microplate Luminometer (Turner designs).
A different 50-μl aliquot was also taken from each sample and analyzed for ?-galactosidase activity with a ?-galactosidase enzyme assay system (Promega). For the luciferase reporter assay in MEF cells, 3 x 104 cells/well were seeded in 12-well plates 20 h before transfection with the Fugene reagent (Roche) according to the manufacturer's instructions with 200 ng of IFN-?-pGL3, 300 ng of PRLTK (Renilla luciferase, internal control), and 1 μg of expression plasmids. At 8 h posttransfection, cells were left untreated or infected with Sendai virus for 16 h. Cells were harvested and lysed in passive lysis buffer and assayed for reporter gene activities with the dual-luciferase reporter assay system (Promega).
Real-time RT-PCR analysis. Total cellular RNA was extracted by acid-guanidinium thiocyanate-phenol chloroform with RNAble (Eurobio, Les Ulis, France) and according to the manufacturer's instructions. The sequences of the primers used for the different reverse transcription (RT)-PCRs are shown in Table 1. The sets of primers were either synthesized with published sequences for IFN-? (42) and HCV (HCV.I, HCV.II, KY78, and KY80 [13]) or designed with the software LC Probe design (Roche) by choosing primers on each side of an intron when possible for human glyceraldehhyde-3-phosphate dehydrogenase (GAPDH), gi:182980 and human IKK, gi: 7288877. The reverse transcription step was performed on 1 μg of total RNA as described (13) with the rTth polymerase (Applied Biosystems) and one of the antisense primers (IKK AS1, GAPDH LC.3, IFN-? [-], or HCV.I for the HCV positive strand and HCV.II for the HCV negative strand).
For each reverse transcription, GAPDH reverse transcription was performed in the same tube. The cDNA was then purified with the High Pure PCR product purification kit (Roche Applied Science, Indianapolis, Ind.) in a final volume of 50 μl. PCR amplification was performed at an annealing temperature of 65°C with 2 to 5 μl of the purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA master SYBR Green kit (Roche), 0.5 μM each primer, and different concentrations of MgCl2 (5 mM for IKK and 4 mM for GAPDH). Quantitative PCR was performed on Light Cycler apparatus with SYBR Green primers, as described (13). Standard curves were established with 10-fold serial dilutions of the plasmid pcDNA3/AMP/zeo/flag (IKKwt), of plasmids containing a GAPDH or an IFN-? amplicon, or of HCV synthetic plus and minus strand RNAs transcribed from the pSP73HCV1_584 plasmid provided by C. Sureau (INTS, Paris, France). The measured amounts of each mRNA were normalized to the amounts of GAPDH mRNA.
Immunoblot analysis. HEK 293T cells were seeded at 3.5 x 106 cells/100-mm plate and transfected after 24 h with Lipofectamine 2000. At 24 h after transfection, the cells were scraped in their culture medium, pelleted by centrifugation, washed twice in phosphate-buffered saline, and the final pellet was either stored at –80°C before further processing or processed immediately. The cell pellets were resuspended in 400 μl of lysis buffer L (10 mM Tris-HCl [pH 7.6], 50 mM KCl, 100 mM NaCl, 1 mM EDTA, 0,5% NP-40, 10 mM 2-mercaptoethanol, 1% aprotinin, 0.2 mM phenylmethylsulfonyl fluoride) containing 1 mM sodium orthovanadate (Na3VO4), 10 mM para-nitrophenyl phosphate, 10 mM ?-glycerophosphate, and 5 mM sodium fluoride (NaF) as phosphatase inhibitors. After 20 min of incubation on ice, the cell extracts were centrifuged at 12 000 x g for 20 min at 4°C, transferred to other tubes, and stored at –80°C.
The protein concentration was determined by the Bradford method; 45 μg of protein from each sample was loaded onto 12.5% sodium dodecyl sulfate-acrylamide gels followed by transfer to polyvinylidene difluoride membranes. Membranes were blocked for 1 h at 37°C with phosphate-buffered saline containing 5% dry milk (blocking buffer). The detection of the different proteins was carried out by overnight incubation of the membrane at 4°C with the required dilution of specific antibodies. After washing, the membrane was further incubated for 1 h with blocking buffer at 1/2,000 dilution of horseradish peroxidase-coupled secondary antibodies (Amersham, Buckinghamshire, United Kingdom). Protein bands were detected by ECL chemoluminescence (Amersham).
For IRF-3 analysis, 10 μg of protein was loaded on 7.5% sodium dodecyl sulfate-acrylamide gels and immunoblot analysis was performed as described (56). For the detection of proteins in immunoblots, we used an anti-NS5A monoclonal antibody (Biodesign) and anti-NS3 monoclonal antibody (a gift from D. Moradpour; University of Freiburg) for the HCV proteins, anti-Flag monoclonal antibody (M2; Sigma) for the detection of Flag-tagged IKK and Flag-tagged TBK-1, and rabbit anti-IRF-3 (Sc9082; Santa Cruz) for the detection of IRF-3. The detection of phosphorylated IRF-3 was performed with polyclonal antibodies specific for IRF-3 phosphorylated at Ser396 prepared as described (56). The protein loading control was performed with monoclonal antiactin antibody (Sigma).
RESULTS
IFN production is inhibited in an HCV replicon-expressing cell. HCV replicon clones have proven useful to study HCV-host cell interactions, even though these cultures cannot produce HCV viral particles (8,9,25,27,36,37,48,68). We previously generated an HCV replicon cell line, referred to as Huh-7 Rep (48), that correctly expresses HCV RNA (see Fig. 5) and HCV proteins, as detected by immunofluorescence (Fig. 1A). Since Huh-7 cells may vary in their ability to produce IFN after virus infection or double-stranded RNA treatment (22, 29, 31, 47), we initially demonstrated that the parental Huh-7 cells could induce IFN expression in response to Sendai virus infection. Induction of the endogenous IFN-? gene was detected by RT-PCR in the Huh-7 cells beginning at 4 h after infection and the amount of IFN-? mRNA increased approximately 30-fold by 24 h. In contrast to Huh-7 cells, expression of IFN-? mRNA remained at basal levels in the Huh-7 Rep cells, indicating strong inhibition of IFN expression in the presence of the HCV replicon (Fig. 1B). Furthermore, in transient transfections, expression of an IFN-? promoter-luciferase reporter construct was significantly reduced (fivefold stimulation) in the Huh-7 Rep cells compared to the original Huh-7 cells (50-fold stimulation) (Fig. 1C).
HCV NS3/4A protease does not affect IKK/TBK-1-mediated IRF-3 phosphorylation. To begin to analyze the level at which the HCV NS3/4A protease interfered with IRF-3 phosphorylation and activation (20), the effect of NS3/4A on the activity of the IKK/TBK-1 kinases was evaluated in a series of transient transfections in HEK 293T cells. IRF-3 phosphorylation, as detected by one-dimensional immunoblot, was observed in the presence of wild-type IKK but not in the presence of the catalytically inactive mutant IKK K38A (Fig. 2D). IRF-3 was unaffected by the presence of either NS3/4A or NS5A, in terms of both total protein expression and phosphorylation levels (Fig. 2D); furthermore, the capacity of IKK to phosphorylate IRF-3 was also unaffected by coexpression of the viral proteins (Fig. 2B and C). Similarly, TBK-1 also phosphorylated IRF-3 in the presence of NS3/4A or NS5A (Fig. 2A). NS3/4A and NS5A had no effect on the ability of IKK or TBK-1 to mediate IFN-? promoter induction by IRF-3. Expression of IKK or TBK-1 resulted in 100- and 500-fold stimulation of IRF-3-mediated induction of IFN-? (data not shown). Taken together, these results argue that IKK and TBK-1 are not targeted by the HCV NS3/4A or NS5A protein.
