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Journal of Virology, September 2005, p. 10968-10977, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.10968-10977.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Hepatitis A Virus Suppresses RIG-I-Mediated IRF-3 Activation To Block Induction of Beta Interferon

Volker Fensterl, Dajana Grotheer, Iris Berk, Stefanie Schlemminger,{dagger} Angelika Vallbracht, and Andreas Dotzauer*

Department of Virology, University of Bremen, D-28359 Bremen, Germany

Received 4 April 2005/ Accepted 2 June 2005


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ABSTRACT
 
Hepatitis A virus (HAV) antagonizes the innate immune response by inhibition of double-stranded RNA (dsRNA)-induced beta interferon (IFN-ß) gene expression. In this report, we show that this is due to an interaction of HAV with the intracellular dsRNA-induced retinoic acid-inducible gene I (RIG-I)-mediated signaling pathway upstream of the kinases responsible for interferon regulatory factor 3 (IRF-3) phosphorylation (TBK1 and IKK{varepsilon}). In consequence, IRF-3 is not activated for nuclear translocation and gene induction. In addition, we found that HAV reduces TRIF (TIR domain-containing adaptor inducing IFN-ß)-mediated IRF-3 activation, which is part of the Toll-like receptor 3 signaling pathway. As IRF-3 is necessary for IFN-ß transcription, inhibition of this factor results in efficient suppression of IFN-ß synthesis. This ability of HAV seems to be of considerable importance for HAV replication, as HAV is not resistant to IFN-ß, and it may allow the virus to establish infection and preserve the sites of virus production in later stages of the infection.


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INTRODUCTION
 
Hepatitis A virus (HAV), a member of the positive-stranded RNA picornavirus family, is transmitted by the fecal-oral route, but detours from the intestinal tract through the liver, which represents the site of viral replication (14, 23). At the cellular level, HAV infections result in a persistent noncytopathic infection (13, 21, 51). However, HAV is eliminated from the organism at later stages of the infection, and HAV infections do not result in viral persistence in the liver in vivo. This eradication of HAV from the organism, which is accompanied by massive hepatocyte destruction, is mediated mainly by the action of HAV-specific cytotoxic T lymphocytes (CTL) (18, 50). In addition, data were presented demonstrating that gamma interferon (IFN-{gamma}) is produced after HAV stimulation by HAV-specific HLA-dependent CTL in vitro and may contribute to the elimination of HAV infections in humans by inducing an antiviral state in the later course of the infection (31). Furthermore, the experimental elimination of persistent HAV infections in human fibroblast cultures by exogenously added IFN-{alpha}/ß showed that HAV is not resistant to these interferons (49, 51). But neither measurable IFN-{alpha}/ß levels, which are normally produced rapidly in direct response to viral replication by virus-infected cells, nor interference with the infection by other viruses could be detected in lymphocytes and fibroblasts infected with HAV (51, 52), and the role of IFN-{alpha}/ß in the elimination of HAV during an acute infection was contradictorily discussed.

Recently we confirmed that no IFN-ß is synthesized during an HAV infection in cell culture, as HAV prevents IFN-ß synthesis by interfering with double-stranded RNA (dsRNA) signaling (6). Using chloramphenicol acetyltransferase (CAT) as a reporter linked to the natural IFN-ß enhancer-promoter we could demonstrate that HAV does inhibit dsRNA-induced transcription of IFN-ß. We discussed that this ability of HAV, which, in concert with the ability to prevent apoptosis induced by accumulating dsRNA (6), preserves the sites of virus production for a longer time, allows spreading to neighboring cells, and helps the virus to establish an infection.

The intention of our investigation was to find out at which site HAV interferes with the dsRNA-induced signaling cascade for induction of IFN-ß expression. The dsRNA-induced expression of IFN-ß is strictly regulated by the activation of preexisting transcription factors, such as interferon regulatory factor 3 (IRF-3), nuclear factor {kappa}B (NF-{kappa}B), and activating transcription factor 2/cellular Jun protein (ATF-2/c-Jun) (32). After activation by specific kinases, these factors translocate to the nucleus and interact with the coactivators p300/CBP (CREB binding protein) and bind to specific IFN-ß enhancer elements, termed positive regulatory domains III-I (IRF-3), II (NF-{kappa}B), and IV (ATF-2/c-Jun), resulting in assembly of the transcription-inducing enhanceosome complex (47, 57; reviewed in references 1 and 32). In transfection experiments with reporter constructs containing the specific binding sites for the transcription factors, we found that HAV interferes with IRF-3 function. It has been shown that IRF-3 is activated by the TANK (TRAF family member-associated NF-{kappa}B activator)-binding kinase 1 (TBK1) or the inhibitor of NF-{kappa}B kinase {varepsilon} (IKK{varepsilon}) (16, 24, 34, 43). Recent work suggests that these kinases are activated through two independent signaling pathways, which are initiated by double-stranded RNA formed as a result of viral replication (see Fig. 8).



