Hepatitis B Virus X Protein Induced Expression of the Nur77 Gene

Biochemical and Biophysical Research Communications 288, 1162–1168 (2001) doi:10.1006/bbrc.2001.5910, available online at http://www.idealibrary.com on

Hepatitis B Virus X Protein Induced Expression of the Nur77 Gene Mi-Ock Lee,* ,1,2 Hyo-Jin Kang,† ,1 Hyeseong Cho,‡ Eui-Cheol Shin,† Jeon Han Park,† and Se Jong Kim† *Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Korea; †Department of Microbiology, Yonsei University College of Medicine, Seoul 120-752, Korea; and ‡Department of Biochemistry, Ajou University School of Medicine, Suwon 442-749, Korea

Received October 10, 2001

Hepatitis B virus (HBV) X protein (HBx) plays an essential role in development of HBV-associated hepatocellular carcinoma (HCC). Recently, we reported that HBx induces Fas Ligand (FasL) expression, which may help HCC cells to evade host-immune surveillance. The aim of this study was to investigate the role of HBx in expression of Nur77, an orphan nuclear receptor implicated in the upregulation of FasL. When Chang X-34 expressing HBx under the control of a doxycycline-inducible promoter was examined, induction of Nur77 was observed following HBx expression. Blocking of Nur77 function by introduction of an antisense or a dominant negative mutant Nur77 significantly inhibited the induction of FasL, indicating that Nur77 plays critical roles in FasL expression. Further, a high-level expression of transcripts and DNA binding of Nur77 were observed in the HBVintegrated cell lines established from HCC patients that express HBx. These results suggested that Nur77 may contribute to leading the HBx-induced Fas/FasL signaling pathway which eliminates invading Fas-expressing lymphocytes. © 2001 Academic Press

Hepatitis B virus (HBV) is the major cause of the development of hepatitis, cirrhosis, and hepatocellular carcinoma (HCC); however, the mechanism involved in pathogenesis of HBV infection remains largely unknown. Among the HBV viral proteins, the hepatitis B virus X protein (HBx) is likely to be the important determinant in mediating pathological effects of HBV (1, 2). HBx is a multifunctional transactivator of 16.5 kDa that is required for transcription of the viral genome (3). It also transactivates a wide range of cellular genes through host-related transcription factor path1

Both authors contributed equally to this work. To whom correspondence and reprint requests should be addressed. Fax: (82)-2-3408-3768. E-mail: [email protected]. 2

0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

ways. In the nucleus, HBx functions as a co-activator by interacting with elements of the transcription machinery, such as transcription factor IIB and RNA polymerase II (4 – 6). In the cytoplasm, it activates signal transduction cascades including the ras/raf/mitogen activated protein kinase (MAP kinase), MAP kinase kinase/c-Jun N-terminal kinase, src-dependent pathways and Jak1 tyrosine kinase (7–10). Thus, HBx results in the transcriptional activation of AP-1, NF-␬B, and STATs, the major transcriptional factors associated with the proliferation and differentiation signals of liver cells (8, 10, 11). Such pleiotropic transcription activity of HBx might lead to the widespread dysregulation of gene expression that in turn, may contribute to HBx-induced pathogenesis and carcinogenesis. Surface expression of Fas ligand (FasL) in tumors such as hepatoma and melanoma was shown to lead elimination of invading Fas-expressing lymphocytes that may induce tumorigenesis (12–14). Others and we previously demonstrated that HBx plays a role in the induction of FasL expression in HBV-associated hepatoma cells (15, 16), however, the associated molecular details are not clearly understood. The present study was undertaken to elucidate the role of HBx in the transactivation of Nur77 that has implicated in FasL induction as well as apoptosis in a variety of normal and neoplastic cells (17–20). For this purpose, we used a doxycyclin-induced HBx-expressing Chang liver cell line (Chang X-34) and HBV-integrated cell lines established from HCC patients as models. MATERIALS AND METHODS Hepatocellular carcinoma cell lines and cell culture. Chang liver cells (ATCC CCL13) and Chang X-34 was as described previously (21). Human hepatocellular carcinoma cell line, Hep3B (ATCC HB 8064), was obtained from American type culture collection. HCC cell lines, SNU-354, -368, -387, -398, and -475 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Hep3B, SNU-354 and SNU-368 express HBx mRNA but others do not (22, 23). Hepatoma