NS3/4A affects IFN signaling via TRIF-dependent and TRIF-independent pathways. Since IKK and TBK-1 were not directly affected by NS3/4A (Fig. 2), the possibility that HCV protease may target upstream components of IFN signaling was examined by analyzing the effect of NS3/4A on the TRIF adaptor, an essential component of the TLR3-double-stranded RNA-dependent pathway. Transfection of HEK 293T cells with a TRIF-expressing vector in the absence of NS3/4A resulted in >500-fold stimulation of the IFN-? promoter; addition of NS3/4A reduced TRIF-mediated activation by 50%, suggesting that the viral protease may interfere, albeit moderately, with a TRIF-dependent pathway leading to IFN promoter transactivation (Fig. 3A). The inhibition of TRIF by NS3/4A was confirmed by real-time RT-PCR of endogenous IFN-? mRNA, and inhibition was more pronounced (fourfold inhibition), whereas NS5A had no inhibitory effect on TRIF-mediated activity (Fig. 3B).
Interestingly, in TRIF+/+ and TRIF–/– mouse embryo fibroblasts, Sendai virus infection stimulated the IFN-? promoter to similar levels and NS3/4A again inhibited IFN induction by 50% (Fig. 3C), indicating that the TRIF-dependent pathway was not the only pathway targeted by NS3/4A. Recently, retinoic acid-inducible gene I (RIG-I) was identified as an intracellular sensor of viral infection. Presumably, double-stranded RNA binds and activates the RIG-I RNA helicase domain, leading to a change in protein conformation that permits recruitment of signaling adaptors to the caspase recruitment domain (CARD) of RIG-I and results in the activation of IRF-3 and NF-B (66). Furthermore, the RIG-I-mediated pathway to IFN activation functions independently of the TLR3/TRIF pathway. Since the data in Fig. 3C suggested the existence of a TRIF-independent pathway that was sensitive to NS3/4A, activation of the IFN promoter by RIG-I and inhibition by NS3/4A was examined with a constitutively active form of RIG-I (RIG-I), which contains only the CARD signaling domain.
In the absence of NS3/4A (or NS5A as a control), the IFN promoter was strongly activated by RIG-I, as previously shown (66). Strikingly, coexpression of RIG-I with NS3/4A resulted in complete inhibition of IFN-? promoter activity. Inhibition was specific, since no inhibition was observed in the presence of NS5A (Fig. 3D). Coexpression of RIG-1 with dominant negative IKK—IKKC—completely abolished RIG-I-mediated activation of the IFN promoter, indicating that RIG-I is an important upstream component of the signaling pathway leading to IRF-3 activation via IKK and/or TBK-1. Together, these data demonstrate that NS3/4A interferes to a minor extent with the TRIF-dependent pathway but has a major effect upon the RIG-I pathway. Although both the N terminus of TRIF and the CARD region of RIG-I contain potential cleavage sites for NS3/4A (63), no cleavage of either protein by NS3/4A was detected by immunoblot (data not shown).
Restoration of IFN activation in HCV replicon-expressing cells. Since NS3/4A can interfere with IFN induction at the level of TRIF and RIG-I, we next sought to determine whether different components of the IFN signaling pathway could restore IFN activation in the presence of HCV. Huh-7 Rep cells were transfected with different signaling components and adaptors: IKK, TBK-1, TRIF, TANK, TRAF6, and TRAF2, and analyzed for activation of IFN promoter activity (10, 14). In an initial experiment, expression of IKK, TBK-1, and TRIF resulted in stimulation of the IFN-?-luciferase reporter (53-, 20-, and 20-fold, respectively), whereas TANK, TRAF2, and TRAF6 were inactive (1.1- to 1.6-fold induction) (data not shown)
Next, the capacity of IKK, TRIF, TBK-1, RIG-I, and RIG-I to restore the IFN signaling pathway was compared in Huh-7 Rep (Fig. 4B) and Huh-7 cells (Fig. 4A), in the presence or absence of Sendai virus infection. Expression of IKK, TBK-1, TRIF, and RIG-I alone stimulated the IFN-? promoter, whether the cells expressed the HCV replicon (Fig. 4B) or not (Fig. 4A). In Huh-7 cells, the induction of the IFN-? promoter by the different signaling components was between 110- and 250-fold (Fig. 4A), with the exception of wild-type RIG-I, which provided only 20-fold activation in Huh-7 cells. However, in Huh-7 cells, RIG-I expression together with Sendai virus infection resulted in a dramatic 1,000-fold stimulation of the IFN-? promoter (Fig. 4A).
In Huh-7 Rep cells, the levels of transactivation by IKK, TRIF, TBK-1, RIG-I, and RIG-I were generally lower (Fig. 4B), with the exception of IKK, which produced the strongest IFN-? transactivation effect (190-fold) compared with TBK-1 and TRIF (35-fold in each case). RIG-I alone, in the absence of virus infection, also induced IFN-? promoter activation about 85-fold in Huh-7 Rep cells, whereas wild-type RIG-I had no stimulatory effect on its own (Fig. 4B). Again, Sendai virus infection together with wild-type RIG-I resulted in a 120-fold induction of the IFN-? promoter (Fig. 4B), clearly a strong activation but significantly less (about 8-fold) than that observed in Huh-7 cells. The lower level of RIG-I-mediated transactivation is likely related to the expression of NS3/4A in the Huh-7 Rep cells. Importantly, expression of IKK resulted in the most efficient activation of the IFN-? promoter in both Huh-7 and HCV replicon-expressing cells, and this IFN induction was related to kinase activity, since wild-type IKK expression but not kinase-dead IKK K38A induced specific Ser396 phosphorylation of IRF-3 (Fig. 4C).
Overexpression of IKK inhibits the HCV replicon and partially restores IFN induction. Since IKK produced the strongest stimulation of the IFN-? promoter activity and functioned independently of HCV-mediated inhibition of the TRIF and RIG-I pathways, we next investigated whether IKK overexpression interfered with the HCV replicon. The presence of HCV negative- or positive-strand RNA was measured by real-time RT-PCR at 24 to 72 h after IKK or IKK K38A transfection. HCV RNA levels in the Huh-7 Rep cells were comparable to those observed by others (48), with more than one log difference between the levels of positive and negative/replicative strand (6 x 107 and 1.7 x 106 copies/μg of total RNA) (5). A progressive inhibition of expression of both viral RNA strands was observed in cells overexpressing IKK, with 80% inhibition at 72 h (Fig. 5).
Inhibition of HCV RNA expression may reflect the production of low levels of IFN as a consequence of IKK expression; therefore, RT-PCR analysis was used to measure endogenous IFN-? RNA levels in the Huh-7 Rep cells. Cells expressing wild-type IKK but not IKK K38A exhibited a threefold increase in IFN-? mRNA at 24 h after transfection. Although the levels are low compared to Huh-7 cells infected with Sendai virus (Fig. 1), IKK expression appears to restore, at least in part, IFN induction in HCV-expressing cells.