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  		FIG. 8. Model of HAV interaction with the dsRNA-induced IRF-3 activation pathway. Intracellular dsRNA binds to RIG-I, which regulates downstream activation of IKK{varepsilon} or TBK1, which form a complex with adapters, such as TANK (9), by still unknown intermediate components. Activated IKK{varepsilon}/TBK1 then phosphorylate IRF-3, leading to its dimerization, nuclear translocation, and transactivity. Extracellular dsRNA binds to TLR3 associated with intracellular vesicles, inducing recruitment of TBK1 and IRF-3 via its adapter TRIF, resulting in IRF-3 phosphorylation and activation. Hepatitis A virus suppresses intracellular dsRNA-induced RIG-I-mediated IRF-3 phosphorylation by a mechanism acting upstream of IKK{varepsilon}/TBK1. Additionally, HAV negatively affects TRIF-induced IRF-3 activation, probably inhibiting TLR3 signaling as well.

Extracellular dsRNA binds to Toll-like receptor 3 (TLR3) (2, 33), which then recruits TBK1 via the adaptor protein TRIF (TIR domain-containing adaptor inducing IFN-ß) (37, 41, 56). For intracellular dsRNA, which accumulates during viral infections, two members of the caspase recruitment domain (CARD)-containing DExD/H box RNA helicases, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA-5), have been identified as dsRNA receptors, which regulate downstream activation of the kinases TBK1/IKK{varepsilon} (3, 8, 19, 58). Both pathways lead to IRF-3 phosphorylation, which results in formation of IRF-3 homodimers and in cytoplasmic to nuclear translocation (28, 30, 38, 45, 58, 59).

We investigated the influence of HAV infection on the IRF-3 activation pathway and found that HAV interferes with TRIF signaling and blocks RIG-I signaling upstream of the IKK{varepsilon}/TBK1 kinases, resulting in inhibition of IFN-ß transcription.


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MATERIALS AND METHODS
 
Cells and transfections. Fetal rhesus monkey kidney cells (FRhK-4) and human embryonic lung fibroblasts (MRC-5) were maintained as continuous cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% (FRhK-4) or 3% (MRC-5) fetal calf serum. In order to split the cells at a ratio of 1:2 (MRC-5) and 1:5 (FRhK-4), they were detached from the tissue culture plate with trypsin supplemented with 0.2 g of sodium-EDTA/liter and cultivated with Dulbecco's modified Eagle's medium-10% fetal calf serum as the growth medium.

To establish stable cell lines containing the respective plasmids, the cells were transfected by calcium phosphate or jetPEI (Qbiogene) and cultured with selection medium containing 600 µg of G418 per ml.

Transient transfections were performed in 58-mm dishes by jetPEI transfection of 5 µg plasmid as instructed by the manufacturer (Qbiogene).

Polyinosinic-polycytidylic acid [poly(I-C)] transfection was performed with serum-free Dulbecco's modified Eagle's medium supplemented with 20 µg of poly(I-C)/ml in the presence of 100 µg of DEAE-dextran/ml for 2 h at 37°C. After one wash with phosphate-buffered saline, the cells were incubated for the times indicated.

Viruses. HAV/7 (10), which is a variant of strain HM175, was propagated in FRhK-4 cells as described previously (7). HAV strain GBM (17) was propagated in MRC-5 cells. HAV-GBM, which is adapted to growth in MRC-5 cells, was used to investigate nuclear translocation of NF-{kappa}B in MRC-5 cells. In all other experiments we used HAV/7. Newcastle disease virus (NDV) strain R 05/93, obtained from the Federal Research Center for Animal Health, Insel Riems, Germany, was passaged on FRhK-4 cells.

The 50% tissue culture infective doses (TCID50) of HAV were determined by indirect immunofluorescence with the monoclonal mouse anti-HAV immunoglobulin G (IgG) antibody 7E7 (Mediagnost, Reutlingen, Germany), and the titer of NDV was determined by cytopathic effect. The titers were calculated by the Kärber method (26).

For experiments, HAV infections were carried out 10 days prior to analysis with a multiplicity of infection (MOI) of 1. In order to guarantee that all cells were infected, the cells were split 1 day prior to the experiments and infection of 100% of the cells was proved by indirect immunofluorescence.

