A 5′ Extended IFN-Stimulating Response Element Is Crucial for IFN- −γ Induced Tripartite Motif 22 Expression via Interaction with IFN Regulatory Factor-1 This information is current as of October 1, 2021. Bo Gao, Yaxin Wang, Wei Xu, Zhijian Duan and Sidong Xiong J Immunol 2010; 185:2314-2323; Prepublished online 14 July 2010;

doi: 10.4049/jimmunol.1001053 Downloaded from http://www.jimmunol.org/content/185/4/2314

References This article cites 49 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/185/4/2314.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

by guest on October 1, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A59 Extended IFN-Stimulating Response Element Is Crucial for IFN-g–Induced Tripartite Motif 22 Expression via Interaction with IFN Regulatory Factor-1

Bo Gao,* Yaxin Wang,* Wei Xu,* Zhijian Duan,* and Sidong Xiong*,†

Interferon-g is crucial for the noncytopathic clearance of hepatitis B virus. In our previous study, we demonstrated that an IFN-g– inducible molecule, tripartite motif (TRIM) 22, played an important role in antiviral immunity against hepatitis B virus. However, the molecular mechanism of TRIM22 induction by IFN-g is still unclear. In this study, we identified a novel cis-element termed 59 extended IFN-stimulating response element (59 eISRE) that was crucial for IFN-g inducibility of TRIM22 through transfection assays with luciferase reporter constructs and EMSAs. The 59 eISRE consists of an ISRE-like motif (ACTTTCGTTTCTC) and a 6-bp sequence (AATTTA) upstream of it, and all three thymine triplets of this cis-element (AATTTAACTTTCGTTTCTC) were revealed to contribute to the IFN-g inducibility of TRIM22 by site-directed mutagenesis. Further studies showed that upon IFN-g Downloaded from stimulation, the 59 eISRE could be bound by IFN regulatory factor-1 (IRF-1), but not by STAT1, as demonstrated by supershift analysis and an ELISA-based assay. Moreover, overexpression of IRF-1 significantly induced TRIM22 expres- sion, whereas silencing of IRF-1 with specific short interference RNA abolished IFN-g–induced TRIM22 expression in HepG2 cells, indicating an IRF-1–dependent expression of TRIM22. Taken together, it was demonstrated in this study that a novel cis-element, 59 eISRE, was crucial for the IFN-g–induced transcriptional activity of the TRIM22 via interaction with IRF-1. The Journal of Immunology, 2010, 185: 2314–2323. http://www.jimmunol.org/

ripartite motif (TRIM) family are involved in In our previous study, we demonstrated that one of the TRIM diverse processes, including , differentiation, family members, TRIM22 (also named Staf50), could inhibit T and transcriptional regulation (1, 2). They are characterized hepatitis B virus (HBV) and replication efficiently by a combination of RING, B-box, and coiled-coil domains (1). The by significantly inhibiting the activity of HBV core in RING domain of many TRIM proteins has been shown to possess a RING domain-dependent manner (13). We also demonstrated that E3 ligase activity (2–5), whereas the B-box and coiled- TRIM22 was a RING finger E3 (4), and its E3 coil domains may be involved in interactions and homo/ ligase activity was responsible for its antiviral activity against ence- heterodimerization (1, 2). Recent studies have demonstrated that phalomycocarditis virus as reported by another research group (14). by guest on October 1, 2021 many members of the TRIM family play important roles in innate Additionally, several studies indicated that TRIM22 possessed anti- antiviral immunity. For example, TRIM5a has been shown to block retroviral activity depending on certain cell types (15–17). the infectivity of a range of different in a species- IFNs have an important role in immune system to defend against specific manner (6, 7); TRIM25 is essential for RNA helicase viral infections. They consist of two main classes of related cyto- RIG-I–mediated antiviral activity (5, 8); TRIM28 can inhibit the kines: type I IFNs and type II IFNs. Although there are many type I replication of murine leukemia viruses or related retroelements in IFNs, including IFN-a, IFN-b, IFN-l, etc., IFN-g is the only type II embryonic cells by transcriptional silencing (9, 10). It is speculated IFN family member (18–20). The biological effects of both IFN- that the TRIM family may represent a new, widespread class of a/b and IFN-g are mediated by IFN-stimulated (ISGs), but proteins involved in antiviral innate immunity (11, 12). the induction of ISGs by IFN-g is often more complex than that of IFN-a/b, largely due to the fact that many of the IFN-g–stimulated *Institute for Immunobiology, Department of Immunology, Shanghai Medical Col- genes are induced with variable kinetics unlike those stimulated by lege of Fudan University, Shanghai; and †Institute of Biology and Medical Sciences, IFN-a/b, and the expression of some IFN-g–regulated genes Soochow University, Suzhou, People’s Republic of China requires de novo protein synthesis (20–23). Received for publication March 31, 2010. Accepted for publication June 6, 2010. To date, nearly 70 TRIM family members have been identified, This work was supported by grants from the National Natural Science Foundation of and two classes of TRIMs can already be distinguished: IFN- China (30890141, 30872355, 30671952), the Science and Technology Commission of Shanghai Municipality (09JC1401800), the Doctoral Fund of Youth Scholars of inducible TRIMs and constitutive TRIMs (12). The IFN-inducible Ministry of Education of China (2009071120060), the Major State Basic Research TRIMs are involved in a broad range of biological processes that Development Program of China (2007CB512401), and the Program for Outstanding Medical Academic Leader of Shanghai (LJ06011, 07JC14004). are associated with innate immunity, and the constitutive TRIMs, such as TRIM1 and TRIM28, can also block virus replication at Address correspondence and reprint requests to Dr. Sidong Xiong, Institute of Bi- ology and Medical Sciences of Soochow University, 199 Ren ai Road, Suzhou, early stages. The two classes of TRIMs may contribute to the over- 215006, P.R. China. E-mail address: [email protected] all antiviral defense (11, 12). TRIM22 was demonstrated to be one Abbreviations used in this paper: Act.D, actinomycin D; ChIP, chromatin immuno- of the most strongly induced TRIMs in HepG2 cells after treatment precipitation; CHX, cycloheximide; eISRE, extended IFN-stimulating response ele- ment; GAS, IFN-g activation site; GBP, guanylate-binding protein; HBV, hepatitis B with IFNs in our previous study (13). Similar to our results, Barr virus; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; ISRE, IFN-stimulating et al. (15) found that TRIM22 was the most upregulated TRIM response element; NP-40, Nonidet P-40; siRNA, short interference RNA; TRIM, queried on the microarray prepared from IFN-treated human tripartite motif. osteosarcoma cells. Additionally, TRIM22 was also reported to Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 be strongly induced by IFNs in several other cell lines, such as www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001053 The Journal of Immunology 2315