DISCUSSION
In the present study, we demonstrate that inhibition of IFN induction by the HCV NS3/4A protease occurs upstream of the noncanonical IKK-related kinases IKK and TBK-1 and interferes with both RIG-I-mediated as well as the TRIF-mediated pathways leading to IRF-3 phosphorylation and activation. Furthermore, overexpression of IKK but not RIG-I could restore IFN induction and hence the ability of cells to mount an innate response against HCV. Finally, IKK expression inhibited the replication of an HCV replicon, an effect that was at least in part attributed to the restoration of IFN production.
The host cellular signaling response to virus infection likely reflects the activation of several redundant and nonredundant cascades that sense different viral pathogen-associated molecular patterns during the initial binding and subsequent entry of virus and then recognize viral nucleic acid structures such as single- and double-stranded RNA in the cytoplasm of infected cells. Thus, IFN induction in response to virus infection likely differs significantly from the double-stranded RNA or lipopolysaccharide response, which uses TLR3 and TLR4, respectively. Several observations support the diversity of the host response to virus: IFN induction by double-stranded RNA through TLR3 involves binding of TRIF to the intracellular domain of TLR3 and the subsequent activation of TBK-1 kinase, which culminates with IRF-3 activation (54); induction of IFN is abolished in TLR3–/–cells after poly(I)-poly(C) treatment but not after virus infection (26); and single-stranded RNA of vesicular stomatitis virus and Sendai virus is sensed through a distinct TLR7-Myd88-dependent pathway (40).
A crucial component of the TRIF-independent pathway was identified recently as the DexD/helicase RIG-I (66), a retinoic acid-inducible gene that encodes an adapter protein with a C-terminal RNA helicase domain as well as an N-terminal caspase recruitment domain (CARD) interaction module. The CARD domain alone is capable of stimulating IRF-3, NF-B, and IFN-? production, while the helicase activity appears to function in a regulatory capacity. At this point, it is thought that interaction of the helicase domain with viral RNA or double-stranded RNA may induce a conformational change and promote protein-protein interactions between the RIG-I CARD domain and other CARD-containing adaptor proteins, resulting in activation of TBK and IKK kinases.
The involvement of RIG-I in IFN induction was confirmed in the present study with two observations: the NS3/4A protease of HCV interfered with RIG-I-mediated IFN induction and RIG-I-mediated induction was also blocked by a dominant negative mutant of IKK. The exact mechanism by which NS3/4A prevents the RIG-I-mediated activation of signaling leading to IRF-3 phosphorylation is not known. It was not possible to detect RIG-I cleavage by NS3/4A, and there was no strong association between NS3/4A and RIG-I in coimmunoprecipitation assays (data not shown). Although NS3/4A strongly inhibited the ability of RIG-I to transactivate the IFN-? promoter in transient expression assays, RIG-I remained partly active in Huh-7 Rep cells, where NS3/4A expression occurred from the endogenous replicon; this apparent discrepancy suggests that NS3/4A interference may depend on the relative expression level of NS3/4A in cells.
IKK overexpression in the HCV replicon-expressing cells resulted in IFN-? promoter induction, and of the signaling components examined in the present study, IKK was the most efficient mediator in bypassing the HCV-induced blockade of IFN. The functional role of IKK in response to pathogens remains to be determined. IKK is expressed in several tissues and is particularly abundant in the thymus, spleen, and peripheral blood leukocytes. IKK shares over 60% identity with TBK-1 and limited (30%) identity with IKK and IKK? and can interact with itself through a coiled-coil multimerization domain (46), thus allowing autoactivation upon overexpression. IKK binds weakly to IKK and IKK? but may be functionally linked to the classical IKK complex through the association between the TANK and NEMO adaptor molecules (14). While IKK? can phosphorylate IB on Ser32 and Ser36, IKK phosphorylates Ser36 in vitro; furthermore, purified TBK-1 and IKK both phosphorylate the distal Ser in the sequence SxxxS. At this point the physiological relevance of this specificity is not known.
Another significant difference between IKK and TBK-1 as well as classical IKK and IKK? kinases is that IKK is an inducible kinase, with expression inducible in response to lipopolysaccharide in macrophages (58) or in response to phorbol myristate acetate in Jurkat T cells (46). Upregulation of IKK first requires the NF-B-mediated activation of the ? and forms of the C/EBP transcription factor family. In addition, IKK is necessary for the activation of C/EBP. Both C/EBP? and C/EBP bind to the IKK promoter, indicating that IKK regulates its own induction through a feedback loop (30). The ability of IKK to autoactivate as a result of homodimerization and to induce its own synthesis through phosphorylation of C/EBP (30) could contribute to the restoration of IFN induction in Huh-7 Rep cells through an amplification mechanism.
With Huh-7 Rep cells, a strong inhibition of IFN production was demonstrated in the presence of the replicon via NS3/4A-mediated inhibition of both the TRIF-dependent and RIG-I dependent pathways. In vivo, one of the primary evasive strategies of HCV may be to prevent the local activation of the IFN antiviral cascade in de novo-infected hepatocytes. Recently, low-level expression of IFN-?- and IFN--specific mRNAs was detected in the liver of HCV-infected patients, as assessed by semiquantitative PCR, but these levels were significantly lower than the levels found in patients suffering from liver damage unrelated to HCV (1).
HCV infects the liver and replicates predominantly in hepatocytes, although with poor efficiency. Direct evidence of its replication is the detection of negative-strand RNA recovered from hepatocytes prepared from in vivo HCV-infected chimpanzees (32) and from in vitro HCV-infected human primary hepatocytes (19, 53). There is also good evidence that HCV can infect cells of extrahepatic origin and particularly cells of hematopoietic origin, such as T and B lymphocytes and dendritic cells (reviewed in references 5, 55, and 60). Due to extensive blood flow, the liver is in contact with peripheral blood dendritic cells, and indeed dendritic cells were found in HCV-infected livers in the vicinity of the lymphatic vessels (23). Dendritic cells represent a heterogeneous population of cells of either myeloid or plasmacytoid lineage which are potent antigen-presenting cells and activators of the Th1/Th2 response (11).
Although HCV envelope glycoprotein was shown to bind to dendritic cells through the dendritic cell SIGN surface receptor (39, 49), it is still unclear whether HCV infection impairs dendritic cell maturation (3, 38, 43). HCV infection of dendritic cells could potentially abolish IFN production though early expression of NS3/4A. Such a mechanism could explain the failure of HCV-infected individuals to mount an efficient immune response, since production of IFN- is necessary for the maturation of dendritic cells. Because IKK can restore IFN induction in the presence of an HCV replicon, this kinase may be important in sustaining IFN production in the face of HCV infection. Identification of therapeutic agents capable of boosting IKK expression may be a beneficial antiviral strategy to explore, with important implications for HCV-infected patients.
ACKNOWLEDGMENTS
This work was supported in part by grants from the Agence Nationale pour la Recherche contre le Sida (ANRS) to E.F.M. and the Canadian Institutes for Health Research (CIHR), National Cancer Institute, and CANVAC, Network Centres of Excellence, to J.H. and R.L. A.B. was supported by a fellowship from the ANRS, N.G. by a postdoctoral fellowship from the Fonds pour la Recherche Scientifique du Quebec (FRSQ), R.L. by an FRSQ chercheur boursier, and J.H. by a CIHR Senior Investigator award.