NDV replication kinetics under one-step growth conditions in FRhK-4 cells infected with HAV/7. FRhK-4 cells were infected with HAV/7 at an MOI of 1; 10 days after infection, when 100% of the cells were infected, as proved by immunofluorescence, cells were superinfected with NDV at an MOI of 10 and 0, 4, 8 and 24 h after NDV infection the 50% tissue culture infective dose titer was determined in FRhK-4 cells 6 days after inoculation by cytopathic effect. The titers were calculated by the Kärber method (26). As controls for NDV replication, FRhK-4 cells not infected with HAV were used. This time frame corresponds to that used during the other experiments.

CAT reporter assay. Cell extracts were prepared by triple freeze-thaw cycles, and expression of the CAT reporter gene was analyzed by subjecting 100 µg of protein to CAT-specific enzyme-linked immunosorbent assay (ELISA) (Roche) as described by the manufacturer. The detection limit of the assay was 10 pg/ml.

Luciferase reporter assay. Cell extracts were prepared by triple freeze-thaw cycles, and luciferase reporter gene expression was analyzed by subjecting 20 µg of protein to the luciferase assay system (Promega) as described by the manufacturer by use of a luminometer (Wallac Microbeta).

Immunoblot analysis. For analysis of IRF-3 phosphorylation, protein extracts were prepared with lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1% NP-40) supplemented with complete protease inhibitor cocktail (Roche). Under these conditions the phosphorylation status of proteins is maintained. Immunoblot analysis was conducted with 20 µg of protein as described earlier (12). The antibodies used were rabbit anti-IRF-3 (no. sc-9082, Santa Cruz) and bovine anti-rabbit-horseradish peroxidase (Santa Cruz). This procedure is a standard procedure described in the literature (30, 59) allowing the simultaneous detection of the two not-activated IRF-3 forms (IRF-3) and the activated, C-terminally phosphorylated IRF-3 (IRF-3-P). For Flag-RIG-I detection, mouse anti-Flag M2 (Sigma) and goat anti-mouse-horseradish peroxidase (Santa Cruz) were used. Visualization was performed with the ECL Western blotting analysis system from Amersham and Kodak Biomax films.

Immunofluorescence analysis. Cells were cultured on Labtek chamber slides, then fixed and probed for HAV with mouse anti-HAV 7E7 (Mediagnost, Reutlingen, Germany), goat anti-mouse-fluorescein isothiocyanate or -Texas Red (KPL), for NF-{kappa}B with rabbit anti-NF-{kappa}B p65 (Santa Cruz), goat anti-rabbit-Biotin (Santa Cruz), and ExtrAvidin-fluorescein isothiocyanate (Sigma).

Plasmids. The (–110-IFN-ß)-CAT, (PRDIII-I)3-CAT, (PRDII)2-CAT and (PRDIV)6-CAT plasmids were kindly provided by T. Maniatis, Harvard University, Cambridge, MA (47); pcDNA3/GFP-IRF-3(wt) by N. C. Reich, SUNY at Stony Brook, Stony Brook, NY (28); pCMVßL/IRF-3(wt) by R. Lin, Lady Davis Institute, Montreal, Canada (30); (PRDIII-I)4-Luc by S. Ludwig, Heinrich Heine University, Düsseldorf, Germany (15); pcDNA3.1/IKK{varepsilon}-myc by U. Siebenlist, National Institutes of Health, Bethesda, MD (9); pcDNA3/Flag-TBK1 (pcDNA3/Flag-NAK) by M. Nakanishi, Nagoya City University Medical School, Nagoya, Japan (48); pEF-Flag-RIG-Ifull, pEF-Flag-RIG-IC and p125-Luc (IFN-ß-Luc) by T. Fujita, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (58); and PL 1004 TRIF (Flag-TRIF) by J. Tschopp, University of Lausanne, Epalinges, Switzerland (35). Empty vectors were obtained by removing the inserted sequences.


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RESULTS
 
HAV differentially affects transcriptional activity of the individual IFN-ß enhancer domains and interacts with IRF-3 signaling to inhibit dsRNA-induced IFN-ß synthesis. In former investigations it has been demonstrated by using a CAT reporter gene linked to the intact IFN-ß enhancer-promoter that HAV completely inhibits activation of the IFN-ß enhancer in response to dsRNA and therefore inhibits induction of IFN-ß mRNA transcription (6). In order to identify the target of HAV-mediated inhibition of IFN-ß transcription in the IFN-ß enhancer and thus the transcription factor with whose activity HAV interferes, we tested the ability of HAV to inhibit dsRNA-activated transcription from the individual regulatory domains of the IFN-ß enhancer. For this purpose, fetal rhesus monkey kidney cells (FRhK-4) were stably transfected with a CAT reporter gene linked to repeated individual enhancer elements (PRDII)2 (NF-{kappa}B binding sites), (PRDIII-I)3 (IRF-3 binding sites), and (PRDIV)6 (ATF-2/c-Jun binding sites).

