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J. Biol. Chem., Vol. 281, Issue 4, 2095-2103, January 27, 2006

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Negative Regulation of the Retinoic Acid-inducible Gene I-induced Antiviral State by the Ubiquitin-editing Protein A20^*

Rongtuan Lin1, Long Yang¶, Peyman Nakhaei¶, Qiang Sun, Ehssan Sharif-Askari2, Ilkka Julkunen||, and John Hiscott¶3

From the Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, and Departments of ^¶Microbiology & Immunology and Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada and the ^||National Public Health Institute and University of Helsinki, Helsinki FIN-00300, Finland

Received for publication, September 20, 2005 , and in revised form, November 14, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Activation of the interferon regulatory factors (IRFs) 3 and 7 transcription factors is essential for the induction of type I interferon (IFN) and development of the innate antiviral response. Retinoic acid-inducible gene I has been shown to contribute to virus-induced IFN production independent of the Toll-like receptor pathways in response to a variety of RNA viruses and double-stranded RNA. In the present study, we demonstrate that the NF-B-inducible, anti-apoptotic protein A20 efficiently blocks RIG-I-mediated activation of NF-B-, IRF-3-, and IRF-7-dependent promoters but only weakly interferes with TRIF-TLR-3-mediated IFN activation. Expression of A20 completely blocked CARD domain containing RIG-I-induced IRF-3 Ser-396 phosphorylation, homodimerization, and DNA binding. The level of A20 inhibition was upstream of the TBK1/IKK kinases that phosphorylate IRF3 and IRF7 and paradoxically, A20 selectively degraded the TRIF protein but not RIG-I. A20 possesses two ubiquitin-editing domains, an N-terminal deubiquitination domain and a C-terminal ubiquitin ligase domain consisting of seven zinc finger domains. Deletion of the N-terminal de-ubiquitination domain had no significant effect on the inhibitory effect of A20, whereas deletion or mutation of zinc finger motif 7 ablated the inhibitory function of A20 on IRF- or NF-B-mediated gene expression. Furthermore, cells stably expressing the active form of RIG-I induced an antiviral state that interfered with replication of vesicular stomatitis virus, an effect that was reversed by stable co-expression of A20. These results suggest that the virus-inducible, NF-B-dependent activation of A20 functions as a negative regulator of RIG-I-mediated induction of the antiviral state.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Upon recognition of specific molecular components of viruses or other pathogens, the host cell activates multiple signaling cascades through Toll-like receptor-dependent and -independent pathways, culminating in the production of cytokines and chemokines that disrupt virus replication and initiate innate and adaptive immune responses (1–3). Rapid induction of type I interferon (IFN)⁴ expression is a central event in establishing the innate antiviral response and requires pathogen-inducible, activation of transcription factors that function in a synergistic fashion to induce gene expression (reviewed in Refs. 4–9). Among the members of the interferon regulatory factor (IRF) family, IRF-3 and IRF-7 play essential roles in the virus-induced type I IFN gene activation following virus infection (10–17). IRF-3 is activated by C-terminal phosphorylation, which promotes dimerization, cytoplasmic to nuclear translocation, DNA binding, association with CBP/p300 histone acetyltransferases, and transactivation of downstream early genes such as IFNB, IFNA1, and RANTES (regulated on activation normal T cell expressed and secreted). In contrast, IRF-7 protein is synthesized de novo upon IFN stimulation and contributes to the expression of delayed-type genes, including other IFNA subtypes. As with IRF-3, virus infection induces C-terminal phosphorylation and activation of IRF-7 (15, 16). The IKK-related kinases, IKK (18) and TBK1 (19–21), were shown to be essential signaling components required for IRF-3 and IRF-7 phosphorylation (22–24).

Among the eleven members of the human Toll-like receptor (TLR) family, TLR3, TLR4, TLR7, TLR8, and TLR9 are involved in the initial sensing of viral components. In mice, viral single- and double-stranded RNA, fusion protein of respiratory syncytial virus, single-stranded RNA, and genomic DNA from herpes and cytomegalovirus are recognized by TLR3, TLR4, TLR7, and TLR9, respectively (25–30). Although MyD88 is commonly used by all TLRs, other adapter proteins, including MAL/TIRAP, TRIF/TICAM1, and TRAM/TICAM2, are involved in MyD88-independent pathways (31, 32). TLR3 and TLR4 engage the adapter TRIF/TICAM-1, leading to TBK1 and IKK activation, which in turn activates IRF3 and IFNA/B transcription (33–35).