We thank Céline Gracia, Julien Thorgue, Rachel Couderc, and Florence Bayard for excellent technical assistance and Klaus Schwamborn for critical review of the manuscript.
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Lady Davis Institute for Medical Research and Departments of Microbiology & Immunology and Medicine, McGill University, Montreal, Canada
Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka
Department of Tumor Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Bunkyo-ku, Japan
ABSTRACT
Interferon (IFN) is one important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor (TLR)-dependent and -independent mechanisms. As part of its pathogenic strategy, hepatitis C virus (HCV) interferes with the innate immune response and induction of IFN-? via the HCV NS3/4A protease activity which inhibits phosphorylation of IRF-3, a key transcriptional regulator of the IFN response. In the present study, we demonstrate that inhibition by the protease occurs upstream of the noncanonical IKK-related kinases IKK and TBK-1, which phosphorylate IRF-3, through partial inhibition of the TLR adapter protein TRIF/TICAM1-dependent pathway. Use of TRIF–/– mouse embryo fibroblasts however revealed the presence of a TRIF-independent pathway involved in IFN induction that was also inhibited by NS3/4A. Importantly, we show that NS3/4A can strongly inhibit the ability of the recently described RIG-I protein to activate IFN, suggesting that RIG-I is a key factor in the TRIF-independent, NS3/4A-sensitive pathway. Expression of IFN signaling components including IKK, TBK-1, TRIF, and wild type or constitutively active forms of RIG-I in the HCV replicon cells resulted in IFN-? promoter transactivation, with IKK displaying the highest efficiency. Subsequently, overexpression of IKK resulted in 80% inhibition of both the positive and negative replicative strands of the HCV replicon. The partial restoration of the capacity of the host cell to transcribe IFN-? indicates that IKK expression is able to bypass the HCV-mediated inhibition and restore the innate antiviral response.
INTRODUCTION
The cellular innate immune response is triggered in response to a variety of pathogens, such as bacteria or viruses, and is essential to limit the initial spread of these pathogens. Microbial agents are recognized through specific motifs or pathogen-associated molecular patterns by different members of the Toll-like receptor (TLR) family. After pathogen-associated molecular pattern recognition, the cytosolic Toll/interleukin (IL)-1 receptor domain (TIR) of the TLRs interacts with adaptor proteins, including Myd88, TIRAP/Mal, and TRIF, and lead sto activation of mitogen-activated protein kinases, NF-B, and IRF-3 transcription factors by several distinct or overlapping signaling pathways (6).
Alpha-beta interferon (IFN-/?) is one of the important elements of the innate immune response. The initial signaling events in IFN activation involve at least three distinct TLR components: TLR3, TLR4, and TLR7, which are activated in response to viral double-stranded RNA (2), lipopolysaccharide (28), and single-stranded viral RNA (16), respectively. In response to double-stranded RNA, the cytoplasmic TIR domain of TLR3 recruits the adapter molecule TICAM-1/TRIF (65), while with lipopolysaccharide, the TLR4 receptor complex recruits the adapter molecule TICAM2/TRAM, which may serve as a bridging adapter to recruit TICAM-1/TRIF (17, 44). Whereas TLR3 andTLR4 act through MyD88-independent pathways, the recently described TLR7 response to single-stranded RNA appears to use the MyD88 and Mal adapter proteins.
IFN induction requires the phosphorylation of the transcription factors IRF-3 and IRF-7 through an amplification process in which IRF-3 is required in an early phase and IRF7 in a late phase (35, 41). Two kinases, TBK-1 (TANK-binding kinase1), also known as T2K or NAK (10, 50, 62), and IKK (46), initially isolated as IKK-i (58), were recently shown to be responsible for IRF-3 and IRF7 phosphorylation (18, 57). TBK-1 associates with the N terminal domain of TRIF, suggesting a relatively direct pathway to IRF-3/IRF7 activation (54). On the other hand, TRIF also associates with TRAF6 to mediate activation of NF-B and mitogen-activated protein kinases (54). Ultimately, the TLR-dependent pathways converge at the level of the classical IKK/IKK? complex to activate NF-B and at the level of TBK-1 and IKK to activate IRF-3.
Several human pathogenic viruses have evolved strategies to inhibit the early signaling events leading to IFN activation at distinct steps in the pathway, illustrating the importance of IFN production in the early host response to viral infection. The NS1 protein of influenza virus, the E3L protein of vaccinia virus, and the Ebola virus VP35 protein all inhibit IRF-3 activation (7, 59, 61); the human papillomavirus 16 E6 oncoprotein interacts directly with IRF-3 and facilitates degradation (51), and the human herpesvirus 8 IRF homologue blocks IRF-3 recruitment of the CBP and p300 coactivators (34). Recently, the viral protease NS3/4A encoded by hepatitis C virus (HCV) has been added to the list of viral inhibitors of the IRF-3 phosphorylation and IFN induction pathway (20). The NS3/4A protein is responsible for cleavage of the majority of the HCV nonstructural proteins after translation of the single polyprotein precursor from the 9.6-kb positive-strand RNA genome (4).
HCV is an enveloped positive-strand virus which belongs to the genus Hepacivirus in the Flaviviridae family (15, 52) and is a serious human pathogen that infects 3% of the world population. Approximately 20% of HCV-infected individuals recover from acute infection, and the cell-mediated immune response has been recognized as the major factor allowing this recovery. In contrast, a defective immune response is associated with persistent infection in 80% of infected individuals, leading to the development of chronic hepatitis infection and the occurrence of cirrhosis in 0.5 to 30% of cases and development of hepatocellular carcinoma at a rate of 1 to 3% per year (reviewed in reference 24). The current standard treatment is pegylated IFN and ribavirin, although positive results are obtained in only 40 to 60% cases, depending on the HCV genotype. A significant fraction of HCV-infected patients are therefore resistant to IFN treatment, and particular attention has focused on the ability of HCV to interfere with the IFN system, with the goal of improving therapeutic intervention.
In addition to its direct antiviral effect in cells through the JAK/STAT-mediated induction of several genes, IFN-/? is important for the maturation and activation of dendritic cells and thus links the innate immune pathway to the adaptive immune pathway (33). The ability of HCV to inhibit the early events of the IFN induction pathway may therefore represent one of the mechanisms by which virus infection compromises the host immune response and favors the establishment of chronic infection.
The objective of the present study was to investigate the molecular basis of NS3/4A interference with IRF-3 activation. We demonstrate that the IFN signaling pathway is inhibited in the presence of NS3/4A in an HCV replicon cell line and delineate that both TRIF-dependent and TRIF-independent pathways are inhibited by NS3/4A. Interestingly, the recently described RIG-I protein which has been implicated in TLR-independent, virus- and double-stranded RNA-mediated induction of IFN expression (66) is a major target of inhibition for NS3/4A. IKK expression partially restored the innate antiviral response in HCV-expressing cells. These results have important implications for virus-host interactions in HCV pathogenesis.