In order to examine the influence of HAV on reporter gene expression, cells infected with HAV were transfected with 20 µg of the synthetic dsRNA analogon poly(I-C)/ml by DEAE-dextran. Extracellular poly(I-C) treatment without DEAE-dextran did not result in detectable reporter gene expression (not shown). In these experiments, HAV infection resulted in enhanced NF-{kappa}B (PRDII)-dependent CAT expression compared with poly(I-C) induction alone (Fig. 1A). Additionally, HAV infection alone also resulted in significant reporter gene expression. These findings indicate that HAV infection induces activation of the pleiotropic transcription factor NF-{kappa}B and show that inhibition of IFN-ß expression by HAV is not due to an interruption of NF-{kappa}B signaling.



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  		FIG. 1. HAV inhibits dsRNA-induced activation of IRF-3 but not of NF-{kappa}B. FRhK-4 cells stably transfected with PRDII-CAT (A) or PRDIII-I-CAT (B) reporter plasmids were infected with HAV/7 at an MOI of 1 and transfected with poly(I-C) by DEAE-dextran (DD) 9 days postinfection; 16 h later, cell extracts were analyzed for NF-{kappa}B-dependent (A) or IRF-3-dependent (B) CAT expression. Noninfectedcells and cells treated with medium or DEAE-dextran alone were used as controls. Data represent means of two replicates, and experiments were carried out twice. (C) Nuclear translocation of NF-{kappa}B was examined by immunofluorescence. MRC-5 cells infected with HAV-GBM were transfected with poly(I-C) by DEAE-dextran (DD); after 3 h, cells were fixed and permeabilized with 4% paraformaldehyde/methanol, and immunostained for NF-{kappa}B p65 (fluorescein isothiocyanate) and HAV (Texas Red). Nontransfected and/or noninfected cells were used as controls. Results are representative of two independent experiments. Magnification, x400 (Axioskop II, Zeiss).

Confirmation was obtained by investigating the nuclear translocation of this factor in cells infected with HAV and after poly(I-C) transfection by indirect immunofluorescence using antibodies specific for NF-{kappa}B/p65. In cells neither infected with HAV nor transfected with poly(I-C), NF-{kappa}B is localized mainly in the cytoplasm, whereas after induction with poly(I-C) as well as after poly(I-C) induction of HAV infected cells, NF-{kappa}B is translocalized into the nucleus, which indicates NF-{kappa}B activation (Fig. 1C). In cells solely infected with HAV NF-{kappa}B translocation was also observed. HAV infection was confirmed by immunofluorescence using antibodies specific for HAV. In contrast to the experiment investigating PRDII-dependent CAT expression, in which we used HAV/7 and FRhK-4 cells, here we used HAV-GBM and MRC-5 cells. We could thus confirm that the influence of HAV on NF-{kappa}B activation is independent of the virus strain and cell line used. This corresponds to our previous findings (6) that the influence of HAV on the induction of IFN-ß does not depend on the virus strain and the cell line.

Induction of ATF-2/c-Jun (PRDIV)-controlled CAT expression by poly(I-C) was not affected by HAV (data not shown).

Induction of IRF-3 (PRDIII-I)-dependent reporter gene expression was not detectable after HAV infection (Fig. 1B). Furthermore, IRF-3 activity, which was strongly induced by poly(I-C) transfection, was completely inhibited by HAV. Thus, HAV does not induce IRF-3 transcriptional activity but even interferes with IRF-3 signaling, consequently resulting in suppression of IFN-ß synthesis.

HAV interferes with nuclear translocation and phosphorylation of IRF-3. In order to clarify whether HAV inhibits binding of IRF-3 to the corresponding PRDIII-I or whether intervention by HAV occurs further upstream in the signaling cascade, preventing nuclear translocation, FRhK-4 cells were stably transfected with IRF-3 linked to the green fluorescent protein (GFP) (FRhK-4/GFP-IRF-3) by calcium phosphate transfection and the subcellular localization of the protein was analyzed by fluorescence microscopy (Fig. 2A to D). GFP-IRF-3 was detected in the cytoplasm of these cells as well as in cells infected with HAV at an MOI of 1 10 days prior to analysis (Fig. 2A and B). After transfection of the cells with poly(I-C) (not shown) as well as after infection with Newcastle disease virus (NDV), which is a strong inducer of IRF-3 activity, at an MOI of 10 4 h prior to analysis, GFP-IRF-3 was mainly detected in the nucleus (Fig. 2C). NDV induction obviously occurs by viral dsRNA and therefore induction by NDV and by poly(I-C) transfection is functionally equivalent, which will be demonstrated below. In cells infected with HAV, which does not interfere with NDV replication (Fig. 3), translocation of IRF-3 to the nucleus could never be detected after stimulation with NDV (Fig. 2D) or transfection with poly(I-C). HAV infection of 100% of the cells was visualized by immunofluorescence. These results imply that HAV already prevents activation of IRF-3 in the cytoplasm.