Hela, MCF-7, and Daudi, etc. (17, 24). However, at present, little is plasmid was mixed with 3 ml Lipofectamine 2000 in serum-free medium known about how IFNs regulate TRIM22 gene expression. and added into HepG2 cells. In all transfection assays, pCMV–b-gal was cotransfected to normalize the transfection efficiency. Cells were incubated In this paper, we mainly focused our study on the molecular for 6 h at 37˚C in the presence of the transfection complex, washed twice mechanisms of transcriptional regulation of TRIM22 gene by with serum-free medium, and grown in fresh supplemented medium. For IFN-g. We identified a novel cis-element termed 59 extended IFN- IFN-g–treated samples, IFN-g was added 24 h posttransfection, and the stimulating response element (eISRE), which was crucial for the cultures were incubated for another 24 h. The cells were harvested, and the IFN-g inducibility of TRIM22. In addition, the 59 eISRE was also luciferase activity in the cell lysates was determined with the Luciferase Reporter Assay System (Promega). Reporter activities are presented as implicated in the constitutive transcriptional activity of TRIM22. means 6 SD of at least three independent experiments. Furthermore, we demonstrated that IFN regulatory factor-1 (IRF-1) could bind to the 59 eISRE both in vitro and in vivo and played a key Nuclear extract preparation role in the transcriptional regulation of TRIM22. A total of 5 3 106 IFN-g–treated or untreated HepG2 cells were washed in cold PBS and resuspended in 500 ml buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). Materials and Methods Postincubation for 15 min on ice, cells were lysed in 0.6% Nonidet P-40 Cell culture and stimulation (NP-40) by vigorous vortex for 10 s and centrifuged at 5000 rpm for 5 min. The human hepatoma HepG2 cells were maintained in DMEM supplemented Pellets contained the nuclei and were washed in buffer A two times and resuspended in 50 ml buffer C (20 mM HEPES [pH 7.9], 400 mM NaCl, with 10% FBS, 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin. 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF). After shaking for HepG2 cells were incubated at 37˚C with 100% humidity in 5–7% CO2 and passaged using standard cell-culture techniques. HepG2 cells were stimu- 30 min at 4˚C and centrifugation for 10 min at 10 000 rpm, supernatants were lated by IFN-g for various times or with different concentrations and then used as nuclear extracts. prepared for RT-PCR and immunoblot analysis. Downloaded from EMSA Semiquantitative RT-PCR and quantitative real-time RT-PCR A total of 5 mg nuclear proteins was preincubated on ice with 2 mg poly HepG2 cells were harvested, and total RNAwas isolated with TRIzol reagent (deoxyinosine-deoxycytosine) as an unspecific competitor and 1 mg soni- (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. One ficated sperm DNA in band shift buffer (50 mM Tris, 150 mM KCl, 5% microgram total RNA was reverse transcribed using a commercially avail- glycerol, 10 mM MgCl2, and 0.1% NP-40, 5 mM EDTA, 2.5 mM DTT) for able cDNA synthesis and oligo(dT) primer (MBI Ferments, St. Leon- 15 min. Biotin-labeled oligonucleotides (+28 to +52, GAGAATTTAAC-

Roth, Germany). Subsequently, cDNA was subjected to PCR in a 20 ml TTTCGTTTCTCACT) were then added in a total volume of 20 ml, in- http://www.jimmunol.org/ reaction mixture with the following primers: TRIM22, forward, 59-AC- cubated on ice for 20 min, and loaded onto 5% native polyacrylamide gels CAAACATTCCGCATAAAC-39 and reverse, 59-GTCCAGCACATTCAC- in 0.53 Tris-borate–EDTA buffer. The gels were blotted on nylon mem- CTCAC-39; and GAPDH, forward, 59-ATCCCATCACCATCTTCCAG-39 brane, and the blot was cross-linked by UV irradiation. Biotin-labeled and reverse, 59-GAGTCCTTCCACGATACCAA-39. The cycling parame- probe was detected by a Lightshift Chemiluminescent EMSA kit (Pierce) ters were as follows: denaturation at 94˚C for 5 min, amplification at 94˚C according to the manufacturer’s recommendations. In competition assays, for 30 s, 58˚C for 30 s, and 72˚C for 30 s for 32 cycles. PCR products were a 5 or 25 M excess of unlabeled competitor oligonucleotides was added subjected to electrophoresis by using 2% agarose gels in Tris-borate–EDTA prior to the addition of the probe to the mixture, which was then preincu- and visualized by ethidium bromide staining. Real-time quantification of bated on ice for 15 min. For supershift experiments, 2 mg STAT1, STAT2, cDNA targets was performed using a Light Cycler 480 and SYBR Green STAT3, IRF-1, IRF-2, or IRF-9 was added to the preincubation mixture, system (Roche Diagnostic Systems, Mannheim, Germany) according to the and the preincubation time was extended to 30 min. manufacturer’s instructions. TRIM22 expression was calculated following by guest on October 1, 2021 normalization to GAPDH levels by the comparative DD threshold cycle ELISA-based transcription factor assay method. The specificity of the amplification reactions was confirmed by The binding of above-mentioned transcription factors to the 59 eISRE melt curve analysis. Results were representative of three independent was further assessed with the ELISA-based transcription factor assay ac- experiments. cording to the method as described by McKay et al. (26). In brief, 200 ng biotin-labeled oligonucleotides (+28 to +52) was added into each well of Western blotting a streptavidin-coated microtiter plate (Boehringer, Mannheim, Germany). Protein was denatured in SDS, electrophoresed on SDS-PAGE (8 or 10% The plate was incubated at room temperature for 1 h, then immersed in wash gel), and transferred onto a nitrocellulose membrane. Nonspecific binding buffer (0.05% Tween in PBS) and washed three times for 10 min in a shaking bath at room temperature. Each well of the plate was then incubated at 37˚C was blocked with TBST [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% 3 Tween 20] containing 5% (w/v) nonfat milk for 2 h at room temperature. for 30 min with 30 mg nuclear extract in 60 ml1 protein binding buffer After overnight incubation at 4˚C with indicated Ab, membranes were (25 mM HEPES–KOH [pH 7.5], 50 mM KCl, 4 mM MgCl2, 20% glycerol, washed three times with TBST and incubated at room temperature for 250 mg/ml BSA, 250 mg/ml poly[deoxyinosine-deoxycytosine]). After 2 h with the corresponding HRP-conjugated secondary Ab diluted washing, 60 ml primary Ab (1:1000) in Ab dilution buffer-2% BSA, 0.05% 1:1000 in blocking buffers. After washing, signals were detected by an Tween in PBS was incubated with each well at room temperature for 1 h. The ECL kit (Pierce, Rockford, IL). bound Abs were detected with HRP-labeled secondary Ab (1:5000) followed by measurement of enzymatic color reaction in a standard microplate reader. Plasmid constructions Chromatin immunoprecipitation A series of deletions of the TRIM22 promoter were generated by PCR Chromatim immunoprecipitation (ChIP) experiments were performed amplification using Takara Ex Taq DNA polymerase (Takara, Dalian, China) according to the manufacturer’s recommendations (Upstate Biotechnology, with the TRIM22 promoter dependent luciferase plasmid [which has been Lake Placid, NY). In brief, 5 3106 HepG2 cells were incubated with or donated by Professor Urban Gullberg (Lund University, Lund, Sweden) (25), without IFN-g (1000 U/ml) for 6 h. The cells were fixed with 1% designated as pLuc-500 in this study] as a template. All of the PCR frag- formaldehyde for 10 min at room temperature to cross-link transcription ments were inserted into the SacI and HindIII sites of the pGL3 Basic vector factors to DNA. The cross-linking reaction was stopped by adding glycin (Promega, Madison, WI), and these reporter plasmids are designated as with a final concentration of 0.125 M. After washing by cold PBS, the cells pLuc-400 (2254 to +146), pLuc-320 (2174 to +146), pLuc-240 (294 to were resuspended in cell lysis buffer [10 mM Tris-HCl (pH 8), 10 mM +146), pLuc-160 (214 to +146), and pLuc-80 (+67 to +146) according to NaCl, 0.2% NP-40 and protease inhibitors), and nuclei were centrifuged at the length of the remaining 59-flanking region. of ISRE1, IFN-g 2500 rpm for 5 min. The nuclei were isolated and sonicated on ice to shear activation site (GAS), and 59 eISRE were performed using QuikChange the DNA to 200–1000 bp. A small aliquot (20 ml) was saved as input DNA Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). for PCR analysis by reversing histone-DNA cross-links by heating at Transient expression and the promoter reporter assay 65˚C for 4 h. Chromatin was immunoprecipitated from 200 ml aliquots at 4˚C by mild agitation overnight with 5 mg Abs specific for IRF-1, IRF-9, HepG2 cells were transfected using Lipofectamine 2000 as recommended STAT1, STAT2, STAT3, IRF-2, or IRF-3. Immune complexes were col- by the manufacturer (Invitrogen). Briefly, HepG2 cells were plated at 1.5 3 lected by incubation with protein A agarose. To analyze the target region, 105 cells per well in a six-well plate 24 h pretransfection. A total of 1.5 mg the immunoprecipitated chromatin DNA samples were amplified by PCR 2316 A 59 eISRE CONFERRED IFN-g INDUCIBILITY OF TRIM22 using the primer pair forward, 59-TTCCCCAGGGTTTATTGTTATG-39,and of ISRE2 within the region (214 to +66) abolishes reverse, 59-GCCG GTGTAAACCAGATTCAC-39,spanningthe59-flanking IFN-g inducibility and significantly decreases the basal 2 region of TRIM22 ( 21 to +93) containing 59 eISRE. transcriptional activity of TRIM22 Short interference RNA assay Toidentify thecis-acting elementsrequiredfortheIFN-g inducibility Transfection of short interference RNAs (siRNAs) targeting IRF-1 or of TRIM22, a series of DNA sequences derived from the 59 flanking IRF-9 was performed using Lipofectamine 2000 with a final concentration region of the TRIM22 gene were produced and cloned into the of siRNA of 100 nM according to the manufacturer’s instructions. The promoterless luciferase reporter plasmid pGL3-Basic (Fig. 2A, left siRNAs targeting IRF-1 or IRF-9 and the scrambled siRNA were pur- panel). These luciferase reporter plasmids were then transiently chased from Santa Cruz Biotechnology (Santa Cruz, CA). transfected into HepG2 cells, treated with IFN-g or left untreated, Statistics and assayed for luciferase activity. Reporter assays revealed 6 a significantly increased transcriptional activity of the construct Results were reported as means SD. t test was applied to comparisons 2 between groups; a p value ,0.05 was considered statistically significant. pLuc-500 ( 354 to +146) as compared with the empty vector, and an induction of the transcriptional activity by ∼5-fold after IFN-g stimulation, indicating the 500-bp 59 flanking region of TRIM22 Results gene was sufficient to ensure high levels of expression and IFN-g TRIM22 is transcriptionally regulated by IFN-g, requiring de inducibility. It was also found that the IFN-g inducibility was not novo protein synthesis significantly altered by the 59 deletions leading to the 400, 320, 240, We first examined the expression of TRIM22 gene induced by and 160 bp fragments. In contrast, the IFN-g inducibility was