MATERIALS AND METHODS
Plasmids. The pcDNA3/AMP/zeo/flag (IKKwt), pcDNA3/AMP/zeo/flag (IKKK38A), pcDNA3/AMP/zeo/flag (IKKC), pcDNA3/AMP/zeo/flag (wild-type TBK-1), pcDNA3/AMP/zeo/flag (TBK-1 K38A), pCMV-flagIRF3, IFN-?-pGL3, pEF-flag-RIG-I, and pEF-flag-RIG-I plasmids were described previously (57,66). pcDNA1/AMP (NS5A) was a gift of G. Duverlie (Centre Hospitalier Universitaire-H?pital Sud, Amiens, France). The NS5A sequence was initially cloned by PCR from HCVJ (genotype 1b) serum. The pCMV-TRIF-flag, pRK5-TRAF6, pRK5 TRAF2, and pcDNA3.1-TANK expression plasmids (12) were a kind gift of K. Fitzgerald (University of Massachusetts Medical School, Worcester).
HR'TRIPU3(CMV), HR'TRIPU3 (CMV)-NS3/4A and HR'TRIPU3 (CMV)-NS3. The HR'TRIPU3 (CMV)-NS3/4A plasmid was constructed with the lentiviral vector HR'TRIPU3(CMV) GFP (67) in order to express NS3/4A, not only after transfection of cell lines but also eventually after transduction of primary hepatocytes. The HR'TRIPU3(CMV)GFP plasmid was first cut with BamHI and XhoI to remove green fluorescent protein (GFP). An adaptor sequence was then inserted with the sense oligonucleotide, 5'-GATCCCGGGAAGGGGTTCC, and the antisense oligonucleotide, 5'-TCGAGGAACCCCTTCCCGG, to recreate the BamHI and XhoI sites and create a SmaI site in addition.
The sequence coding for NS3 was amplified from a vaccine-derived construct expressing NS2 to 5B (a gift from C.Wychowsky; CNRS-UPR 2511, IBL, Lille, France) with the sense primer 5'-CGCGGATCCGCCACCATGGCGCCCATCACGGCGTACGCC-3', presenting a BamHI restriction site and an ATG start codon immediately upstream of the beginning of the NS3 sequence, and the antisense primer 5'-CGGGGTACCGATATCTTACGTGACGACCTCCAGGTC-3', containing the TAA stop codon at the end of the NS3 coding sequence and an additional KpnI site. The amplified product was subcloned into PCR2.1 TOPO, cut out with BamHI and KpnI (after verification of sequence) and inserted into HR'TRIPU3(CMV) previously digested by the same enzymes. NS4A was then amplified from the same vaccine construct with the sense primer 5'-ACTACAGTTAGGCTACGAGCG-3', starting at position 4603 in the HCV NS3 sequence, which allows us to include an adjacent SmaI site present in the NS3 sequence at position 4634 of NS3, and the antisense primer: 5'-CGGGGTACCGATATCTTAGCACTCTTCCATCTCATC-3', corresponding to the 3' end of NS4A with an additional TAA stop codon and a KpnI site. After subcloning into PCR 2.1 TOPO and sequence verification, the PCR product was cut out with SmaI and KpnI and inserted into HR'TRIPU3 (CMV-NS3) digested by the same enzymes.
HR'TRIPU3 CMV-NS5A. the NS5A coding sequence was amplified from pcDNA1/AMP-NS5A, with the sense primer 5'-TCCCCGGGCCAGCCATGGGTTCCGGCTCGTGG-3', designed to contain a SmaI restriction site, and the antisense primer 5'-CGGGGTACCTCAGCAGCAGACGACGTC-3', designed to contain a KpnI restriction site. After subcloning in the vector PCR 2.1 TOPO and verification of the sequence, the NS5A cDNA was cut out by SmaI and KpnI digestion and inserted into the HR'TRIPU3(CMV) vector previously digested by the same enzymes.
Cell culture. HEK 293T cells were cultured in Glutamax1-Dulbecco's modified Eagle's medium (with 110 mg of sodium pyruvate/liter and 4,500 mg of glucose/liter; Gibco BRL) containing 10% heat-inactivated fetal bovine serum, 50 U of penicillin G/ml, and 50 μg of streptomycin/ml. The Huh-7 cell line was cultured in the same medium with addition of nonessential amino acids (Gibco BRL).
For the preparation of the full-length HCV replicon cell line, in vitro transcription was first performed on the plasmid pFK-I398/neo/Core-3' containing the SfiI fragment from replicon 5.1 provided by R. Bartenschlager (48); 2 μg of the RNA was then introduced by electroporation into 2 x 106 Huh7 cells. After 24 h, the cells were cultured for 2 days in medium containing 100 μg of G418 (PAA Laboratories, Linz, Austria) per ml and then for 8 days in medium containing 400 μg of G418 per ml and subsequently grown in medium containing 100 μg of G418 per ml. Approximately 35 colonies were selected, and one of the clones (Huh-7Rep) was further isolated by limiting dilution, amplified, and used for subsequent experiments. Murine embryonic fibroblasts (MEFs) from wild-type mice (MEF TRIF+/+) and TRIF knockout mice (TRIF–/–) were described previously (64). MEF cells were cultured in minimal essential medium (Wisent) containing 10% heat-inactivated fetal bovine serum and antibiotics.
Virus. The Sendai virus (2,000 HAU/ml; Cantell strain, ATCC VR-907 Parainfluenza 1) was a gift of Veronique Kruys (Université Libre de Bruxelles, Brussels, Belgium) (45) (Fig. 1 and 4) or obtained from Charles River Laboratories (Fig. 3).
Immunofluorescence. Cells were incubated in eight-well chamber slides (Lab-Tek II; Nalge Nunc International, Naperville, Ill.). Fixation, incubation with different antibodies, and immunofluorescence analysis were as described (21). Observation was performed on a Zeiss Axioplan2 microscope with a x40 lens and digital pictures were collected with a Cool Snap HQ camera (Photometrics) and the Simple PCI software (Compix, Inc.).
Transfection and reporter assay. Eighteen to 24 h before transfection, the cells were seeded in 24-well plates (Falcon; Becton Dickinson) at different concentrations depending on the cell types, 100,000/well for HEK293T cells and Huh-7Rep cells, 80,000/well for Huh-7 cells. Three hours before transfection, the cells were washed and incubated in 300 μl of culture medium deprived of antibiotics and containing 10% serum. The required amount of Lipofectamine 2000 (from a 1 mg/ml stock solution; InVitrogen) was diluted with 75 μl of antibiotic-free and serum-free Dulbecco's modified Eagle's medium and mixed for 20 min at room temperature with 75 μl of the same medium containing DNA in the required combinations, to give a final ratio of 1.2 μl of Lipofectamine/μg of DNA.
For each sample, the DNA mix was adjusted to 1/10th of the total content with a plasmid expressing Rous sarcoma virus ?-galactosidase for normalization of the data; 150 μl of the medium covering the cells was removed and replaced with the 150 μl of the Lipofectamine-DNA mix. After 3 h of incubation with the mix, the cells were washed twice with 10% serum-antibiotic-free medium and further incubated in this medium in the presence of antibiotics. At 24 h after transfection, the culture medium was aspirated and the cells were washed once in phosphate-buffered saline. The cells in each well were then scraped and lysed in 200 μl of luciferase lysis buffer (25 mM Tris-phosphate [pH 7.6], 8 mM MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100, 15% glycerol) per well. For each sample, 50 μl was mixed with 50 μl of Bright-Glo luciferase assay reagent (Promega) and analyzed for luciferase activity with the Reporter Microplate Luminometer (Turner designs).