A separate signaling pathway utilizes the retinoic acid-inducible gene I (RIG-I) to recognize a variety of RNA viruses and trigger the innate antiviral response, independent of the TLR-dependent pathways. RIG-I contains a DEX(D/H) box RNA helicase domain and two caspase recruitment domains (CARDs); full-length RIG-I can interact with dsRNA through its DEX(D/H) box within C terminus and augment IFN production in response to viral infection in an ATPase-dependent manner, and the two copies of the CARD at its N terminus transduce signals leading to the activation of IRF-3 and NF-B. The constitutively active form of RIG-I (CARD domain alone) is capable of activating IRF-3 and NF-B and stimulating IFN- production (36). Recent studies demonstrated that the hepatitis C virus (HCV) gene product NS3/4A protease complex efficiently blocks RIG-I signaling pathway and contributes to the establishment of HCV persistence (37–39). The generation of RIG-I-deficient mice revealed that RIG-I, but not the TLR system, plays an essential role in antiviral responses in various cells, except plasmacytoid dendritic cells. Reciprocally, the TLR system, but not RIG-I, was indispensable to IFN secretion in plasmacytoid dendritic cells (40).

Regulation of the TLR-independent, RIG-I signaling pathway leading to IRF-3 and NF-B has not been well defined, although recent experiments suggest that the NF-B-inducible ubiquitin-editing protein A20 negatively regulates IRF-3 activation (41, 42). We therefore sought to define the involvement of A20 in the regulation of RIG-I signaling. Activation of the IFN antiviral state by RIG-I was completely inhibited by A20 expression, and the C-terminal zinc finger domain of the ubiquitin ligase region was required for the inhibitory function of A20. Furthermore, cells stably expressing the active form of RIG-I induced an antiviral state that significantly blocked replication of VSV, an effect that was reversed by stable co-expression of A20. These results demonstrate that virus-mediated activation of A20 functions as a negative regulator of RIG-I mediated induction of the antiviral state.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmid Constructions and Mutagenesis—Plasmids encoding IKK and TBK1, P2 (2)-TK pGL3, IFNB/pGL3, IFNA14/pGL3, and pRLTK were described previously (23, 43). The FLAG-A20-expressing plasmid and ISRE-luc reporter construct were provided by Hong-Bing Shu (42). The A20 RNAi pSuper constructs were provided by Peter Storz (44). Human TRIF cDNA was amplified from T cell cDNA library and cloned into MYC pcDNA3.1/Zeo (Myc-TRIF). The cDNAs encoded full-length and 1–229 amino acids of human RIG-I were amplified from FLAG-RIG-I expression plasmid (36). Both cDNAs were cloned into Myc pcDNA3.1/Zeo (Myc-RIG-I and Myc-RIG-I). To generate Myc-RIG-I IRES puro expression plasmid, the cDNA encoded Myc-RIG-I was released from Myc-RIG-I pcDNA3.1 zeo by NheI/BamHI and subcloned into NheI/BamHI sites of pIRES puro2 vector (Clontech Laboratories, Inc.). Full-length human A20 cDNA was amplified from FLAG-A20 expression plasmid and cloned into FLAG pcDNA3.1/Zeo (FLAG-A20). The A20 point mutants, including 1) A103, 2) zinc finger domain (ZNF) 4 MT, and 3) ZNF7 MT, were generated by overlap PCR-mediated mutagenesis. The A20 deletion mutants, including 1) 1–380, 2), 1–517, 3) 1–680, 4) 1–740, 5) 373–790, 6) ZNF4 (537–624), 7) ZNF4/5 (537–674), 8) ZNF2/3 (424–536), 9) ZNF2–4 (424–624), and 10) ZNF2–5 (424–674), were generated by PCR. DNA sequencing was performed for confirmation of mutations.

Cell Culture, Transfections, and Luciferase Assays—Transfections for Luciferase assay were carried out in human embryonic kidney (HEK) 293 cells grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, glutamine, and antibiotics. Subconfluent HEK293 cells were transfected with 100 ng of pRLTK reporter (Renilla luciferase for internal control), 200 ng of pGL-3 reporter (firefly luciferase, experimental reporter), 200 ng of RIG-I, TRIF, IKK, or TBK1 expression plasmids, and 500 ng of pcDNA3 or FLAG A20 pcDNA3 plasmid as indicated by calcium phosphate coprecipitation method. The reporter plasmids were: IFNB pGL3, ISRE-luc, P2 (2)-TK pGL3, and IFNA14 pGL-3 reporter genes; the transfection procedures were previously described (45). At 24 h after transfections, the reporter gene activities were measured by Dual-Luciferase Reporter Assay, according to manufacturer's instructions (Promega). Where indicated cells were treated with Sendai virus (40 hemagglutination units/ml) for the indicated time or 15 h for luciferase assays.