IFN-g in human hepatoma HepG2 cells. It was found that the completely lost in the case of the construct pLuc-80 (+67 to +146), Downloaded from expression of TRIM22 could be induced by IFN-g in a dose- and this construct only retained ∼25% of the transcriptional activity dependent manner at both the mRNA and protein level (Fig. 1A, of the construct pLuc-160 (214 to +146) (Fig. 2A, right panel), in- 1B). To determine whether the induction of TRIM22 gene by IFN-g dicating the region (–14 to +66) was of critical importance not only was a transcriptional response, we treated HepG2 cells with for the IFN- g inducibility, but also for the constitutive expression of actinomycin D (Act.D), an inhibitor of gene transcription. It was TRIM22 gene. found that pretreatment with Act.D could abolish the IFN-g– The region (214 to+66) was then subjected to computer searchand http://www.jimmunol.org/ induced TRIM22 mRNA expression at the indicated time points, manual analysis based on sequence homologies to identify potential implying that the induction was at the transcriptional level (Fig. 1C). transcription factor binding sites. As shown in Fig. 2B, one region was To further characterize the induction of TRIM22 by IFN-g, found matching GAS consensus sequence TTCNNNG/TAA, GAS cycloheximide (CHX), an established inhibitor of protein syn- (+24 to +32, TTCTGAGAA), and two regions that match ISRE thesis, was added to cells pretreatment with IFN-g. As shown in consensus sequence, AGTTTCN(1–2)TTTCNY: ISRE1 (+16 to +28, Fig. 1D, pretreatment of HepG2 cells with CHX dramatically TCTTTCACTTCTG) and ISRE2 (+37 to+49, ACTTTCGTTTCTC). decreased levels of TRIM22 mRNA at the indicated time points, To investigate the role of each putative IFN-g–responsive element in suggesting that de novo protein synthesis was essential for IFN-g– the transcriptional regulation of TRIM22 gene, mutations were induced TRIM22 gene expression. introduced into the above sites with reporter plasmid pLuc-500 or by guest on October 1, 2021

FIGURE 1. The TRIM22 gene is transcrip- tionally regulated by IFN-g, requiring de novo protein synthesis. A, HepG2 cells were treated with the indicated concentrations of IFN-g for 24 h. TRIM22 mRNA levels were determined by semiquantitative PCR (bottom panel) and quantitative real-time PCR (top panel) with GAPDH used as an internal control. B, TRIM22 protein levels were determined by Western blot using anti-TRIM22 or anti-actin in HepG2 cells posttreatment with the indicated concentrations of IFN-g for 24 h. C and D, HepG2 cells were pretreated for 1 h with 1 mg/ml Act.D or 20 mg/ ml CHX, followed by treatment with 1000 U/ml IFN-g for 6, 12, and 24 h or left untreated. TRIM22 mRNA levels were determined by semiquantitative PCR (bottom panels) and quantitative real-time PCR (top panels) as men- tioned above. The Journal of Immunology 2317