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cells were maintained in either RPMI or minimal essential medium containing 10% fetal bovine serum at 37°C in an atmosphere of humidified incubator with 5% CO 2 and 95% air. Northern blot analysis. Northern blot analysis was performed essentially as described previously (21, 24). Total RNA was prepared using Qiagen RNeasy kit (Qiagen Inc., Chatsworth, CA) from hepatoma cells and 20 ␮g of total RNA was fractionated on 1% agarose gel and transferred to nylon membrane. A 1.2-kb EcoRI/PstI fragment of Nur77 cDNA or the HBx cDNA was labeled with [ 32P]dATP and used as a probe. To determine that equal amounts of RNA were used, the expression of GAPDH or 18S ribosomal RNA was examined. Immunoprecipitation/Western blot analysis. To detect Nur77 protein, cells were lysed in a lysis buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, 10% NP-40, 1 mM PMSF, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 10 ␮g/ml pepstatin. Five hundred ␮g of whole cell lysate was incubated with 2-␮g anti-Nur77 antibodies (Santa Cruz Biotech., Inc., Santa Cruz, CA). The resulting immunocomplex was precipitated by adding 50-␮l protein G–Sepharose slurry, washed four times with lysis buffer, subjected to 7.5% SDS–PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). The membrane was probed with anti-Nur77 antibody (PharMingen, San Diego, CA). To detect HBx protein, 40-␮g protein of whole cell lysate protein was subjected to 15% SDS–PAGE, transferred to a PVDF membrane and proved with specific antibodies against HBx (25). The protein concentration of the lysate was quantified by bicinchoninic acid assay (Pierce, Rockford, IL). Electromobility shift assays. Nuclear extracts were obtained by the method previously described (25, 26). Five micrograms of nuclear extract was incubated with 32P-labeled oligonucleotide in a 20-␮l reaction mixture containing 10 mM Tris buffer (pH 7.5), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mM PMSF, 1 mg/ml BSA, 5% glycerol, and 1 ␮g of poly(dI– dC) at 25°C for 20 min. The oligonucleotides used as probes in this experiment were NBRE, 5⬘GGAGTTTTAAAAGGTCATGCTCA-3⬘; mNBRE, 5⬘-GATCATAGGACA-3⬘; NurRE, 5⬘-GTGATA TTTACCTCCAAATGCCAG-3⬘; and SP-1, 5⬘-GATCGATCGGGGCGGGGCGAGC-3⬘. Transient transfection assays. The NurRE-pomc-Luc, Nur77 promoter (⫺454 to 57)-Luc, AP-1-RE-Luc, RSRF-RE-Luc, and the eukaryotic expression vector for wild type HBx, HpSVX, and the vector encoding frameshift mutant of HBx, pSVXkB, have been described elsewhere (15, 25, 27). Hepatoma cells (2 ⫻ 10 5 cells/well) were seeded in a 6-well culture plate and transfected with reporter plasmid (0.75-␮g), ␤-gal expression vector (0.25-␮g) in the presence or absence of HBx expression vector using LipofectaminePlus (GIBCO BRL, Grand Island, NY). After 24 h of incubation, luciferase activity was determined using an Analytical luminescence luminometer according to the manufacturer’s instructions. Luciferase activity was normalized for transfection efficiency using the corresponding ␤-gal activity. For statistical analysis, one-way analysis of variance was performed using GraphPad Instat (GraphPad Software, San Diego, CA). A value of P ⬍ 0.05 was considered statistically significant. Reverse-transcriptase polymerase chain reaction for FasL expression. To construct the eukaryotic expression vector encoding dominant negative Nur77, pCDNA-Nur77 Hga, a mouse Nur77 cDNA fragment encoding aa 252– 601 (20) was subcloned into the EcoRI/XhoI sites of the pCDNA3.1 (Invitrogen, Groningen, The Netherlands) by PCR, using EcoRI and XhoI-bearing primers. The vector encoding anti-sense Nur77, i.e., pCDNA-AS-Nur77, was constructed by inserting a 2.5-kb EcoRI fragment of pECE-Nur77 (26) into the corresponding restriction site of pCDNA3.1 in an anti-sense orientation. To analyze the role of Nur77 in the induction of FasL, either pCDNANur77 Hga or pCDNA-AS-Nur77 construct was transfected in Chang X-34 cells using LipofectaminePlus (GibcoBRL, Grand Island, NY). After 24 h of incubation, total RNA was obtained and PCR was performed as described previously with specific primers for FasL and