Generation of RIG-I-, A20-, and NS3/4A-expressing Cell Lines—Plasmids pCDNA3 zeo, Myc RIG-I, and Myc-RIG-I were introduced into HEK293 cells by the calcium phosphate method. Cells were selected beginning at 48 h for 3 weeks in Dulbecco's modified Eagle's medium containing 10% heat-inactivated calf serum, glutamine, antibiotics, and 100 µg/ml Zeocin (Invitrogen). To generate Myc-RIG-I/FLAG A20 and Myc-RIG-I/FLAG NS3/4A cell lines, the Myc-RIG-I IRES puro expression plasmid was co-transfected with FLAG A20 pcDNA3.1 zeo or FLAG NS3/4A pcDNA3.1 zeo plasmid into HEK293 cells by the calcium phosphate method. Cells were selected beginning at 48 h for 2 weeks in Dulbecco's modified Eagle's medium containing 10% heat-inactivated calf serum, glutamine, antibiotics, and 2 µg/ml puromycin (Sigma).

Antibody Preparation—RIG-I-(1–228) was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified by glutathione-Sepharose column chromatography. The recombinant proteins were injected into rabbits to produce antisera against RIG-I-(1–228).

Co-immunoprecipitation and Western Blot Analysis—Transient transfection, co-immunoprecipitation, and Western blot analysis were performed as previous described (10).

Analysis of IRF-3 Dimerization by Native PAGE—Whole cell extracts were prepared in Nonidet P-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 30 mM NaF, 5 mM EDTA, 10% glycerol, 1.0 mM Na₃VO₄, 40 mM -glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml of each leupeptin, pepstatin, and aprotinin, and 1% Nonidet P-40), and then were subjected to electrophoresis on 7.5% native acrylamide gels, which were pre-run for 30 min at 4 °C. The electrophoresis buffers were composed of an upper chamber buffer (25 mM Tris, pH 8.4, 192 mM glycine, and 1% sodium deoxycholate) and a lower chamber buffer (25 mM Tris, pH 8.4, 192 mM glycine). Gels were soaked in SDS running buffer (25 mM Tris, pH 8.4, 250 mM glycine, 0.1% SDS) for 30 min at 25 °C and were then electrophoretically transferred on Hybond-C nitrocellulose membranes (Amersham Biosciences) in 25 mM Tris, pH 8.4, 192 mM glycine, and 20% methanol for 1 h at 4°C. Membranes were blocked in phosphate-buffered saline containing 5% nonfat dry milk and 0.05% Tween 20 for 1 h at 25°Cand then were blotted with an antibody against IRF3 (1 µg/ml) in blocking solution for 1 h at 25 °C. After washing the membranes five times in phosphate-buffered saline/0.05% Tween, they were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000) in blocking solution. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences).