FIGURE 2. Mutation of ISRE2 within the region (214 to +66) abolishes the IFN-g inducibility and decreases the constitutive expression of TRIM22 gene. A, PCR pro- ducts from the 59 flanking region of TRIM22 gene were cloned into SacI and HindIII sites of the pGL3 basic and named pLuc-500, pLuc- 400, pLuc-320, pLuc-240, pLuc-160, and pLuc-80, respectively. HepG2 cells trans- fected with these constructs were then treated with or without 1000 U/ml IFN-g for 24 h. Luciferase activity in the cell lysate was measured according to the manufacturer’s instructions. In all transfection assays, pCMV– b-gal was cotransfected to normalize the trans- Downloaded from fection efficiency. The activity of unstimulated pLuc-500 was set to 1. The data are repre- sentative of three independent experiments. B, Schematic representation of the ISRE1, GAS, or ISRE2 within the region (214 to +66) in the 59-flanking region of TRIM22 http://www.jimmunol.org/ gene. C, Mutations at these cis-elements were introduced to pLuc-500 and pLuc- 160, respectively, and HepG2 cells trans- fected with these constructs were stimulated with or without IFN-g (1000 U/ml) for 24 h. Luciferase activity in the cell lysate was measured as mentioned above. The activity of unstimulated pLuc-500 was set to 1. The data are representative of three independent experiments. by guest on October 1, 2021

pLuc-160 as the template, respectively (Fig. 2B), and the transcrip- transcriptionally active sequences can be cloned upstream of the tional activity of the resulting constructs was examined by transient SV40 promoter. The reporter plasmid was then assayed for the transfection experiments. Results showed that mutation at ISRE1 had ability to respond to IFN-g as described above. The result showed no influence on the IFN-g inducibility of TRIM22. In contrast, that the region (+35 to +52), which harbors ISRE2, failed to confer mutation at ISRE2 abolished the IFN-g inducibility and led to the the IFN-g inducibility (Fig. 3A, right panel), therefore we added decrease in the basal transcriptional activity of TRIM22 gene. nucleotides upstream of the region (+35 to +52) according the Unexpectedly, mutation at GAS did not have significant influence sequence in the 59 flanking region of TRIM22 gene. It was found on the IFN-g inducibility (Fig. 2C). These results suggested that that addition of nucleotides to the position +33 could increase the IRES2 might be required for the basal and IFN-g–induced tran- IFN-g inducibility markedly, and addition of nucleotides to the scriptional activity of TRIM22 gene. position +31 could further increase the effect, but further addition of nucleotides upstream of the region (+31 to +52) to the position ISRE2 per se is not sufficient, whereas a 6-bp sequence 59 +26 failed to further increase the effect (Fig. 3A). We also tested the adjacent to ISRE2 is required to confer IFN-g inducibility of contribution of the nucleotides at the 39 end of the region (+26 to TRIM22 gene +52) to the IFN-g inducibility by deletion analysis and found that Transient transfection experiments suggested but did not prove that deletion of three nucleotides at the 39 end to the position +49 had no the IRES2 contributed to the IFN-g–induced transcriptional activity influence on the IFN-g inducibility, but further deletion of of TRIM22 gene. To corroborate this suggestion, a synthetic nucleotides at the 39 end nearly abolished the IFN-g inducibility double-stranded oligonucleotide spanning the region (+35 to +52) (Fig. 3A). We also tested the IFN-g inducibility of the ISRE of and containing the ISRE2 motif (ACTTTCGTTTCTC) was inserted human 6-16 gene upon the SV40 promoter and found that the ISRE into the pGL3-Promoter reporter plasmid (Fig. 3A, left panel). This of human 6-16 gene also conferred IFN-g inducibility upon a het- plasmid contains a luciferase reporter gene under the control of erologous promoter (Fig. 3A), consistent with the results reported an SV40 promoter without enhancer sequences, and putative by Reid et al. (27). 2318 A 59 eISRE CONFERRED IFN-g INDUCIBILITY OF TRIM22 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. The 59 eISRE, not ISRE2, confers the IFN-g inducibility. A, Double-stranded synthetic oligonucleotides as shown in left panel but with SacI and BglII overhands were inserted into the pGL3-Promoter reporter plasmids. HepG2 cells transfected with these constructs were then treated with or without 1000 U/ml IFN-g for 24 h. Luciferase activity in the cell lysate was measured according to the manufacturer’s instructions. In all transfection assays, pCMV–b-gal was cotransfected to normalize the transfection efficiency. The activity of the unstimulated construct that harbors the region (+35 to 52) was set to 1. The data are representative of three independent experiments. B, Mutations of the first or third thymine of 59 eISRE and nucleotides downstream of 59 eISRE were introduced to pLuc-160, as indicated in the left panel, and HepG2 cells transfected with these constructs were stimulated with or without IFN-g (1000 U/ml) for 24 h. Luciferase activity in the cell lysate was measured as mentioned above. The activity of unstimulated pLuc-160 was set to 1. The data are representative of three independent experiments.

The thymine triplets of ISRE have been reported to play important of TRIM22 gene. Using site-directed mutagenesis analysis, we found roles in the IFN-inducibility (28, 29), and the above-mentioned result the thymine triplet (+33 to +35) was also crucial for the IFN-g also demonstrated that mutation of a thymine triplet of IRES2 induciblity of the TRIM22 gene (Fig. 3B). We also investigated (GAGAATTTAACTTTCGTTTCTCATC, ISRE2 was shown in the contribution of the thymine triplet (+44 to +46, GAGAATTT- bold) disrupted the IFN-g responsiveness of TRIM22 (Fig. 2B,2C). AACTTTCGTTTCTCATC) and the nucleotides downstream of As there is an additional thymine triplet (+33 to +35) upstream of IRES2 (+50 to +51, GAGAATTTAACTTTCGTTTCTCATC) to ISRE2 (GAGAATTTAACTTTCGTTTCTCATC), we suspected that the IFN-g inducibilty of TRIM22. As shown in Fig. 3B, the thymine this thymine triplet might also contribute to the IFN-g responsiveness triplet (+44 to +46) was crucial for the IFN-g inducibility of the The Journal of Immunology 2319