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  		FIG. 2. HAV inhibits IRF-3 nuclear translocation and phosphorylation. (A to D) FRhK-4/GFP-IRF-3 cells infected with HAV/7 were infected with NDV (MOI, 10); 4 h postinfection, cells were fixed with 4% paraformaldehyde and 0.2% Triton X-100 and immunostained for HAV (Texas Red) (D). Noninfected cells (A) and cells infected only with HAV or NDV (B and C) were used as controls. Magnification, x400 (Axioskop II, Zeiss). (E) IRF-3 C-terminal phosphorylation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. FRhK-4/IRF-3 cells noninfected or infected with HAV/7 were inoculated with NDV at an MOI of 10 for 2 h. At different times postinfection, whole-cell extracts were prepared. Phosphorylated IRF-3 was detected as an additional, slower-migrating form. All results shown are representative of a series of three independent experiments.



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  		FIG. 3. NDV replication kinetics under one-step growth conditions in FRhK-4 cells infected or not infected with HAV/7. The kinetics show the total NDV titers in the course of 24 h. Titers were determined in FRhK-4 cells by cytopathic effect 6 days after inoculation. Each data point is an average obtained from duplicate titrations of two separate experiments. Error bars indicate standard deviations of the mean. HAV infection of 100% of the cells was proved by indirect immunofluorescence prior to infection with NDV (MOI of 10) (lower half).

To further clarify this, we stably transfected FRhK-4 cells with IRF-3 (FRhK-4/IRF-3) and analyzed the phosphorylation status of IRF-3 at different times after induction by NDV (MOI 10) in cells infected with HAV by immunoblot analysis. Cells overexpressing IRF-3 were used as due to low concentrations, phosphorylated endogenous IRF-3 could not be detected by immunoblot analysis; 4 h after infection with NDV, phosphorylation of IRF-3 was observed by the occurence of an additional, slower migrating form (Fig. 2E) compared to the two not-activated IRF-3 forms (30, 59). In cells infected with HAV, NDV-mediated phosphorylation of IRF-3 was completely inhibited. HAV infection was confirmed by immunofluorescence, and NDV infection by appearance of cytopathic effects. The results demonstrate that HAV prevents phosphorylation and thus activation and nuclear translocation of IRF-3.

HAV does not directly interfere with the activity of IKK{varepsilon} and TBK1. In order to further analyze the stage at which HAV interferes with phosphorylation of IRF-3, we first investigated whether HAV is able to inhibit the activity of the kinases responsible for IRF-3 phosphorylation (see Fig. 8). FRhK-4 cells infected with HAV were cotransfected with CAT or luciferase as a reporter gene linked to the intact IFN-ß enhancer-promoter (IFN-ß-CAT and IFN-ß-Luc) or to repeated PRDIII-I domains for specific detection of activated IRF-3 (PRDIII-I-CAT, and PRDIII-I-Luc) and a plasmid for constitutive expression of IKK{varepsilon} or TBK1, respectively, 10 days postinfection. Noninfected cells were used as references.

As demonstrated by different laboratories, overexpression of the kinases results in kinase activity without additional activation (8, 16, 43) and 42 h after transfection reporter gene expression was analyzed. In cells overexpressing IKK{varepsilon} (Fig. 4A) or TBK1 (Fig. 4B), significant reporter gene expression was detected. This shows that each of the individual kinases activates IRF-3 and induces IFN-ß under these experimental conditions. In cells infected with HAV, reporter gene expression and therefore the activity of the kinases were not affected. In order to prove functional HAV infection of the cells, they were cotransfected with the reporter plasmid and the empty vectors for the kinases. As expected, NDV-induced reporter gene expression was completely inhibited in HAV-infected cells. In addition, with cells overexpressing TBK1 and infected with NDV it was shown by using PRDIII-I-controlled luciferase expression that HAV is able to inhibit NDV-induced IRF-3 activation independently of TBK1 activity resulting from kinase overexpression (Fig. 4B, right). In these experiments luciferase expression was reduced by HAV to levels observed with overexpressed TBK1 alone.