␤-actin (15). Genes were analyzed under the same conditions used to exponentially amplify the PCR products. Flow cytometric analysis for FasL expression. Cells were detached by trypsinization and washed twice with buffer containing 0.5% BSA in PBS. After cells were stained with 1-␮g mouse antiFasL antibody (PharMingen, Santa Cruz, CA) for 1 h at 4°C, cells were washed and incubated with FITC-conjugated goat anti-mouse IgG for 1 h at 4°C. Normal mouse IgG was run in parallel as negative control. Stained cells were acquired in the FACStar PLUS flow cytometer. Data were presented on histograms plotted as fluorescence intensity against cell number and analyzed using a PC-lysis software program (Becton–Dickinson, Mountain View, CA). Immunocytochemistry. To detect the expression of HBx, we performed immunofluorescence study. Hepatoma cells were cultured on a 11-mm coverslips for overnight and fixed with a mixture of methanol:acetone (1:1) for overnight at ⫺20°C. Then cells were stained with anti HBx-specific antibody (24) (1:500 in PBS/2% BSA, 1.5% horse serum), followed by anti-mouse biotin (1:1000 dilution, Vector Laboratories, Inc., Burlingame, CA) and streptavidin–fluorescein isothiocyanate (1:200 dilution, Vector Laboratories). Immunofluorescent cells were washed again with PBS and visualized with a confocal microscopy (Nikon, Japan).

RESULTS Induction of Expression and Transactivation Function of Nur77 by HBx Protein Given the possibility that Nur77 might be involved in HBx-induced FasL expression and thereby in the development of HBV-related liver diseases, we investigated regulation of Nur77 gene expression by the HBx. First, we employed the Chang X-34, in which the expression of HBx gene is under control of an inducible doxycycline promoter (21). As shown in Fig. 1A, doxycycline treatment remarkably induced the expression of HBx at both transcript- and protein-level. Interestingly, doxycycline resulted in an induction of Nur77 transcripts, which started after 4 h of treatment (Fig. 1B). Consistently, the induction of Nur77 was also observed at the protein-level (Fig. 1C). Next, we measured DNA binding of Nur77 by electromobility shift assays with an oligonucleotide probe encoding the specific target sequences of Nur77, i.e., NBRE (27, 28). Nur77 has been shown to bind DNA as a monomer to NBRE (5⬘-AAAGGTCA-3⬘) and to bind as a homodimer to NurRE (5⬘-TGATATTTACCTCCAAATGCCA3⬘) (27–29). As shown in Fig. 2A, the NBRE binding was strongly enhanced by doxycycline treatment, reaching a maximum level after 9 h, while the DNA binding of SP-1 remained unchanged. An excess of cold oligonucleotide completely inhibited the DNA binding complex and the specific anti-Nur77 antibodies abolished the binding, indicating that the NBRE binding was specific. Transactivation of Nur77 by HBx protein was further confirmed using the reporter construct coding NurRE. The inducible expression of HBx by doxycycline activated the NurRE-Luc reporter in a time- and dose-dependent manner (Fig. 2B). However, doxycyclin treatment in parental Chang liver cells did not induce

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transcriptional activation of these transcriptional factors using reporter constructs containing AP-1 and RSRF binding sites present in the Nur77 promoter (25). Overexpression of HBx induced the reporter activities of AP-1-RE-Luc and RSRF-RE-Luc (Fig. 3B), which suggests that both AP-1 and RSRF may be involved in HBx-induced induction of Nur77. Expression of a Dominant Negative Nur77 or an Antisense Nur77 Repressed FasL Expression The role of Nur77 in FasL expression has been speculated, since a constitutive expression of Nur77 led to massive apoptosis with a high level expression of FasL in thymocytes of transgenic mice (30). Therefore, a high level expression of Nur77 in HBx expressing hep-