TRIM22 gene, whereas the nucleotides downstream of ISRE2 to the 59 eISRE, we performed supershift assay with Abs against seemed to be dispensable. STAT1, STAT2, STAT3, IRF1, IRF2, IRF3, and IRF9. As shown in Taken together, these results indicated that at least six additional Fig. 5A, the major DNA–protein complex, C1, was shifted by the nucleotides (AATTTA) upstream of ISRE2 were required, whereas anti–IRF-1 Ab, and the minor complex, C2, was shifted by anti- the nucleotides downstream of ISRE2 seemed to be dispensable, and IRF9 Ab, whereas other Abs failed to shift the protein complexes. all three thymine triplets contributed to the IFN-g inducibility of To further confirm the results, a more sensitive ELISA-based assay TRIM22 gene. As this novel IFN-g response element consisted of was used to detect the transcription factors binding to the biotin- ISRE2 plus six additional nucleotides upstream of it, we therefore labeled probe. The results showed that IRF1 could bind to the immo- named it 59 eISRE. bilized probe very efficiently. Consistent with the EMSA results, the binding occurred within 30 min with its peak value at 6 h after the The 59 eISRE, not ISRE2 or GAS, binds to nuclear proteins IFN-g stimulation, and the binding could be continuously detected efficiently in response to IFN-g treatment over the 24 h after the IFN-g addition. IRF9 could also bind the To analyze the binding of nuclear proteins to the 59 eISRE in response probe, although to a lesser extent, whereas other transcription to IFN-g treatment, we prepared nuclear extracts from HepG2 cells factors did not show significant binding (Fig. 5B). at various times after the addition of 1000 U/ml IFN-g and To investigate the expression of these transcription factors in performed EMSA with the biotin-labeled fragment from bp +28 to HepG2 cells after IFN-g stimulation, Western blot was performed +52 which contains the 59 eISRE as a probe (Fig. 4A). As shown in to detect their expression in nuclear extracts from HepG2 cells at Fig. 4B, two DNA–protein complexes, C1 and C2, were formed various times after the addition of 1000 U/ml IFN-g.Wellin- within 30 min of IFN-g stimulation with its peak value at 6 h post- duction of these transcription factors by IFN-g was demonstrated stimulation, and the DNA-protein complexes could be continuously (Fig. 5C), thus excluding the possibility that the inability of other Downloaded from detected over the 24 h after the IFN-g addition. The binding of transcription factors, such as STAT1, to bind to the 59 eISRE was due nuclear proteins to the probe was specific because the binding was to their weak expression in IFN-g–induced HepG2 cells or the poor abolished by an excess of wild-type competitor, but not by an excess quality of the Abs. of competitor (Fig. 4C). We also performed competition To further determine which transcription factor might bind to the assays with an excess of ISRE2 or GAS competitors, and it was 59 eISRE in living cells, ChIP assays were used to precipitate found that both ISRE2 and GAS competitors failed to block the transcription factor–DNA complexes with various Abs as men- http://www.jimmunol.org/ interaction between the IFN-g–induced proteins and the biotin- tioned above. The immunoprecipitated DNA was then subjected to labeled 59 eISRE (data not shown). Furthermore, both biotin-labeled PCR analysis using primers flanking the 59 eISRE sequence. As ISRE2 and GAS (Fig. 4A)failedtobindtotheIFN-g–induced shown in Fig. 5D, IRF-1 (and to a lesser extent IRF-9) could bind to nuclear proteins efficiently as shown in Fig. 4D. the 59-flanking region, which contains 59 eISRE of the TRIM22 gene, whereas other transcription factors, such as STAT1, could not. IRF-1 binds to the 59 eISRE The ISRE motif was originally described in the promoters of several The critical role of IRF-1 in the transcription activity of the IFN-inducible genes and is the recognition site for multiprotein TRIM22 gene complex IFN-stimulated gene factor 3, composed of IRF-9 and To investigate the role of IRF-1 and IRF-9 in the expression of by guest on October 1, 2021 the phosphorylated STAT1 and STAT2 (30, 31). The ISRE motif TRIM22, we constructed the expression plasmid for IRF-1 or also binds to other IRF family members, such as IRF-1 and IRF-2 IRF-9, respectively, by cloning the appropriate PCR fragments in (32–34). To further characterize the protein complexes binding the pCDNA3.1 vector between NheI–HindIII. The expression of

FIGURE 4. The 59 eISRE, not ISRE2 or GAS, binds to the IFN-g–induced nuclear proteins. A, Biotin-labeled probes or competitors used in EMSA are illustrated. B, Nuclear extracts from HepG2 cells treated with 1000 U/ml IFN-g for the indicated times were analyzed by EMSA with the pISRE-E probe. The arrows indicate specific DNA–protein complexes induced by IFN-g and the free probe. C, Competitive EMSA was performed with 5- or 25-fold molar excess of wild-type competitor (cISRE-E) or mutant competitor (cISRE-Emut) to determine the binding specificity. D, Nuclear extracts from HepG2 cells treated with 1000 U/ml IFN-g for 6 h, and EMSA was performed with probe pISRE-E, pISRE2, and pGAS, respectively. 2320 A 59 eISRE CONFERRED IFN-g INDUCIBILITY OF TRIM22

FIGURE 5. Identification of transcription factors binding to the 59 eISRE. A, Supershift assays were performed with the pISRE-E Downloaded from probe and nuclear extracts from HepG2 cells treated with 1000 U/ml IFN-g for 6 h. The Abs against STAT1, STAT2, STAT3, IRF1, IRF2, IRF3, and IRF9 used for the supershift are indicated at the top of the gel. The IRF-1 or IRF-9 complex and super-

shifted complexes were indicated by the http://www.jimmunol.org/ arrows. B, The binding of above-mentioned transcription factors to the pISRE-E probe was confirmed with an ELISA-based assay as described in Materials and Methods. C, Western blot assays of STAT1, STAT2, STAT3, IRF1, IRF2, IRF3, and IRF9 were performed in the nuclear extracts from HepG2 cells treated with IFN-g for the indicated times with histone H1 as loading control. D, Formaldehyde cross-linked chro- by guest on October 1, 2021 matin was prepared from control and IFN-g– treated HepG2 cells. ChIP assays were performed using the indicated Abs. Immu- noprecipitated chromatin was subjected to PCR analysis using TRIM22-specific primer. The equivalent amount of chromatin in the immunoprecipitations was monitored by PCR amplification of input chromatin as an internal control. ChIP assay was performed at least three times. The Journal of Immunology 2321 rIRF-1 or IRF-9 was confirmed by immunoblotting (Fig. 6A). We into HepG2 cells together with 100 ng IRF-1, IRF-9 specific, or then tested the effect of IRF-1 or IRF-9 on the transcriptional activity control siRNA, respectively. Twenty-four hours posttransfection, of TRIM22 promoter by cotransfecting the luciferase reporter plas- the transfected HepG2 cells treated with IFN-g for additional mid pLuc-160 into the HepG2 cells together with increasing 24 h. Results showed that the silence of IRF-1 abolished the IFN-g amounts of pcDNA-IRF-1 or pcDNA-IRF-9. It was found that over- inducibility and even could lead to the decrease in the constitutive expression of IRF-1 increased the transcriptional activity of transcriptional activity of the TRIM22 gene. However, silencing the pLuc-160 in a dose-dependent manner, whereas overexpression of expression of IRF-9 did not have significant influence on the IFN-g IRF-9 did not have significant influence on the activity of pLuc-160 inducibility of the TRIM22 gene (Fig. 6E). Furthermore, IRF-1 (Fig. 6B). Cotransfection with the reporter plasmid pLuc-160 siRNA, but not IRF-9 siRNA, completely inhibited the induction ISRE2mut and IRF-1 or IRF-9 demonstrated the failure of both of TRIM22 mRNA by IFN-g in HepG2 cells (Fig. 6F). IRF-1 and IRF-9 to stimulate the transcriptional activity of pLuc-160 Taken together, these results indicated the critical role of ISRE2mut in HepG2 cells (Fig. 6B). Further study showed that over- IRF-1 in the transcriptional activation of TRIM22 gene, which may expression of IRF-1 significantly upregulated the mRNA level of explain the dependence on new protein synthesis for IFN-g– TRIM22 in HepG2 cells, whereas IRF-9 could not (Fig. 6C). induced TRIM22 expression as shown in Fig. 1D. To further investigate the role of IRF-1 and IRF-9 in the IFN-g inducibility of the TRIM22 gene, we silenced their expression via Discussion RNA interference. Functionality of the siRNAs directed against HBV replication is blocked noncytolytically by both IFN-a/b and IRF-1 and IRF-9 in HepG2 cells was confirmed by Western blot IFN-g (35–37). Although IFN-a/b is not strongly induced during analysis: protein levels of IRF-1 and IRF-9 following IFN-g acute HBV infection of chimpanzees, IFN-g from intrahepatic Downloaded from treatment were strongly reduced in the presence of specific immune cells plays a central role in controlling virus replication siRNA compared with the unspecific control siRNA (Fig. 6D). (38, 39). TRIM22, for which expression is correlated with HBV We then cotransfected the luciferase reporter plasmid (pLuc-160) clearance in acutely infected chimpanzees (38, 39), was found to http://www.jimmunol.org/