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  		FIG. 4. HAV does not inhibit IFN-ß induction and IRF-3 activation by IKK{varepsilon} or TBK1. (A) In order to analyze IKK{varepsilon}-dependent IFN-ß induction (IFN-ß-CAT) or IRF-3 activity (PRDIII-I-CAT), noninfected or HAV/7-infected FRhK-4 cells were cotransfected with CAT reporter gene plasmids and an IKK{varepsilon} expression vector. NDV infection (MOI of 0.1) was used as a control for functional HAV infection in cells cotransfected with the empty vector. (B) For analysis of TBK1-dependent IFN-ß induction (IFN-ß-Luc) or IRF-3 activity (PRDIII-I-Luc), noninfected or HAV/7-infected FRhK-4 cells were cotransfected with luciferase reporter gene plasmids and a TBK1 expression vector. NDV infection (MOI of 0.1) was used as a control for functional HAV infection in cells cotransfected with the empty vector. The right panel includes TBK1-transfected plus NDV-infected controls to demonstrate the integrity of the inhibitory mechanism of HAV independently of TBK1 overexpression. In all experiments, NDV infection was performed 24 h after transfection, and reporter gene expression was analyzed 42 h after transfection. Data represent means of at least two replicates, and experiments were carried out at least twice.

These results demonstrate that HAV does not interfere either directly with IKK{varepsilon} and TBK1, thus not inhibiting kinase activity, or directly with IRF-3, thus not inhibiting IRF-3 phosphorylation and activity, but imply that inhibition of IRF-3 phosphorylation is the result of an intervention of HAV with dsRNA signaling upstream of the kinases, preventing their activation.

HAV inhibits RIG-I-mediated signaling. The results described above indicate that the target site of inhibition of dsRNA-induced IFN-ß synthesis by HAV is located upstream of the activator kinase complex for IRF-3. Therefore, we investigated the influence of HAV on signaling initiated by RIG-I, which acts as an intracellular dsRNA receptor regulating IRF-3 activation and thence IFN-ß induction. Constitutive RIG-I expression was detected at low basal levels in FRhK-4 cells by reverse transcription-PCR (not shown). FRhK-4 cells cotransfected with a RIG-I expression plasmid and a CAT reporter construct containing the complete IFN-ß enhancer or repeated IRF-3 binding sites (PRDIII-I) were analyzed for CAT expression 42 h after transfection. As shown in Fig. 5, overexpression of RIG-I, proved by immunoblot detection, resulted in significant CAT expression. In cells infected with HAV, which does not influence RIG-I expression (Fig. 5, lower panels), however, CAT expression controlled by the complete IFN-ß enhancer (Fig. 5A) as well as by PRDIII-I (Fig. 5B) was inhibited completely. As RIG-I overexpression did not complement the inhibitory effect of HAV, it can be assumed that HAV does interact with signaling downstream of RIG-I.



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  		FIG. 5. HAV inhibits IFN-ß induction and IRF-3 activation by RIG-I. In order to analyze RIG-I-dependent IFN-ß induction (IFN-ß-CAT) (A) or IRF-3 activity (PRDIII-I-CAT) (B), noninfected or HAV/7-infected FRhK-4 cells were cotransfected with CAT reporter gene plasmids and a RIG-I expression vector (upper panels). Empty vector cotransfection was used as a control. CAT expression was analyzed 42 h after transfection. Flag-RIG-I overexpression was verified by immunoblotting (lower panels). Data represent means of at least two replicates, and experiments were carried out at least twice.

Furthermore, we were able to show that NDV-mediated reporter gene induction, which we used in some parts of our experiments, is mediated by NDV dsRNA. FRhK-4 cells were cotransfected with an IFN-ß-CAT reporter construct and with a RIG-IC expression plasmid, which encodes the dsRNA-binding helicase domain but not the signal-transducing CARD domain and exhibits a dominant negative effect by sequestering dsRNA molecules. As expected (58), RIG-IC overexpression prevented IFN-ß-CAT expression induced by poly(I-C) transfection (Fig. 6A) or NDV infection (Fig. 6B), suggesting functional equivalence of the two inductors and even more demonstrating exclusively intracellular, TLR3-independent dsRNA signaling.



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  		FIG. 6. RIG-I N-terminal deletion mutant (RIG-IC) suppresses IFN-ß enhancer activation induced by poly(I-C) transfection or NDV infection. FRhK-4 cells were cotransfected with IFN-ß-CAT and a RIG-IC expression vector encoding the RIG-I dsRNA-binding helicase domain; 24 h later, cells were transfected with poly(I-C) via DEAE-dextran (DD) (A) or infected with NDV (MOI of 0.1) (B) and 18 h later, cell extracts were analyzed for CAT expression. Data represent means of two replicates, and experiments were carried out twice.

These experiments demonstrate that HAV prevents IFN-ß synthesis by inhibiting activation of endogenous IRF-3 by blocking intracellular dsRNA-induced RIG-I-mediated downstream signaling.