FIG. 1. Induction of Nur77 expression in Chang liver cells that stably express HBx. (A) The time-dependent expression of HBx in Chang X-34 cells upon doxycycline (Doxy) (2 ␮g/ml) treatment was examined by Northern (upper panel) and Western (lower panel) blot analyses as described under Materials and Methods. (B) The expression of Nur77 transcripts after the indicated time period with 2 ␮g/ml doxycyclin treatment was analyzed by Northern blot analysis. The expression of 18S ribosomal RNA was shown as a control. (C) The expression of Nur77 protein after the indicated time period with 2 ␮g/ml doxycyclin treatment was analyzed by immunoprecipitation/ Western blot analysis as described in the Material and Methods. Representative figures of at least three independent experiments with similar results were shown.

Nur77 expression when tested by any of the above experimental procedures (data not shown). Expression of HBx-Induced Nur77-Promoter Activity To test whether the functional induction of Nur77 was due to transcriptional activation of the gene, we employed a Nur77 promoter-luciferase reporter gene containing 511 bp (⫺454 to ⫹57) DNA sequence upstream of the transcription start site of Nur77, which is sufficient for the transcriptional activation of the Nur77 gene (19). As shown in Fig. 3A, when the plasmid encoding HBx gene was co-transfected into Chang liver cells, the Nur77 promoter reporter activity was induced as strong as that induced by PMA/ionomycin, a known potent inducer of the reporter (19). In contrast, coexpression of the nonfunctional mutant HBx (15) did not show any activation. The induction of the reporter was also observed in other hepatoma cell lines such as HepG2 and PLC/PRF/5 with a similar degree (data not shown). Consistent with these results, HBx expression also induced transcriptional activity of the reporter gene, NurRE-Luc (Fig. 3A). The Nur77 promoter contains several potential cis-acting regulatory elements, such as binding sites for AP-1 and related serum response factor (RSRF) (19, 25). We tested whether HBx transactivated Nur77 promoter through

FIG. 2. Induction of DNA binding and transcriptional activity of Nur77 by HBx protein in Chang X-34. (A) The amount of active Nur77 protein that binds DNA was determined by gel shift assays using P 32-labeled oligonucleotide encoding NBRE as probe as described under Materials and Methods. The reaction mixtures contained 5-␮g nuclear extract obtained from Chang X-34 treated with doxycyclin (Doxy) (2 ␮g/ml) for the indicated time period. C indicates an excess amount of unlabeled probe used for competition to show specificity of the binding. When anti-Flag antibodies were used, it was incubated with the receptor protein for 20 min at room temperature before performing gel shift assays. SP-1 binding was shown as control. Representatives of at least three independent experiments with similar results were shown. (B) The NurRE-Luc reporter was transfected into Chang X-34 as described under Materials and Methods. Cell lysate was obtained after cells were treated with different concentration of doxycyclin for 9 h (left) or with 2 ␮g/ml doxycycline for different time period (right). Luciferase activity was normalized by corresponding ␤-gal activity. Data shown are means ⫾ SD of three independent experiments.

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differentially express HBx. The chromosomal integration status and mRNA expression of HBx in each cell line was reported previously (22, 23). In Fig. 5A, we showed the expression of HBx protein in the cell lines by immunofluorescence staining. HBx was detected in SNU-368, which was predominantly distributed in cytosol. The HBx was also detected in the cytosol of SNU-354 with less intensity; however, the protein was not detected in the other cell lines in the condition we employed. Under stringent hybridization conditions, we were able to detect a high level of Nur77 transcripts in SNU-354 and SNU-368. However, the expression of Nur77 was detected in a low level or undetected in other cell lines (Fig. 5B). Next, we measured the amount of active Nur77 protein, which binds DNA by