FIGURE 6. IRF-1 plays a crucial role in the transcriptional activity of TRIM22. A,HepG2 cells were transfected with empty vector pcDNA, pcDNA/IRF-1, or pcDNA/IRF-9, re- spectively. Twenty-four hours posttransfection, the expression level of IRF-1 or IRF-9 in the transfected cell lysate was determined by im-

munoblotting with actin as the loading control. by guest on October 1, 2021 B, HepG2 cells were cotransfected with lucif- erase reporter plasmid, pLuc160, or pLuc160- mut, with increasing amount of pcDNA/IRF-1 or pcDNA/IRF-9. Twenty-four hours post- transfection, the cell lysate was tested for lucif- erase activity, and the luciferase activity of pcDNA transfected cells was set to 1. The data are representative of three independent experiments. C, HepG2 cells were transfected with increasing amount of pcDNA/IRF-1 or pcDNA/IRF-9. Twenty-four hours posttrans- fection, levels of TRIM22 mRNA were deter- mined by semiquantitative RT-PCR. D,HepG2 cells were transfected with scrambled siRNA (control), IRF-1–, or IRF-9–specific siRNA. After 96 h, cells transfected with each type of siRNA were treated with IFN-g or control medium for 24 h. Protein expression of IRF-1 or IRF-9 was detected by immunoblotting. E, HepG2 cells were cotransfected with reporter plasmid, pLuc160, and control,IRF-1, or IRF-9 siRNA, respectively. Posttreatment with 1000 U/ml IFN-g for 24 h, the transfected cells were lysed and tested for the luciferase activity. F, HepG2 cells were transfected with control, IRF-1, or IRF-9 siRNA. After 96 h, cells trans- fected with each type of siRNA were treated with IFN-g or control medium for 24 h, levels of TRIM22 mRNA were determined by semiquantitative RT-PCR. 2322 A 59 eISRE CONFERRED IFN-g INDUCIBILITY OF TRIM22 play an important role in antiviral immunity against HBV in our to the IFN-a–induced TRIM22 expression. We found that, similar previous study (13). Interestingly, we also found that TRIM22 was to its role in IFN-g–induced TRIM22 expression, the 59 eISRE also strongly induced by IFN-g in human hepatoma HepG2 cells, played an important role in the IFN-a inducibility of the TRIM22 indicating its role as a downstream effector in IFN-g–mediated anti- gene, as mutation of any of the thymine triplets of the 59 eISRE HBV immune responses. However, how IFN-g induces the expression abolished this inducibility. Upon IFN-a stimulation, it was of TRIM22 remains obscure. In this study, we investigate the IRF-1, not STAT1, that bound to the 59 eISRE, and IRF-1 siRNA molecular mechanism of TRIM22 induction by IFN-g. could abolish the IFN-a–induced TRIM22 expression (data not With deletion analysis, we narrowed down the IFN-g responsive shown). Interestingly, several other ISGs, such as 9-27 gene, human region of the TRIM22 gene to an 80-bp region (214 to +66). In- ISG20, and human STAT1 (27, 32, 46), are also induced by both terestingly, this region also contributed to the basal transcriptional IFN-a and IFN-g via a similar molecular mechanism, which may activity of TRIM22 gene. Inspection of the region (214 to +66) by be due to the cross talk between the signaling pathways activated by computer search and manual sequence analysis revealed three puta- both types of IFN. tive regulatory motifs, including one GAS and two ISRE elements. It TRIM22 gene is located in 11p15 in a cluster with is known that the majority of responsive genes are induced by IFN-g other TRIM genes including TRIM3, TRIM5, TRIM6, TRIM21, and through the interaction of STAT1a homodimers with GAS element, TRIM34. It is of interest that all of these TRIMs are IFN-inducible and, in some cases, ISRE can also contribute to the IFN-g inducibility genes, suggesting they may have coevolved to coordinate important (18, 21). In this study, however, we found the GAS element seemed to antiviral functions (11, 47). TRIM5 was particularly related with be dispensable for the IFN-g induction of TRIM22 gene, as mutation TRIM22, as TRIM5 is adjacent to TRIM22 in a head-to-head di- of the GAS element could not significantly decrease the IFN-g– rection in with only a 4.8-kb distance, and they have induced transcriptional activity, and this cis-element was unable to evolved under positive selection in a mutually exclusive fashion (48). Downloaded from bind to the IFN-g–induced nuclear proteins as demonstrated by Asaoka et al. (49) have investigated the molecular mechanisms of EMSA experiments. TRIM5a induction by IFNs. They identified a functional ISRE in the A more interesting finding of this study is that a novel cis-element TRIM5a promoter that could be bound by STAT1 upon IFN-b 59 eISRE, which consists of ISRE2 (AGTTTCGTTTCTC) and six stimulation. However, the ISRE of TRIM5a does not have the nucleotides (AATTTA) upstream of it, was crucial for the IFN-g characteristics of an eISRE, and the interaction between this ISRE inducibility. First, when cloned into the reporter plasmid pGL3- and other transcriptional factors, such as IRF-1, has not been inves- http://www.jimmunol.org/ Promoter immediately upstream of SV40 promoter, 59 eISRE, not tigated. As the majority of TRIM family members are IFN-inducible ISRE2, was found to be sufficient to confer IFN-g inducibility genes, and the molecular mechanisms of the induction of most TRIM on a heterologous promoter. Second, site-directed mutagenesis dem- family members by IFNs remain largely unknown (11, 12, 47), it is onstrated that besides two thymine triplets in ISRE2 (AATT- of interest to investigate whether the ISRE of other TRIM family TAAGTTTCGTTTCTC), the thymine triplet upstream of ISRE2 members possesses the features of eISRE and whether IRF-1 plays (AATTTAAGTTTCGTTTCTC) was also required for IFN-g induci- roles in IFN-induced expression of other TRIM family molecules. bility of TRIM22. Third, EMSA experiments showed that it was 59 Because TRIM22 has been demonstrated with strong inhibitory eISRE, not ISRE2, that could bind to the IFN-g–induced nuclear effect on HBV replication, the 59 eISRE might possibly be involved by guest on October 1, 2021 proteins efficiently. Similar to our present findings, the ISRE of in the anti-HBVactivity of the IFN-g–induced TRIM22. It is known several ISGs, such as guanylate-binding protein (GBP), IL-12, that the abilities of spontaneous clearance of viral infection b2-microglobulin, and caspase-8 (34, 40–42), also has the features of after HBV exposure differ significantly among various subjects an eISRE, although the contribution of the sequence adjacent to (37). Our preliminary data showed that TRIM22 expression varied the conventional ISRE for the IFN inducibility of those ISGs has not among different individuals, and more strikingly, the IFN-induced been determined. Additionally, there also exist differences in DNA TRIM22 expression was individual specific (data not shown). Such sequence between the 59 eISRE of TRIM22 and the eISRE of other variations in TRIM22 expression and IFN inducibility in liver cells ISGs. For example, the eISRE of GBP comprises a conventional may contribute to differences in response to treatment of HBV ISRE plus another TTTC repeat and that of caspase-8 consists of a con- infections and different courses of viral pathogenesis in infected ventional ISRE and an additional TTTTTG motif. Interestingly, these individuals. We think the 59 eISRE may contribute in several ways eISREs seem to be bound preferentially by IRF members: the eISRE to such variations. First, there may exist nucleotide mutations in the of all above-mentioned ISGs can be bound by IRF-1 and that of IL-12 59 eISRE of TRIM22 in IFN low-response individuals. Second, the or b2-microglobulin can also be bound by IRF-8 in immune cells. transcription factors, such as IRF-1, are crucial for the IFN-induced With supershift analysis, an ELISA-based transcriptional factor TRIM22 expression through binding with the 59 eISRE. Varied assay, and ChIP analysis, we found the 59 eISRE of TRIM22 could expression levels of 59 eISRE-binding transcription factors among be efficiently bound by IRF-1, but not by STAT1, both in vitro and individuals may also influence the expression of antiviral protein in vivo. It is known that IRF-1 is involved in diverse biological TRIM22. So it would be of interest to design chemical compounds processes, such as antiviral immunity, antiproliferative response, or small peptides to upregulate TRIM22 expression via mimicking apoptosis, and inflammation, etc., and is critical for the IFN-g– or stimulating the expression of those key transcription factors, with induced expression of several ISGs, such as iNOS, gp91 phox, the aim to enhance the clearance of HBV infection. and GBP, etc. (43–45). Our study also founded that IRF-1 was cru- cial for both the basal and IFN-g–inducible expression of TRIM22, Disclosures as overexpression of IRF-1 cDNA in HepG2 cells resulted in high- The authors have no financial conflicts of interest. level expression of TRIM22, whereas knocking down IRF-1 expres- sion abolished the IFN-g inducibility and significantly downreg- ulated the basal expression of TRIM22. The involvement of IRF-1 References may also explain the dependence on new protein synthesis for IFN- 1. Reymond, A., G. Meroni, A. Fantozzi, G. Merla, S. Cairo, L. Luzi, D. Riganelli, g–inducible expression of TRIM22. E. Zanaria, S. Messali, S. Cainarca, et al. 2001. The identifies cell compartments. EMBO J. 20: 2140–2151. As TRIM22 expression can be strongly induced by IFN-a,we 2. Meroni, G., and G. Diez-Roux. 2005. TRIM/RBCC, a novel class of ‘single have also investigated whether the 59 eISRE or IRF-1 contributed protein RING finger’ E3 ubiquitin ligases. Bioessays 27: 1147–1157. The Journal of Immunology 2323