In order to investigate whether HAV also impairs the TLR3 pathway-associated TRIF-mediated induction of IFN-ß and IRF-3 activation, FRhK-4 cells were cotransfected with a TRIF expression plasmid and a CAT reporter construct containing the complete IFN-ß enhancer or repeated IRF-3 binding sites (PRDIII-I) (Fig. 7). Analysis of CAT expression was performed 42 h after transfection. As with RIG-I, overexpression of TRIF, which is constitutively expressed in FRhK-4 cells at basal levels (not shown), also resulted in significant CAT expression, but in cells infected with HAV CAT expression controlled by the complete IFN-ß enhancer (Fig. 7 A) as well as by PRDIII-I (Fig. 7 B) was not inhibited completely, but reduced up to 50%. In addition to the presence of HAV in 100% of the cells, as proved by immunofluorescence, functional HAV infection of the cells was confirmed by the demonstration that reporter gene expression induced by NDV (Fig. 7B) or by RIG-I (shown in Fig. 5A; data shown in Fig. 5A and 7A represent results of a combined experiment) was completely inhibited. This shows that HAV is able to reduce TRIF-mediated activation of IRF-3, but seems to be well adapted to interfere with the RIG-I signaling pathway, which means with intracellular dsRNA-induced IFN-ß synthesis.



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  		FIG. 7. HAV negatively affects IFN-ß induction and IRF-3 activation by TRIF. TRIF-dependent IFN-ß induction (IFN-ß-CAT) (A) or IRF-3 activity (PRDIII-I-CAT) (B) was examined in noninfected or HAV/7-infected FRhK-4 cells after cotransfection with CAT reporter gene plasmids and a TRIF expression vector. Empty vector cotransfection was used as a control. CAT expression was analyzed 42 h after transfection. Data represent means of at least two replicates, and experiments were carried out at least twice.


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DISCUSSION
 
Previously we proved that HAV infections in general do not result in IFN-ß expression and that this is due to the ability of HAV to inhibit dsRNA-mediated transcriptional induction of IFN-ß (6). In this study, we investigated with what section of the dsRNA-signaling cascade HAV interferes to prevent dsRNA-induced transcription of IFN-ß, thereby antagonizing the innate antiviral immune response.

Using plasmid constructs with the individual domains of the IFN-ß enhancer linked to reporter genes, we showed that inhibition of IFN-ß transcription by HAV occurs through a mechanism influencing IRF-3 activity. Whereas transcription factor ATF-2/c-Jun activation is not affected by HAV and transcription factor NF-{kappa}B activation is even enhanced, the activity of transcription factor IRF-3 is completely inhibited. We demonstrated that HAV prevents IRF-3 phosphorylation, which is necessary for IRF-3 dimerization and cytoplasm-to-nucleus translocation (Fig. 8). Obviously HAV does not interact either with IRF-3 or with the IKK{varepsilon}/TBK1 kinases directly, because in the case of IKK{varepsilon} and TBK1 overexpression, HAV does not alter IRF-3 activity. Based on our data, we conclude that HAV prevents IKK{varepsilon}/TBK1 kinase activation by inhibiting dsRNA-controlled and RIG-I-mediated downstream signaling to the kinases, which represents a pathway essential for viral triggering of IRF-3 activation and IFN-ß induction in hepatocytes as well as in HeLa cells and L929 mouse fibroblasts (29, 46, 58).

As RIG-I activity obtained by overexpression of the protein and not by dsRNA induction is completely inhibited by HAV, it appears that HAV does not mask dsRNA. This is further supported by two observations. First, large amounts of intracellular dsRNA obtained by poly(I-C) transfection or NDV infection do not compete with the inhibition by HAV. Second, activation of NF-{kappa}B by dsRNA is not suppressed by HAV. Also, HAV does not seem to interact directly with RIG-I, as overexpression of RIG-I does not compete with the inhibitory effect caused by HAV. Based on our experimental findings we can conclude that HAV interacts with intracellular dsRNA-induced signal transduction downstream of RIG-I or a protein functionally similar to RIG-I, such as MDA-5 (3), and upstream of the IKK{varepsilon}/TBK1 kinase complex, thus preventing IRF-3 phosphorylation and in consequence IFN-ß synthesis. At present, it is not possible to determine the site of HAV intervention more exactly, as the components of the pathway connecting RIG-I with the kinases have not been identified. Recently, Balachandran et al. (4) proposed an involvement of the adaptor Fas-associated death domain (FADD) and the RIP1 kinase in intracellular dsRNA signaling, providing possible targets for HAV.