Electrophoretic Mobility Shift Assay—Briefly, cell pellets were treated with 10 mM HEPES, 50 mM NaCl, 10 mM EDTA, 5 mM MgCl₂, 0.5 mM spermidine, 0.15 mM spermine, 0.5 mM phenylmethylsulfonyl fluoride, leupeptin (10 µg/ml), pepstatin (10 µg/ml), aprotinin (10 µg/ml), and 1 mM Na₃VO₄. Suspension was held on ice for 30 min and brought to 0.1% Nonidet P-40 and 10% glycerol concentration. Samples were spun for 5 min at 5,000 rpm at 4 °C. Supernatant was removed, and the pellet was washed in 50 mM NaCl. Nuclear extracts were obtained in a 10 mM HEPES, 400 mM NaCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, leupeptin (10 µg/ml), pepstatin (10 µg/ml), aprotinin (10 µg/ml), and 1 mM Na₃VO₄ solution. Samples were left to rotate at 4 °C for 30 min and spun at 15,000 rpm for 10 min at 4 °C. Whole cell extracts were assayed for IRF-3 binding in gel shift analysis using a ³²P-labeled double-stranded oligonucleotide corresponding to the interferon-stimulated response element (ISRE) of the IFNA/B-inducible ISG15 gene (5'-GATCGGAAAGGGAAACCGAAACTGAAGCC-3'). Complexes were formed by incubating the probe with 10 µg of nuclear extract for 20 min at room temperature in 10 mM Tris-Cl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 2 mM dithiothreitol, 5% glycerol, 0.5% Nonidet P-40, 1 mg of bovine serum albumin per ml, and poly(dI-dC) (1.0 mg/ml). Extracts were run on a 5% polyacrylamide gel (60:1 crosslink) prepared in 0.25x Tris borate-EDTA. After running at 160 V for 3 h, the gel was dried and exposed to a Kodak film at –70 °C overnight. To demonstrate the specificity of the detected signal, 1 mg of anti-IRF-3 (Santa Cruz Biotechnology FL-425) antibody was incubated for 30 min on ice prior to the addition of the probe to observe a supershift in the complex formation.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. A20 inhibits RIG-I-mediated transactivation. HEK293 cells were transfected with pRLTK control plasmid, IFNB-pGL3 (A), ISRE-Luc (B), IFNA4-pGL3 (C), or P2 (2)-TK-pGL3 (D) reporter plasmid and the pcDNA3 vector or expression plasmid encoding RIG-I as well as IRF-7 expression plasmid as indicated. Luciferase activity was analyzed at 24 h post-transfection by the Dual-Luciferase Reporter assay as described by the manufacturer (Promega). Relative luciferase activity was measured as -fold activation (relative to the basal level of reporter gene in the presence of pcDNA3 vector after normalization with co-transfected RLU activity); values are mean ± S.D. for three experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (37K): [in this window] [in a new window]   FIGURE 2. A20 completely blocks RIG-I-mediated transactivation but only partially inhibits TRIF- or IKK/TBK1-mediated transactivation. HEK293 cells were transfected with pRLTK control plasmid (100 ng), ISRE-Luc reporter plasmid (200 ng), RIG-I (A)-, TRIF (B)-, IKK (C)-, or TBK1(D)-expressing plasmid (200 ng) together with an increase amount of A20 expression plasmid (0, 40, 200, and 1000 ng) as indicated. In all transfections, the pcDNA3 vector was added to bring the total plasmids to 1500 ng. Luciferase activity was analyzed at 24-h post-transfection by the Dual-Luciferase Reporter assay as described by the manufacturer (Promega). Relative luciferase activity was measured as -fold activation (relative to the basal level of reporter gene in the presence of pcDNA3 vector after normalization with co-transfected RLU activity); values are mean ± S.D. for three experiments.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Silencing of A20 expression enhances virus-mediated activation of ISRE promoter. A, HEK293 cells were transfected with pSuper-control-RNAi vector or pSuper-A20-RNAi vector. Approximately 36 h after transfection, cells were infected with Sendai virus for 8 or 12 h as indicated. Whole cell extracts (60 µg) were prepared and subjected to SDS-PAGE and probed with anti-A20 and anti-actin antibodies. B, HEK293 cells were transfected and Sendai infected as described in A. Luciferase activity was analyzed by the Dual-Luciferase Reporter assay as described by the manufacturer (Promega). Relative luciferase activity was measured as -fold activation (relative to the basal level of reporter gene in uninfected cells after normalization with co-transfected RLU activity); values are mean ± S.D. for three experiments.