3. Yamauchi, K., K. Wada, K. Tanji, M. Tanaka, and T. Kamitani. 2008. Ubiq- 27. Reid, L. E., A. H. Brasnett, C. S. Gilbert, A. C. Porter, D. R. Gewert, G. R. Stark, uitination of E3 ubiquitin ligase TRIM5 alpha and its potential role. FEBS J. and I. M. Kerr. 1989. A single DNA response element can confer inducibility by 275: 1540–1555. both alpha- and gamma-. Proc. Natl. Acad. Sci. USA 86: 840–844. 4. Duan, Z., B. Gao, W. Xu, and S. Xiong. 2008. Identification of TRIM22 as 28. Qureshi, S. A., M. Salditt-Georgieff, and J. E. Darnell, Jr. 1995. Tyrosine- a RING finger E3 ubiquitin ligase. Biochem. Biophys. Res. Commun. 374: 502– phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming 506. -stimulated-gene factor 3. Proc. Natl. Acad. Sci. USA 92: 3829–3833. 5. Gack, M. U., Y. C. Shin, C. H. Joo, T. Urano, C. Liang, L. Sun, O. Takeuchi, 29. Kalvakolanu, D. V. 2003. Alternate interferon signaling pathways. Pharmacol. S. Akira, Z. Chen, S. Inoue, and J. U. Jung. 2007. TRIM25 RING-finger E3 Ther. 100: 1–29. ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446: 30. Fu, X. Y., C. Schindler, T. Improta, R. Aebersold, and J. E. Darnell, Jr. 1992. The 916–920. proteins of ISGF-3, the interferon alpha-induced transcriptional activator, define 6. Stremlau, M., C. M. Owens, M. J. Perron, M. Kiessling, P. Autissier, and a gene family involved in signal transduction. Proc. Natl. Acad. Sci. USA 89: J. Sodroski. 2004. The cytoplasmic body component TRIM5alpha restricts 7840–7843. HIV-1 infection in Old World monkeys. Nature 427: 848–853. 31. van Boxel-Dezaire, A. H., M. R. Rani, and G. R. Stark. 2006. Complex 7. Neagu, M. R., P. Ziegler, T. Pertel, C. Strambio-De-Castillia, C. Gru¨tter, modulation of cell type-specific signaling in response to type I interferons. G. Martinetti, L. Mazzucchelli, M. Gru¨tter, M. G. Manz, and J. Luban. 2009. Immunity 25: 361–372. Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered 32. Gongora, C., G. Degols, L. Espert, T. D. Hua, and N. Mechti. 2000. A unique from human components. J. Clin. Invest. 119: 3035–3047. ISRE, in the TATA-less human Isg20 promoter, confers IRF-1-mediated respon- 8. Gack, M. U., A. Kirchhofer, Y. C. Shin, K. S. Inn, C. Liang, S. Cui, S. Myong, siveness to both and type II. Nucleic Acids Res. 28: 2333–2341. T. Ha, K. P. Hopfner, and J. U. Jung. 2008. Roles of RIG-I N-terminal tandem 33. Lefebvre, S., S. Berrih-Aknin, F. Adrian, P. Moreau, S. Poea, L. Gourand, CARD and splice variant in TRIM25-mediated antiviral signal transduction. J. Dausset, E. D. Carosella, and P. Paul. 2001. A specific interferon Proc. Natl. Acad. Sci. USA 105: 16743–16748. (IFN)-stimulated response element of the distal HLA-G promoter binds IFN- 9. Wolf, D., and S. P. Goff. 2007. TRIM28 mediates primer binding site-targeted regulatory factor 1 and mediates enhancement of this nonclassical class I gene by silencing of murine leukemia virus in embryonic cells. Cell 131: 46–57. IFN-beta. J. Biol. Chem. 276: 6133–6139. 10. Wolf, D., K. Hug, and S. P. Goff. 2008. TRIM28 mediates primer binding 34. Ruiz-Ruiz, C., C. Ruiz de Almodo´var, A. Rodrı´guez, G. Ortiz-Ferro´n, site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells. J. M. Redondo, and A. Lo´pez-Rivas. 2004. The up-regulation of human Proc. Natl. Acad. Sci. USA 105: 12521–12526. caspase-8 by interferon-gamma in breast tumor cells requires the induction 11. Nisole, S., J. P. Stoye, and A. Saı¨b. 2005. TRIM family proteins: retroviral re- and action of the transcription factor interferon regulatory factor-1. J. Biol. Downloaded from striction and antiviral defence. Nat. Rev. Microbiol. 3: 799–808. Chem. 279: 19712–19720. 12. Ozato, K., D. M. Shin, T. H. Chang, and H. C. Morse, III. 2008. TRIM family 35. Ganem, D., and A. M. Prince. 2004. Hepatitis B virus infection—natural history proteins and their emerging roles in innate immunity. Nat. Rev. Immunol. 8: 849– and clinical consequences. N. Engl. J. Med. 350: 1118–1129. 860. 36. Wieland, S. F., A. Eustaquio, C. Whitten-Bauer, B. Boyd, and F. V. Chisari. 13. Gao, B., Z. Duan, W. Xu, and S. Xiong. 2009. Tripartite motif-containing 22 2005. Interferon prevents formation of replication-competent hepatitis B virus inhibits the activity of hepatitis B virus core promoter, which is dependent on RNA-containing nucleocapsids. Proc. Natl. Acad. Sci. USA 102: 9913–9917. nuclear-located RING domain. Hepatology 50: 424–433. 37. Guidotti, L. G., A. Morris, H. Mendez, R. Koch, R. H. Silverman, B. R.