A different 50-μl aliquot was also taken from each sample and analyzed for ?-galactosidase activity with a ?-galactosidase enzyme assay system (Promega). For the luciferase reporter assay in MEF cells, 3 x 104 cells/well were seeded in 12-well plates 20 h before transfection with the Fugene reagent (Roche) according to the manufacturer's instructions with 200 ng of IFN-?-pGL3, 300 ng of PRLTK (Renilla luciferase, internal control), and 1 μg of expression plasmids. At 8 h posttransfection, cells were left untreated or infected with Sendai virus for 16 h. Cells were harvested and lysed in passive lysis buffer and assayed for reporter gene activities with the dual-luciferase reporter assay system (Promega).
Real-time RT-PCR analysis. Total cellular RNA was extracted by acid-guanidinium thiocyanate-phenol chloroform with RNAble (Eurobio, Les Ulis, France) and according to the manufacturer's instructions. The sequences of the primers used for the different reverse transcription (RT)-PCRs are shown in Table 1. The sets of primers were either synthesized with published sequences for IFN-? (42) and HCV (HCV.I, HCV.II, KY78, and KY80 [13]) or designed with the software LC Probe design (Roche) by choosing primers on each side of an intron when possible for human glyceraldehhyde-3-phosphate dehydrogenase (GAPDH), gi:182980 and human IKK, gi: 7288877. The reverse transcription step was performed on 1 μg of total RNA as described (13) with the rTth polymerase (Applied Biosystems) and one of the antisense primers (IKK AS1, GAPDH LC.3, IFN-? [-], or HCV.I for the HCV positive strand and HCV.II for the HCV negative strand).
For each reverse transcription, GAPDH reverse transcription was performed in the same tube. The cDNA was then purified with the High Pure PCR product purification kit (Roche Applied Science, Indianapolis, Ind.) in a final volume of 50 μl. PCR amplification was performed at an annealing temperature of 65°C with 2 to 5 μl of the purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA master SYBR Green kit (Roche), 0.5 μM each primer, and different concentrations of MgCl2 (5 mM for IKK and 4 mM for GAPDH). Quantitative PCR was performed on Light Cycler apparatus with SYBR Green primers, as described (13). Standard curves were established with 10-fold serial dilutions of the plasmid pcDNA3/AMP/zeo/flag (IKKwt), of plasmids containing a GAPDH or an IFN-? amplicon, or of HCV synthetic plus and minus strand RNAs transcribed from the pSP73HCV1_584 plasmid provided by C. Sureau (INTS, Paris, France). The measured amounts of each mRNA were normalized to the amounts of GAPDH mRNA.
Immunoblot analysis. HEK 293T cells were seeded at 3.5 x 106 cells/100-mm plate and transfected after 24 h with Lipofectamine 2000. At 24 h after transfection, the cells were scraped in their culture medium, pelleted by centrifugation, washed twice in phosphate-buffered saline, and the final pellet was either stored at –80°C before further processing or processed immediately. The cell pellets were resuspended in 400 μl of lysis buffer L (10 mM Tris-HCl [pH 7.6], 50 mM KCl, 100 mM NaCl, 1 mM EDTA, 0,5% NP-40, 10 mM 2-mercaptoethanol, 1% aprotinin, 0.2 mM phenylmethylsulfonyl fluoride) containing 1 mM sodium orthovanadate (Na3VO4), 10 mM para-nitrophenyl phosphate, 10 mM ?-glycerophosphate, and 5 mM sodium fluoride (NaF) as phosphatase inhibitors. After 20 min of incubation on ice, the cell extracts were centrifuged at 12 000 x g for 20 min at 4°C, transferred to other tubes, and stored at –80°C.
The protein concentration was determined by the Bradford method; 45 μg of protein from each sample was loaded onto 12.5% sodium dodecyl sulfate-acrylamide gels followed by transfer to polyvinylidene difluoride membranes. Membranes were blocked for 1 h at 37°C with phosphate-buffered saline containing 5% dry milk (blocking buffer). The detection of the different proteins was carried out by overnight incubation of the membrane at 4°C with the required dilution of specific antibodies. After washing, the membrane was further incubated for 1 h with blocking buffer at 1/2,000 dilution of horseradish peroxidase-coupled secondary antibodies (Amersham, Buckinghamshire, United Kingdom). Protein bands were detected by ECL chemoluminescence (Amersham).
For IRF-3 analysis, 10 μg of protein was loaded on 7.5% sodium dodecyl sulfate-acrylamide gels and immunoblot analysis was performed as described (56). For the detection of proteins in immunoblots, we used an anti-NS5A monoclonal antibody (Biodesign) and anti-NS3 monoclonal antibody (a gift from D. Moradpour; University of Freiburg) for the HCV proteins, anti-Flag monoclonal antibody (M2; Sigma) for the detection of Flag-tagged IKK and Flag-tagged TBK-1, and rabbit anti-IRF-3 (Sc9082; Santa Cruz) for the detection of IRF-3. The detection of phosphorylated IRF-3 was performed with polyclonal antibodies specific for IRF-3 phosphorylated at Ser396 prepared as described (56). The protein loading control was performed with monoclonal antiactin antibody (Sigma).
RESULTS
IFN production is inhibited in an HCV replicon-expressing cell. HCV replicon clones have proven useful to study HCV-host cell interactions, even though these cultures cannot produce HCV viral particles (8,9,25,27,36,37,48,68). We previously generated an HCV replicon cell line, referred to as Huh-7 Rep (48), that correctly expresses HCV RNA (see Fig. 5) and HCV proteins, as detected by immunofluorescence (Fig. 1A). Since Huh-7 cells may vary in their ability to produce IFN after virus infection or double-stranded RNA treatment (22, 29, 31, 47), we initially demonstrated that the parental Huh-7 cells could induce IFN expression in response to Sendai virus infection. Induction of the endogenous IFN-? gene was detected by RT-PCR in the Huh-7 cells beginning at 4 h after infection and the amount of IFN-? mRNA increased approximately 30-fold by 24 h. In contrast to Huh-7 cells, expression of IFN-? mRNA remained at basal levels in the Huh-7 Rep cells, indicating strong inhibition of IFN expression in the presence of the HCV replicon (Fig. 1B). Furthermore, in transient transfections, expression of an IFN-? promoter-luciferase reporter construct was significantly reduced (fivefold stimulation) in the Huh-7 Rep cells compared to the original Huh-7 cells (50-fold stimulation) (Fig. 1C).
HCV NS3/4A protease does not affect IKK/TBK-1-mediated IRF-3 phosphorylation. To begin to analyze the level at which the HCV NS3/4A protease interfered with IRF-3 phosphorylation and activation (20), the effect of NS3/4A on the activity of the IKK/TBK-1 kinases was evaluated in a series of transient transfections in HEK 293T cells. IRF-3 phosphorylation, as detected by one-dimensional immunoblot, was observed in the presence of wild-type IKK but not in the presence of the catalytically inactive mutant IKK K38A (Fig. 2D). IRF-3 was unaffected by the presence of either NS3/4A or NS5A, in terms of both total protein expression and phosphorylation levels (Fig. 2D); furthermore, the capacity of IKK to phosphorylate IRF-3 was also unaffected by coexpression of the viral proteins (Fig. 2B and C). Similarly, TBK-1 also phosphorylated IRF-3 in the presence of NS3/4A or NS5A (Fig. 2A). NS3/4A and NS5A had no effect on the ability of IKK or TBK-1 to mediate IFN-? promoter induction by IRF-3. Expression of IKK or TBK-1 resulted in 100- and 500-fold stimulation of IRF-3-mediated induction of IFN-? (data not shown). Taken together, these results argue that IKK and TBK-1 are not targeted by the HCV NS3/4A or NS5A protein.