A20 Inhibits RIG-I-mediated IRF-3 Activation—Latent IRF-3 is activated in response to virus infection by phosphorylation events that target a cluster of Ser/Thr residues at the C-terminal end of the protein (23). Ser-396 within the C-terminal Ser/Thr cluster is targeted in vivo for phosphorylation following virus infection and plays an essential role in IRF-3 activation (23, 35). Therefore, the phosphorylation state of IRF-3 following RIG-I expression was evaluated by immunoblot using the phosphospecific Ser-396 antibody. RIG-I induced the accumulation of the Ser-396 phosphorylation (Fig. 4A, lane 3), and RIG-I-induced Ser-396 phosphorylation was completely block by A20 (Fig. 4A, lane 4). Expression of TRIF or TBK1 similarly induced Ser-396 phosphorylation (Fig. 4A, lanes 5 and 7); however, A20 only partially reduced TRIF- or TBK1-induced Ser-396 phosphorylation (Fig. 4A, lanes 6 and 8). Complementing the phosphorylation status, the ability of A20 to inhibit RIG-I-induced IRF-3 dimerization was evaluated using native SDS-PAGE and immunoblot with anti-IRF-3 antibody. RIG-I-induced dimerization of endogenous IRF-3 (Fig. 4B, lane 3) was completely abolished with A20 co-expression (Fig. 4B, lane 4). Again, A20 only partially reduced TRIF- or TBK1-induced IRF-3 dimer formation (Fig. 4B, lanes 5–8). Furthermore, EMSA analysis demonstrated that RIG-I, TRIF, or TBK1 expression induced the formation of an IRF-3 protein-DNA complex (Fig. 4C, lanes 3, 5, and 7) that was supershifted with antibody to IRF-3 (Fig. 4C, lane 8). A20 co-expression completely blocked RIG-I-induced IRF-3 protein-DNA complex formation (Fig. 4C, lane 4) but only partially reduced TRIF- or TBK1-mediated IRF-3 protein-DNA complex formation (Fig. 4C, lanes 5, 6, 9, and 10), strongly arguing that A20 specifically targets the RIG-I pathway upstream of TBK1 and does not significantly affect the TRIF-TLR3 pathway.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. A20 blocks RIG-I-mediated IRF-3 activation. A and B, HEK293 cells were transfected with 1 µg of pcDNA3-, RIG-I-, TRIF-, or TBK1-expressing plasmid together with 2 µg of pcDNA3 or A20-expressing plasmid as indicated above the lanes. Whole cell extracts (20 µg) were subjected to SDS-PAGE (A) or native PAGE (B) and probed with anti-IRF-3 Ser-396 phosphospecific antibody His-5033 to detect IRF-3 phosphorylation (A) or anti-IRF-3 antibody to detect IRF-3 dimerization (B). C, EMSA was performed with whole cell extracts (20 µg) derived from HEK293 cells transfected with 1 µg of pcDNA3-, RIG-I-, TRIF-, or TBK1-expressing plasmid together with 1 µg of IRF-3-expressing plasmid and 2 µg of pcDNA3- or A20-expressing plasmid as indicated above the lanes. The 32P-labeled probe corresponds to the ISRE motif (5'-GATCGGAAAGGGAAACCGAAACTGAAGCC-3') from ISG15 gene promoter. Anti-IRF-3 antibody was added as indicated to demonstrate the presence of IRF-3 in the protein-DNA complex, indicated by the arrows.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. C-terminal zinc finger domain of A20 is both necessary and sufficient to block RIG-I-mediated activation of ISRE promoter. The uppermost hatched bar depicts the 790-amino acid residue A20 open reading frame. The de-ubiquitination domain and seven zinc finger domains are indicated. Deletion mutants 1–380 (1), 1–517 (2), 1–680 (3), 1–740 (4), 373–790 (5), ZNF4 (537–624) (6), ZNF4/5 (537–674) (7), ZNF2/3 (424–536) (8), ZNF2–4 (424–624) (9), and ZNF2–5 (424–674) (10) are represented below the full-length protein (A, top right panel). The amino acids targeted for alanine substitutions are shown in large letters (A103, ZNF4 MT, and ZNF7 MT) (B, top right panel). Western blot analysis of whole cell extracts (20 µg) from HEK293 cells transiently transfected with expressing plasmids as indicated (A and B, top left panel). HEK293 cells were transfected with pRLTK control plasmid, ISRE-Luc reporter plasmid and the pcDNA3 vector or expression plasmid encoding RIG-I as well as A20 expression plasmid as indicated. Luciferase activity was analyzed at 24-h post-transfection by the Dual-Luciferase Reporter assay as described by the manufacturer (Promega). Relative luciferase activity was measured as -fold activation (relative to the basal level of reporter gene in the presence of pcDNA3 vector after normalization with co-transfected RLU activity); values are mean ± S.D. for three experiments (A and B, bottom panel).

To assess the role of the seven zinc finger motifs in inhibiting RIG-I-activation, a series of A20 zinc finger deletions and point mutations were examined. Deletion of one, two, or three internal zinc finger domains had essentially no effect on the inhibitory activity of A20; ZNF4, ZNF4/5, ZNF2/3, and ZNF2–4 were still able to inhibit RIG-I-mediated transactivation from 100- to 250-fold (Fig. 5B). In contrast, deletion of the one, two, or three C-terminal zinc finger domains (A20(aa1–740), A20(aa1–680), and A20(aa1–517)) reduced the capacity of A20 to inhibit RIG-I-mediated transactivation activity, resulting in inhibition of only 4.2-, 2.9-, and 2.8-fold, respectively.