FIG. 3. Expression of HBx induced transcriptional activity of Nur77. (A) The Nur77 promoter (⫺454⬃⫹57)-Luc reporter or the NurRE-Luc reporter was cotransfected with an eukaryotic expression vector for wild type HBx, i.e., HpSVX, or a vector encoding frameshift mutant of HBx, i.e., pSVXkB, into Chang liver cells. (B) The AP-1-RE-Luc reporter or the RARF-RE-Luc reporter was cotransfected with HpSVX into PLC/PRF/5 as described under Materials and Methods. P/I represents a treatment with PMA (10 nM) and ionomycin (0.5 ␮M), which was shown as a positive control. Luciferase activity was normalized using corresponding ␤-gal activity. Data shown are means ⫾ SD of three independent experiments.

atoma, may cause the induction of FasL. To test this hypothesis, we transfected the eukaryotic expression vector encoding a dominant negative Nur77, pCDNANur77 Hga, or an anti-sense Nur77 (17, 18, 20), into Chang X-34. The expression of FasL transcripts in Chang lever cells was undetectable; however, it was detected in Chang X-34 even in the absence of doxycycline, which may be explained by a low level expression of HBx in the Chang X-34 in the absence of doxycycline (Fig. 4A) (21). Inducible expression of HBx in Chang X-34 caused a strong induction of FasL expression upon 4 h of doxycycline treatment. When either the dominant negative Nur77 or the antisense Nur77 construct was transfected, the induction of FasL decreased dramatically. Similarly, the expression of FasL on the surface of Chang X-34 was induced by doxycycline. However, cotransfection of the dominant negative Nur77 or the antisense Nur77 was almost completely blocked the surface expression of FasL (Fig. 4B). Thus, this result provides evidence that Nur77 is involved in HBx-induced FasL expression. Differential Expression of Nur77 Correlated with the Expression of HBx in HBV-Integrated Hepatocellular Carcinoma Cells Next, we examined the expression of Nur77 transcripts in a panel of the HBV DNA-integrated HCC cell lines, i.e., SNU-354, -368, -387, -398, and -475, which

FIG. 4. HBx-induced FasL expression was blocked by the dominant negative Nur77 and anti-sense Nur77. Chang X-34 cells were transfected with the dominant negative Nur77 construct, pRc/CMVNur77 Hga (0.5 ␮g), or the anti-sense Nur77, pcDNA-AS-Nur77 (3.5 ␮g), as indicated. (A) The expression of FasL transcript was analyzed after doxycycline (Doxy) (2 ␮g/ml) treatment for 9 h by RT-PCR as described under Materials and Methods. The expression of ␤-actin was monitored as a control. The intensity of PCR band of FasL was normalized by corresponding ␤-actin and shown as relative intensity. (B) The surface expression of FasL protein in Chang X-34 after doxycycline treatment for 9 h was determined by flow cytometry. One representative of at least three independent experiments with similar results was shown. Filled histogram, stained with normal mouse IgG; open histogram, stained with anti-FasL antibody.

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complex was observed in the other cell lines. The Nur77 binding complexes in the hepatoma cells consisted of two bands; the relative intensities of the two bands were similar in SNU-354 and SNU-368, while the lower band was dominant in Hep3B. Both bands were completely abolished by treatment with specific Nur77 antibodies or a 10-fold excess of probe encoding NBRE or NurRE, but not with mutated NBRE, indicating that both complexes were NBRE specific. These results showed that HBx expression was correlated with the expression of Nur77, at least, in the hepatoma cells we tested, and suggested that HBx may induce Nur77 gene expression in the HCC cell lines. DISCUSSION

FIG. 5. Differential expression of Nur77 in HBV-integrated hepatocellular carcinoma cell lines. (A) Expression of HBx protein was analyzed by immunocytochemistry as described under Materials and Methods. (B) Expression of Nur77 and HBx at the transcriptional level was analyzed by Northern blot analysis as described under Materials and Methods. The expression of GAPDH gene was shown as a control. (C) The amount of active Nur77 protein that binds DNA was determined by gel shift assays using 32P-labeled oligonucleotide encoding NBRE as probe as described under Materials and Methods. The reaction mixture contained 5-␮g nuclear extract obtained from the indicated cell line. NBRE, mNBRE, and NurRE indicate 10-, 30-, and 90-fold excess amount of unlabeled oligonucleotide encoding NBRE, mutated NBRE, NurRE, respectively, used for competition to show specificity of the binding. Representatives of at least three independent experiments with similar results were shown.