NS3/4A affects IFN signaling via TRIF-dependent and TRIF-independent pathways. Since IKK and TBK-1 were not directly affected by NS3/4A (Fig. 2), the possibility that HCV protease may target upstream components of IFN signaling was examined by analyzing the effect of NS3/4A on the TRIF adaptor, an essential component of the TLR3-double-stranded RNA-dependent pathway. Transfection of HEK 293T cells with a TRIF-expressing vector in the absence of NS3/4A resulted in >500-fold stimulation of the IFN-? promoter; addition of NS3/4A reduced TRIF-mediated activation by 50%, suggesting that the viral protease may interfere, albeit moderately, with a TRIF-dependent pathway leading to IFN promoter transactivation (Fig. 3A). The inhibition of TRIF by NS3/4A was confirmed by real-time RT-PCR of endogenous IFN-? mRNA, and inhibition was more pronounced (fourfold inhibition), whereas NS5A had no inhibitory effect on TRIF-mediated activity (Fig. 3B).
Interestingly, in TRIF+/+ and TRIF–/– mouse embryo fibroblasts, Sendai virus infection stimulated the IFN-? promoter to similar levels and NS3/4A again inhibited IFN induction by 50% (Fig. 3C), indicating that the TRIF-dependent pathway was not the only pathway targeted by NS3/4A. Recently, retinoic acid-inducible gene I (RIG-I) was identified as an intracellular sensor of viral infection. Presumably, double-stranded RNA binds and activates the RIG-I RNA helicase domain, leading to a change in protein conformation that permits recruitment of signaling adaptors to the caspase recruitment domain (CARD) of RIG-I and results in the activation of IRF-3 and NF-B (66). Furthermore, the RIG-I-mediated pathway to IFN activation functions independently of the TLR3/TRIF pathway. Since the data in Fig. 3C suggested the existence of a TRIF-independent pathway that was sensitive to NS3/4A, activation of the IFN promoter by RIG-I and inhibition by NS3/4A was examined with a constitutively active form of RIG-I (RIG-I), which contains only the CARD signaling domain.
In the absence of NS3/4A (or NS5A as a control), the IFN promoter was strongly activated by RIG-I, as previously shown (66). Strikingly, coexpression of RIG-I with NS3/4A resulted in complete inhibition of IFN-? promoter activity. Inhibition was specific, since no inhibition was observed in the presence of NS5A (Fig. 3D). Coexpression of RIG-1 with dominant negative IKK—IKKC—completely abolished RIG-I-mediated activation of the IFN promoter, indicating that RIG-I is an important upstream component of the signaling pathway leading to IRF-3 activation via IKK and/or TBK-1. Together, these data demonstrate that NS3/4A interferes to a minor extent with the TRIF-dependent pathway but has a major effect upon the RIG-I pathway. Although both the N terminus of TRIF and the CARD region of RIG-I contain potential cleavage sites for NS3/4A (63), no cleavage of either protein by NS3/4A was detected by immunoblot (data not shown).
Restoration of IFN activation in HCV replicon-expressing cells. Since NS3/4A can interfere with IFN induction at the level of TRIF and RIG-I, we next sought to determine whether different components of the IFN signaling pathway could restore IFN activation in the presence of HCV. Huh-7 Rep cells were transfected with different signaling components and adaptors: IKK, TBK-1, TRIF, TANK, TRAF6, and TRAF2, and analyzed for activation of IFN promoter activity (10, 14). In an initial experiment, expression of IKK, TBK-1, and TRIF resulted in stimulation of the IFN-?-luciferase reporter (53-, 20-, and 20-fold, respectively), whereas TANK, TRAF2, and TRAF6 were inactive (1.1- to 1.6-fold induction) (data not shown)
Next, the capacity of IKK, TRIF, TBK-1, RIG-I, and RIG-I to restore the IFN signaling pathway was compared in Huh-7 Rep (Fig. 4B) and Huh-7 cells (Fig. 4A), in the presence or absence of Sendai virus infection. Expression of IKK, TBK-1, TRIF, and RIG-I alone stimulated the IFN-? promoter, whether the cells expressed the HCV replicon (Fig. 4B) or not (Fig. 4A). In Huh-7 cells, the induction of the IFN-? promoter by the different signaling components was between 110- and 250-fold (Fig. 4A), with the exception of wild-type RIG-I, which provided only 20-fold activation in Huh-7 cells. However, in Huh-7 cells, RIG-I expression together with Sendai virus infection resulted in a dramatic 1,000-fold stimulation of the IFN-? promoter (Fig. 4A).
In Huh-7 Rep cells, the levels of transactivation by IKK, TRIF, TBK-1, RIG-I, and RIG-I were generally lower (Fig. 4B), with the exception of IKK, which produced the strongest IFN-? transactivation effect (190-fold) compared with TBK-1 and TRIF (35-fold in each case). RIG-I alone, in the absence of virus infection, also induced IFN-? promoter activation about 85-fold in Huh-7 Rep cells, whereas wild-type RIG-I had no stimulatory effect on its own (Fig. 4B). Again, Sendai virus infection together with wild-type RIG-I resulted in a 120-fold induction of the IFN-? promoter (Fig. 4B), clearly a strong activation but significantly less (about 8-fold) than that observed in Huh-7 cells. The lower level of RIG-I-mediated transactivation is likely related to the expression of NS3/4A in the Huh-7 Rep cells. Importantly, expression of IKK resulted in the most efficient activation of the IFN-? promoter in both Huh-7 and HCV replicon-expressing cells, and this IFN induction was related to kinase activity, since wild-type IKK expression but not kinase-dead IKK K38A induced specific Ser396 phosphorylation of IRF-3 (Fig. 4C).
Overexpression of IKK inhibits the HCV replicon and partially restores IFN induction. Since IKK produced the strongest stimulation of the IFN-? promoter activity and functioned independently of HCV-mediated inhibition of the TRIF and RIG-I pathways, we next investigated whether IKK overexpression interfered with the HCV replicon. The presence of HCV negative- or positive-strand RNA was measured by real-time RT-PCR at 24 to 72 h after IKK or IKK K38A transfection. HCV RNA levels in the Huh-7 Rep cells were comparable to those observed by others (48), with more than one log difference between the levels of positive and negative/replicative strand (6 x 107 and 1.7 x 106 copies/μg of total RNA) (5). A progressive inhibition of expression of both viral RNA strands was observed in cells overexpressing IKK, with 80% inhibition at 72 h (Fig. 5).
Inhibition of HCV RNA expression may reflect the production of low levels of IFN as a consequence of IKK expression; therefore, RT-PCR analysis was used to measure endogenous IFN-? RNA levels in the Huh-7 Rep cells. Cells expressing wild-type IKK but not IKK K38A exhibited a threefold increase in IFN-? mRNA at 24 h after transfection. Although the levels are low compared to Huh-7 cells infected with Sendai virus (Fig. 1), IKK expression appears to restore, at least in part, IFN induction in HCV-expressing cells.