The deletion analysis indicated that zinc finger domain 7 (ZNF7) was critical to A20-mediated inhibition. To test this idea, full-length A20 with point mutations of conserved cysteines within ZNF4 (C624/627A) or ZNF7 (C779/782A) as well as the A20 OTU mutant (C103A) were generated (Fig. 5B). Expression of A20 OTU mutant (A103) or the A20 ZNF4 mutant (ZNF4 MT) inhibited RIG-I -induced ISRE promoter activation to a level comparable to that obtained with wild-type A20 protein (Fig. 5B); notably, the A20 ZNF7 mutant (ZNF7 MT) had a limited capacity to inhibit RIG-I-mediated transactivation (<2-fold) (Fig. 5B). These results demonstrate that the ZNF7 motif of human A20 is absolutely required for inhibition of RIG-I-induced ISRE activation.

View larger version (46K): [in this window] [in a new window]   FIGURE 6. A20 weakly interacts with full-length RIG-I but not RIG-I. HEK293 cells were transfected with FLAG-tagged A20 together with Myc pcDNA3, Myc-tagged A20C (A20 373–790), Myc-tagged RIG-I, or Myc-tagged RIG-I (3.5 µg of each plasmid, as indicated above the lanes). At 24-h post transfection, whole cell extracts (300 µg) were prepared and immunoprecipitated with anti-Myc antibody 9E10; immunoprecipitated complexes were run on 7.5% SDS-PAGE and subsequently probed with anti-FLAG antibody M2 (top panel). The same membrane as in the top panel was also probed with anti-Myc antibody 9E10 (middle panel). Whole cell extracts (30 µg) were run on 7.5% SDS-PAGE and probed with anti-FLAG antibody (bottom panel) to assess the level of transgene expression.

A20 Does Not Interact with RIG-I—To determine if A20 could interact with RIG-I, TRIF, IKK, or TBK1, co-immunoprecipitation of A20 and RIG-I was performed with Myc- and FLAG-tagged proteins (Fig. 6). After immunoprecipitation of Myc-tagged proteins from cell extracts, immunoblot analysis revealed that FLAG-tagged A20 did not co-precipitate with Myc-tagged RIG-I (Fig. 6, lane 3) and only weakly interacted with full-length RIG-I (Fig. 6, lane 4). As a positive control, FLAG-tagged A20 co-precipitated with Myc-tagged A20 zinc finger domain (Fig. 6, lanes 2).

Because the inhibition of RIG-I-induced activation by A20 is mediated through its C-terminal ubiquitin ligase domain, we then examined whether A20 targeted RIG-I for degradation. Increasing A20 expression significantly reduced the level of TRIF (Fig. 7B, lane 4, bottom panel) and at high levels of A20, the amount of RIG-I was also reduced (Fig. 7A, lane 4, bottom panel). However, A20 had essentially no effect on the level of IKK or TBK1 expression (Fig. 7, C and D, lane 4, bottom panel). The complete loss of TRIF protein by A20 did not, however, correlate with the 2- to 3-fold inhibition of TRIF-mediated transactivation (Fig. 2B); furthermore, because A20 inhibition of RIG-I-mediated transactivation was far greater (>100-fold) than the decrease in RIG-I protein (2- to 3-fold), we speculate that the target of A20 is an as yet unidentified adapter of the RIG-I pathway. Furthermore, the capacity of A20 to inhibit RIG-I signaling but not TRIF-TLR3 signaling and yet target TRIF for degradation, is reminiscent of the effect of the hepatitis C virus NS3/4A protease on these two pathways (37, 38).

View larger version (36K): [in this window] [in a new window]   FIGURE 7. A20 does not significantly reduce the level of RIG-I protein. HEK293 cells were transfected with pRLTK control plasmid (100 ng), ISRE-Luc reporter plasmid (200 ng), RIG-I (A)-, TRIF (B)-, IKK (C)-, or TBK1(D)-expressing plasmid (200 ng) together with an increase amount of A20 expression plasmid (0, 40, 200, and 1000 ng) as indicated. In all of transfection, the pcDNA3 vector was added to bring the total plasmids to 1500 ng. Whole cell extracts (equivalent to 106 RLU) were resolved by SDS-PAGE and analyzed by Western blot with anti-FLAG antibody M2 (top panel) or anti-Myc antibody 9E10 (bottom panel) to assess the level of transgene expression.