14. Eldin, P., L. Papon, A. Oteiza, E. Brocchi, T. G. Lawson, and N. Mechti. 2009. G. Williams, and F. V. Chisari. 2002. Interferon-regulated pathways that control http://www.jimmunol.org/ TRIM22 E3 ubiquitin ligase activity is required to mediate antiviral activity hepatitis B virus replication in transgenic mice. J. Virol. 76: 2617–2621. against encephalomyocarditis virus. J. Gen. Virol. 90: 536–545. 38. Wieland, S., R. Thimme, R. H. Purcell, and F. V. Chisari. 2004. Genomic 15. Barr, S. D., J. R. Smiley, and F. D. Bushman. 2008. The interferon response analysis of the host response to hepatitis B virus infection. Proc. Natl. Acad. Sci. inhibits HIV particle production by induction of TRIM22. PLoS Pathog. 4: USA 101: 6669–6674. e1000007. 39. Wieland, S. F., and F. V. Chisari. 2005. Stealth and cunning: hepatitis B and 16. Bouazzaoui, A., M. Kreutz, V. Eisert, N. Dinauer, A. Heinzelmann, hepatitis C viruses. J. Virol. 79: 9369–9380. S. Hallenberger, J. Strayle, R. Walker, H. Ru¨bsamen-Waigmann, R. Andreesen, 40. Chen, C. J., T. T. Lin, and J. E. Shively. 1996. Role of interferon regulatory and H. von Briesen. 2006. Stimulated trans-acting factor of 50 kDa (Staf50) factor-1 in the induction of biliary glycoprotein (cell CAM-1) by interferon- inhibits HIV-1 replication in human monocyte-derived macrophages. Virology gamma. J. Biol. Chem. 271: 28181–28188. 356: 79–94. 41. Masumi, A., S. Tamaoki, I. M. Wang, K. Ozato, and K. Komuro. 2002. 17. Tissot, C., and N. Mechti. 1995. Molecular cloning of a new interferon-induced IRF-8/ICSBP and IRF-1 cooperatively stimulate mouse IL-12 promoter activity

factor that represses human immunodeficiency virus type 1 in macrophages. FEBS Lett. 531: 348–353. by guest on October 1, 2021 expression. J. Biol. Chem. 270: 14891–14898. 42. Gobin, S. J., P. Biesta, and P. J. Van den Elsen. 2003. Regulation of human beta 18. Platanias, L. C. 2005. Mechanisms of type-I- and type-II-interferon-mediated 2-microglobulin transactivation in hematopoietic cells. Blood 101: 3058–3064. signalling. Nat. Rev. Immunol. 5: 375–386. 43. Lorsbach, R. B., W. J. Murphy, C. J. Lowenstein, S. H. Snyder, and S. W. Russell. 19. Stark, G. R. 2007. How cells respond to interferons revisited: from early history 1993. Expression of the gene in mouse macrophages acti- to current complexity. Growth Factor Rev. 18: 419–423. vated for tumor cell killing. Molecular basis for the synergy between interferon- 20. Uddin, S., and L. C. Platanias. 2004. Mechanisms of type-I interferon signal gamma and lipopolysaccharide. J. Biol. Chem. 268: 1908–1913. transduction. J. Biochem. Mol. Biol. 37: 635–641. 44. Kumatori, A., D. Yang, S. Suzuki, and M. Nakamura. 2002. Cooperation of 21. van Boxel-Dezaire, A. H., and G. R. Stark. 2007. Cell type-specific signaling in STAT-1 and IRF-1 in interferon-gamma-induced transcription of the gp91(phox) response to interferon-gamma. Curr. Top. Microbiol. Immunol. 316: 119–154. gene. J. Biol. Chem. 277: 9103–9111. 22. Gough, D. J., D. E. Levy, R. W. Johnstone, and C. J. Clarke. 2008. IFNgamma 45. Briken, V., H. Ruffner, U. Schultz, A. Schwarz, L. F. Reis, I. Strehlow, T. Decker, signaling-does it mean JAK-STAT? Cytokine Growth Factor Rev. 19: 383–394. and P. Staeheli. 1995. Interferon regulatory factor 1 is required for mouse Gbp 23. Liu, X., L. Ye, Y. Bai, H. Mojidi, N. E. Simister, and X. Zhu. 2008. Activation of gene activation by gamma interferon. Mol. Cell. Biol. 15: 975–982. the JAK/STAT-1 signaling pathway by IFN-gamma can down-regulate functional 46. Wong, L. H., H. Sim, M. Chatterjee-Kishore, I. Hatzinisiriou, R. J. Devenish, expression of the MHC class I-related neonatal Fc for IgG. J. Immunol. G. Stark, and S. J. Ralph. 2002. Isolation and characterization of a human STAT1 181: 449–463. gene regulatory element. Inducibility by interferon (IFN) types I and II and role 24. Sivaramakrishnan, G., Y. Sun, S. K. Tan, and V. C. Lin. 2009. Dynamic local- of IFN regulatory factor-1. J. Biol. Chem. 277: 19408–19417. ization of tripartite motif-containing 22 in nuclear and nucleolar bodies. Exp. 47. Rajsbaum, R., J. P. Stoye, and A. O’Garra. 2008. Type I interferon-dependent Cell Res. 315: 1521–1532. and -independent expression of tripartite motif proteins in immune cells. Eur. J. 25. Obad, S., H. Brunnstro¨m, J. Vallon-Christersson, A. Borg, K. Drott, and Immunol. 38: 619–630. U. Gullberg. 2004. Staf50 is a novel target gene conferring reduced 48. Sawyer, S. L., M. Emerman, and H. S. Malik. 2007. Discordant evolution of the clonogenic growth of leukemic U-937 cells. Oncogene 23: 4050–4059. adjacent antiretroviral genes TRIM22 and TRIM5 in mammals. PLoS Pathog. 3: 26. McKay, I. A., L. Kirby, E. V. Volyanik, V. Kumar, P. W. Wong, and S. A. Bustin. e197. 1998. An enzyme-linked immunosorbent assay for the detection of agents which 49. Asaoka, K., K. Ikeda, T. Hishinuma, K. Horie-Inoue, S. Takeda, and S. Inoue. interfere with the DNA binding activities of transcription factors—exemplified 2005. A restriction factor TRIM5alpha is transcriptionally regulated by NF-IL6. Anal. Biochem. 265: 28–34. by interferons. Biochem. Biophys. Res. Commun. 338: 1950–1956.