DISCUSSION
In the present study, we demonstrate that inhibition of IFN induction by the HCV NS3/4A protease occurs upstream of the noncanonical IKK-related kinases IKK and TBK-1 and interferes with both RIG-I-mediated as well as the TRIF-mediated pathways leading to IRF-3 phosphorylation and activation. Furthermore, overexpression of IKK but not RIG-I could restore IFN induction and hence the ability of cells to mount an innate response against HCV. Finally, IKK expression inhibited the replication of an HCV replicon, an effect that was at least in part attributed to the restoration of IFN production.
The host cellular signaling response to virus infection likely reflects the activation of several redundant and nonredundant cascades that sense different viral pathogen-associated molecular patterns during the initial binding and subsequent entry of virus and then recognize viral nucleic acid structures such as single- and double-stranded RNA in the cytoplasm of infected cells. Thus, IFN induction in response to virus infection likely differs significantly from the double-stranded RNA or lipopolysaccharide response, which uses TLR3 and TLR4, respectively. Several observations support the diversity of the host response to virus: IFN induction by double-stranded RNA through TLR3 involves binding of TRIF to the intracellular domain of TLR3 and the subsequent activation of TBK-1 kinase, which culminates with IRF-3 activation (54); induction of IFN is abolished in TLR3–/–cells after poly(I)-poly(C) treatment but not after virus infection (26); and single-stranded RNA of vesicular stomatitis virus and Sendai virus is sensed through a distinct TLR7-Myd88-dependent pathway (40).
A crucial component of the TRIF-independent pathway was identified recently as the DexD/helicase RIG-I (66), a retinoic acid-inducible gene that encodes an adapter protein with a C-terminal RNA helicase domain as well as an N-terminal caspase recruitment domain (CARD) interaction module. The CARD domain alone is capable of stimulating IRF-3, NF-B, and IFN-? production, while the helicase activity appears to function in a regulatory capacity. At this point, it is thought that interaction of the helicase domain with viral RNA or double-stranded RNA may induce a conformational change and promote protein-protein interactions between the RIG-I CARD domain and other CARD-containing adaptor proteins, resulting in activation of TBK and IKK kinases.
The involvement of RIG-I in IFN induction was confirmed in the present study with two observations: the NS3/4A protease of HCV interfered with RIG-I-mediated IFN induction and RIG-I-mediated induction was also blocked by a dominant negative mutant of IKK. The exact mechanism by which NS3/4A prevents the RIG-I-mediated activation of signaling leading to IRF-3 phosphorylation is not known. It was not possible to detect RIG-I cleavage by NS3/4A, and there was no strong association between NS3/4A and RIG-I in coimmunoprecipitation assays (data not shown). Although NS3/4A strongly inhibited the ability of RIG-I to transactivate the IFN-? promoter in transient expression assays, RIG-I remained partly active in Huh-7 Rep cells, where NS3/4A expression occurred from the endogenous replicon; this apparent discrepancy suggests that NS3/4A interference may depend on the relative expression level of NS3/4A in cells.
IKK overexpression in the HCV replicon-expressing cells resulted in IFN-? promoter induction, and of the signaling components examined in the present study, IKK was the most efficient mediator in bypassing the HCV-induced blockade of IFN. The functional role of IKK in response to pathogens remains to be determined. IKK is expressed in several tissues and is particularly abundant in the thymus, spleen, and peripheral blood leukocytes. IKK shares over 60% identity with TBK-1 and limited (30%) identity with IKK and IKK? and can interact with itself through a coiled-coil multimerization domain (46), thus allowing autoactivation upon overexpression. IKK binds weakly to IKK and IKK? but may be functionally linked to the classical IKK complex through the association between the TANK and NEMO adaptor molecules (14). While IKK? can phosphorylate IB on Ser32 and Ser36, IKK phosphorylates Ser36 in vitro; furthermore, purified TBK-1 and IKK both phosphorylate the distal Ser in the sequence SxxxS. At this point the physiological relevance of this specificity is not known.
Another significant difference between IKK and TBK-1 as well as classical IKK and IKK? kinases is that IKK is an inducible kinase, with expression inducible in response to lipopolysaccharide in macrophages (58) or in response to phorbol myristate acetate in Jurkat T cells (46). Upregulation of IKK first requires the NF-B-mediated activation of the ? and forms of the C/EBP transcription factor family. In addition, IKK is necessary for the activation of C/EBP. Both C/EBP? and C/EBP bind to the IKK promoter, indicating that IKK regulates its own induction through a feedback loop (30). The ability of IKK to autoactivate as a result of homodimerization and to induce its own synthesis through phosphorylation of C/EBP (30) could contribute to the restoration of IFN induction in Huh-7 Rep cells through an amplification mechanism.
With Huh-7 Rep cells, a strong inhibition of IFN production was demonstrated in the presence of the replicon via NS3/4A-mediated inhibition of both the TRIF-dependent and RIG-I dependent pathways. In vivo, one of the primary evasive strategies of HCV may be to prevent the local activation of the IFN antiviral cascade in de novo-infected hepatocytes. Recently, low-level expression of IFN-?- and IFN--specific mRNAs was detected in the liver of HCV-infected patients, as assessed by semiquantitative PCR, but these levels were significantly lower than the levels found in patients suffering from liver damage unrelated to HCV (1).
HCV infects the liver and replicates predominantly in hepatocytes, although with poor efficiency. Direct evidence of its replication is the detection of negative-strand RNA recovered from hepatocytes prepared from in vivo HCV-infected chimpanzees (32) and from in vitro HCV-infected human primary hepatocytes (19, 53). There is also good evidence that HCV can infect cells of extrahepatic origin and particularly cells of hematopoietic origin, such as T and B lymphocytes and dendritic cells (reviewed in references 5, 55, and 60). Due to extensive blood flow, the liver is in contact with peripheral blood dendritic cells, and indeed dendritic cells were found in HCV-infected livers in the vicinity of the lymphatic vessels (23). Dendritic cells represent a heterogeneous population of cells of either myeloid or plasmacytoid lineage which are potent antigen-presenting cells and activators of the Th1/Th2 response (11).
Although HCV envelope glycoprotein was shown to bind to dendritic cells through the dendritic cell SIGN surface receptor (39, 49), it is still unclear whether HCV infection impairs dendritic cell maturation (3, 38, 43). HCV infection of dendritic cells could potentially abolish IFN production though early expression of NS3/4A. Such a mechanism could explain the failure of HCV-infected individuals to mount an efficient immune response, since production of IFN- is necessary for the maturation of dendritic cells. Because IKK can restore IFN induction in the presence of an HCV replicon, this kinase may be important in sustaining IFN production in the face of HCV infection. Identification of therapeutic agents capable of boosting IKK expression may be a beneficial antiviral strategy to explore, with important implications for HCV-infected patients.
ACKNOWLEDGMENTS
This work was supported in part by grants from the Agence Nationale pour la Recherche contre le Sida (ANRS) to E.F.M. and the Canadian Institutes for Health Research (CIHR), National Cancer Institute, and CANVAC, Network Centres of Excellence, to J.H. and R.L. A.B. was supported by a fellowship from the ANRS, N.G. by a postdoctoral fellowship from the Fonds pour la Recherche Scientifique du Quebec (FRSQ), R.L. by an FRSQ chercheur boursier, and J.H. by a CIHR Senior Investigator award.
We thank Céline Gracia, Julien Thorgue, Rachel Couderc, and Florence Bayard for excellent technical assistance and Klaus Schwamborn for critical review of the manuscript.
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