To further evaluate the antiviral state, the stable HEK293 cell lines were infected with VSV, and viral protein expression was measured at different times after infection. In control or RIG-I-expressing cells, VSV proteins (nucleocapsid (N), surface glycoprotein (G), and matrix (M)) were detected at 8 h post-infection, whereas in RIG-I-expressing cells, VSV replication was significantly delayed with viral proteins detected only at a low level beginning at 12 h post-infection (Fig. 9A). Interestingly, in cells that expressed either A20 or NS3/4A, a restoration of the kinetics of VSV expression was observed, with viral proteins again detectable as early as 8 h post-infection (Fig. 9B). Taken together, these results demonstrate that A20 can efficiently block the RIG-I-mediated signaling pathway and down-regulate cellular antiviral response. Although the target of A20 remains unknown, the similarity between A20-mediated inhibition and HCV NS3/4A inhibition suggests that the cellular A20 and the viral NS3/4A may be targeting related components or adapters of the RIG-I pathway.

View larger version (47K): [in this window] [in a new window]   FIGURE 8. A20 represses Sendai virus- and RIG-I-induced expression of RIG-I and ISG56 genes. A, whole cell extracts (30 µg) prepared from uninfected, Sendai virus-infected or recombinant IFN 2b-treated pcDNA3- and A20-expressing HEK293T cells were subjected to SDS-PAGE and probed with anti-RIG-I, anti-ISG56, anti-FLAG (FLAG-A20), and anti-actin antibodies. B, whole cell extracts (30 µg) prepared from control, RIG-I-, RIG-I plus A20-, and RIG-I plus NS3/4A-expressing cell lines were subjected to SDS-PAGE and probed with anti-RIG-I, anti-ISG56, anti-FLAG (FLAG-A20 or FLAG-NS3/4A), anti-MYC (Myc-RIG-I), and anti-actin antibodies.

View larger version (38K): [in this window] [in a new window]   FIGURE 9. A20 repression of the RIG-I-induced antiviral state. The stable HEK293 cell lines, as indicated in A and B, were infected with VSV at a multiplicity of infection of 1 from 0 to 24 h post-infection. Whole cell extract (50 µg) was resolved by SDS-7.5% PAGE and transferred to nitrocellulose. Viral protein expression was measured by immunoblotting with anti-VSV antisera.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A20 was originally characterized as a TNF-inducible gene in human umbilical vein endothelial cells (52). As an NF-B target gene (53), A20 is also induced in many other cell types by a wide range of stimuli, including virus infection. Overexpression of A20 has been shown to protect from TNF--induced apoptosis and functions via a negative-feedback loop to block NF-B activation induced by TNF and other stimuli (54). A20-deficient mice (Tnfaip3^–/–) were generated, and these mice developed severe multiorgan inflammation and were extremely susceptible to TNF due to the enhanced sensitivity to TNF-induced apoptosis (55). A20-deficient fibroblasts displayed prolonged NF-B activity and were unable to properly terminate TNF-induced NF-B activation; A20 was also essential for the termination of TLR-induced NF-B activation macrophages (56). The inhibition of both IRF- and NF-B-dependent pathways suggests that A20 functions in the physiological context as a negative feedback regulator of immune response signaling. It will be of interest to determine the response of A20-deficient mice to pathogenic viruses.

View larger version (30K): [in this window] [in a new window]   FIGURE 10. A20 blocks MAVS/VISA/IPS-1/CARDIF-mediated transactivation. A, HEK293 cells were transfected with pRLTK control plasmid (100 ng), ISRE-Luc reporter plasmid (100 ng), together with an increase amount of NS3/4A or A20 expression plasmid (0, 100, 200, and 400 ng) as indicated. In all transfections, the pcDNA3 vector was added to bring the total plasmids to 600 ng. Luciferase activity was analyzed at 24-h post-transfection by the Dual-Luciferase Reporter assay as described by the manufacturer (Promega). Relative luciferase activity was measured as -fold activation (relative to the basal level of reporter gene in the presence of pcDNA3 vector after normalization with co-transfected RLU activity); values are mean ± S.D. for three experiments. B, NS3/4A but not A20 directly target MAVS/VISA/IPS-1/CARDIF. The soluble lysates and insoluble fractions prepared from A were equilibrated to the same volumes by SDS-PAGE loading buffer and analyzed by immunoblot with anti-Myc antibody 9E10, anti-FLAG antibody M2, and anti-actin antibody as indicated.