IRF-3 activation and IFN-ß induction are also possible through TLR3 signaling, a pathway normally initiated in response to extracellular dsRNA (16). However, in FRhK-4 cells extracellular dsRNA treatment did not result in IFN-ß induction, although TLR3 mRNA could be detected (not shown). As TLR3 is known to be mainly associated with intracellular vesicular structures (20, 36), activation by HAV replication, which also occurs in association with intracellular vesicles (44), seems possible. In this case, signaling would be transduced by the adaptor molecule TRIF, which forms a complex with TLR3, TBK1, and IRF-3, thus activating the kinase and thence IRF-3 (41). We demonstrated that HAV also has the ability to affect this transduction pathway. The only partial inhibition of the TRIF-mediated signal transduction by HAV which we observed in our experiments may result from competition with surplus TRIF, which was overexpressed and may therefore counteract suppression by HAV, implying a direct interaction with TRIF.

The effectiveness of the interferon response has caused many viruses to develop specific mechanisms to antagonize the antiviral effects (for reviews, see references 22 and 42). Viruses vary considerably in their ability to prevent the production or actions of IFNs and several of them interact with IRF-3 function to inhibit IFN-ß synthesis. For example, influenza A virus NS1 protein and vaccinia virus E3L protein mask dsRNA, thus preventing induction of the signaling cascade (53, 55), whereas human papillomavirus E6 protein and rotavirus NSP1 protein bind directly to IRF-3 (5, 39). Sendai virus V protein interacts with MDA-5, thus inhibiting intracellular dsRNA signaling (3). Similar to HAV, hepatitis C virus antagonizes dsRNA-activated RIG-I and TLR3 signaling. Whereas proteolytic cleavage of a not yet identified RIG-I pathway component by NS3/4A protease of hepatitis C virus is assumed, it could be demonstrated that this protease causes specific proteolysis of TRIF (19, 29, 46). As IRF-3 activity is necessary for IFN-ß transcription (40, 59), this factor represents an excellent target for interventions to achieve suppression of IFN-ß synthesis.

The competence of HAV to directly modulate the host cell's antiviral IFN response seems to be of considerable importance for HAV replication, as HAV is not resistant to IFN-ß (49, 51). After entry into host cells, HAV replicates exceptionally slowly. The ability to inhibit dsRNA-induced transcriptional activation of IFN-ß guarantees that no antiviral activities by IFN-stimulated genes are induced in the already infected cells as well as in surrounding noninfected cells. This strategy may allow HAV to establish and preserve infection and may be the basis for the persistent character of HAV infections.

In addition, the inhibition of dsRNA-induced RIG-I signaling by HAV may allow the virus to evade the cellular IFN response for a longer time at later stages of the infection. At that time, RIG-I expression may be upregulated by IFN-{gamma} (11, 25), which is secreted by HAV-specific CTL (31), enhancing the responsiveness of cells to viral dsRNA. As HAV obviously does not interact with RIG-I directly, HAV may be able to further inhibit signaling in this situation, because larger amounts of RIG-I are not able to competitively overcome inhibition at the site where HAV interferes.

Furthermore, since IRF-3 is also involved in induction of apoptosis by dsRNA (54) and since HAV is also able to inhibit dsRNA-induced apoptosis (6), HAV may not only inhibit IFN-ß synthesis by suppression of IRF-3 activation; this mechanism may also support inhibition of apoptosis. We found that HAV enhances activation of transcription factor NF-{kappa}B. As this pleiotropic factor is involved in expression of antiapoptotic genes (reviewed in reference 27), the ability of HAV to activate NF-{kappa}B may play a role in inhibition of apoptosis by this virus.

In summary, we localized the site at which HAV inhibits dsRNA-induced IFN expression, in the RIG-I-mediated signaling cascade upstream of the kinases for IRF-3 phosphorylation. It remains to be shown through which viral factors HAV is able to interfere with the RIG-I and TLR3 signaling pathways and whether suppression of IRF-3 activity by HAV may contribute to inhibition of apoptosis.


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ACKNOWLEDGMENTS
 
We thank T. Fujita, R. Lin, S. Ludwig, T. Maniatis, M. Nakanishi, N. C. Reich, U. Siebenlist, and J. Tschopp for providing plasmids and Mediagnost, Reutlingen, Germany, for providing the monoclonal anti-HAV antibody 7E7.

This work was supported by grant BFK no. 02/105/2 of the University of Bremen, Bremen, Germany, and by the Tönjes-Vagt-Stiftung, Bremen, Germany.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Virology, University of Bremen, Leobener Str. / UFT, D-28359 Bremen, Germany. Phone: 49 421 218 4354. Fax: 49 421 218 4266. E-mail: dotzauer{at}uni-bremen.de. Back

{dagger} Present address: Klinik I für Innere Medizin, University of Cologne, Cologne, Germany. Back


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Journal of Virology, September 2005, p. 10968-10977, Vol. 79, No. 17
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