A20 was shown recently to be involved in negative regulation of TLR-3- and Sendai virus-mediated activation of IRF-3 (42). Wang and colleagues reported that A20 interacted with TRIF and inhibited TRIF- but not TBK1- and IKK-induced activation of ISRE and IFNB promoter (42). Saitoh and colleagues (41) demonstrated that A20 physically interacted with TBK1 and IKK and inhibited TLR-3- and Newcastle disease virus-mediated IRF-3 activation. The present studies confirm that A20-mediated inhibition of ISRE promoter activity correlated with the inhibition of IRF-3 activation; A20 completely blocked the RIG-I-induced IRF-3 phosphorylation, dimerization, and protein-DNA complex formation. However, A20 only minimally reduced TRIF- and TBK1-mediated IRF-3 activation and did not inhibit TBK1- or IKK-induced gene activation (Fig. 3). The fact that A20 had no effect on the stability of either RIG-I itself or on TBK1/IKK suggested that the biological target of A20 may be an as yet unidentified adapter molecule that links sensing of virus infection by RIG-I with the downstream kinase activation. In support of this concept, recent studies demonstrated that hepatitis C virus (HCV) gene product NS3/4A protease strongly inhibited virus- and RIG-I-mediated activation of NF-B and IRF-3 (37, 39), but NS3/4A only weakly inhibited TRIF-mediated induction of NF-B and IRF-3 (37). Paradoxically, TRIF was identified as a proteolytic substrate of NS3/4A (46) despite the fact that the TRIF pathway does not appear to be a major pathway for IFN response to HCV; in vitro, RIG-I was not a proteolytic substrate for NS3/4A, and expression of NS3/4A did not alter the stability of RIG-I, again in contrast to the strong inhibition of the RIG-I pathway by NS3/4A (37, 39). Recently, Meylan et al. (51) has shown that MAVS/VISA/IPS-1/CARDIF is a direct target by NS3/4A. Although A20 strongly inhibited RIG-I- and MAVS/VISA/IPS-1/CARDIF-mediated transactivation of IRF and NF-B pathways, no strong association between RIG-I and A20 or MAVS/VISA/IPS-1/CARDIF and A20 was detected by immunoprecipitation; A20 also only weakly inhibited TRIF-, TBK1-, or IKK-mediated transactivation, further implicating a distinct target for A20 interaction and function. Studies are underway to characterize other components of the RIG-I signaling pathway as potential targets of A20 regulation.

	FOOTNOTES

 
^* This work was supported in part by grants from the Cancer Research Society Inc. (to R. L.), Canadian Institutes of Health Research (to R. L. and J. H.), the Canadian Network for Vaccines and Immunotherapeutics (to J. H.), and by the National Cancer Institute of Canada, with the support of the Canadian Cancer Society (to J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1 and S2.

² Supported by a Post-doctoral Fellowship from the Fonds de la Recherche en Santé du Quebec.

³ Supported by a Canadian Institutes for Health Research Senior Investigator award.

¹ Supported by the Fonds de la Recherche en Santé du Quebec Chercheur-boursier: To whom correspondence should be addressed: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec H3T 1E2, Canada. Tel.: 514–340-8222 (ext. 5272); Fax: 514–340-7576; E-mail: rongtuan.lin{at}mcgill.ca.

⁴ The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; TLR, Toll-like receptor; RIG-I, retinoic acid-inducible gene I; CARD, caspase recruitment domain; HEK293, human embryonic kidney 293 cells; EMSA, electrophoretic mobility shift assay; ISRE, interferon-stimulated response element; VSV, vesicular stomatitis virus; OTU, ovarian tumor; RLU, Renilla luciferase activity units; HCV, hepatitis C virus; ZNF, zinc finger domain; aa, amino acid(s); STAT, signal transducers and activators of transcription; IKK; IKB kinase; TBK1; TANK-binding kinase 1; TRIF; TIR domain-containing adaptor-inducing interferon B.

	ACKNOWLEDGMENTS

 
We thank Hong-Bing Shu, Ganes Sen, and Peter Storz for reagents used in this study and members of the Molecular Oncology Group, Lady Davis Institute for helpful discussions.

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