electromobility shift assays with an oligonucleotide probe encoding NBRE (Fig. 5C). DNA binding complexes were detected strongly in SNU-368 and weakly in SNU-354. Hep3B that expresses HBx transcripts (22) also showed DNA binding. In contrast, no binding

HBx plays an essential role in HBV replication, the development of hepatitis and the subsequent generation of hepatocellular carcinoma (1, 2). However, the mechanism involved in the HBx-associated hepatocarcinogenesis is largely unknown. Surface expression of FasL in tumor cells contributes to the development of malignant transformation, indicating that Fas/FasL could be an efficient way of destroying immune effector cells by inducing apoptotic pathways in host immune cells (12–14). Others and we previously showed that HBx played a role in the induction of FasL expression in HBV-associated hepatoma cells, which may help to eliminate infiltrating T-lymphocytes (15, 16). In the present investigation, we demonstrated that HBx transactivated Nur77 expression that may induce the Fas/FasL signaling pathway. Nur77 (also known as NGFI-B, N10, TIS1, and Nak-1) is an orphan member of the steroid/thyroid receptor superfamily of transcriptional factors that positively or negatively regulates gene expression. It is an immediate-early response gene the expression of which is rapidly induced by a variety of stimuli, such as treatment with growth factors and mitogens (31–33). Nur77 is also rapidly induced by T-cell receptor (TCR) signaling in immature thymocytes and T-cell hybridomas, which is followed by apoptotic cell death (17, 19, 20, 30). Weih et al. (30) showed that FasL was upregulated in Nur77-transgenic thymocytes, which suggested that Nur77 may lead apoptosis via the induction of FasL. Interestingly, we have observed recently that FasL was expressed in HepG2.2.15, into which the HBV genome had been transfected (15). Further, FasL was induced in PLC/PRF/5 cells transfected with HBx. Together, these results implied that Nur77 mediated HBx-induced FasL expression. Our previous finding was further supported by the present result that doxycyclin treatment induced FasL in Chang X-34 (Fig. 4). Both the anti-sense and dominant negative Nur77 significantly blocked the induction of FasL after doxycyclin treatment, strongly suggesting that Nur77 mediates the role of HBx in induction of FasL. However, the

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direct binding site for Nur77 (NBRE or NurRE) was not found in the promoter of FasL (34) indicating that the Nur77 may activate FasL by an indirect mechanism. Instead, the nuclear factors of activated T-cells (NFATs) directly binds an enhancer sequence on FasL promoter that plays a key role on the expression of FasL (36, 37). Recently Lara-Pezzi et al. (35) showed that HBx induced the dephosphorylation of the nuclear factor of activated T cells (NFAT), which caused translocation and transcriptional activation of the protein through a CsA-dependent pathway. Interestingly, we observed that the HBx-induced Nur77 activation was sensitive to CsA (data not shown), raise the possibility that HBx causes transcriptional activation of Nur77 and induce FasL expression through a subsequent transcriptional activation of NFAT. Similar to our observation, Chen et al. (38, 39) showed that the Tax, viral transactivator of human T lymphotropic virus Type 1 (HTLV-1), caused transcriptional activation of Nur77 and FasL in HTLV-1 infected T cells, which implies the involvement of Nur77 in HTLV-1-induced FasL expression. Also, Nef, a viral protein of the Simian immunodeficient virus, induced FasL expression (40). These similarities between viral transforming proteins that are evolutionary different indicates that Nur77 may be involved in a wide spectrum of viral pathogenesis. The expression of FasL-induced by Nur77/HBx may serve as a tool for virus-infected cells to escape the immune surveillance of the host by eliminating T-lymphocytes. Alternatively, Nur77 may induce apoptotic self-destruction of virus-infected host cells immediately upon entry of viruses. Apoptotic cell death of host cells is considered as one of the primary anti-viral defense mechanisms, and many animal viruses are known to encode one or more proteins that interact with apoptotic regulatory pathways. In fact, several lines of evidence suggest that the HBx protein triggers apoptotic cell death in HBV infected hepatocytes (41– 43). However, the experimental models we employed did not show significant apoptotic changes upon expression of either HBx or Nur77 (data not shown), supports the former possibility. To better understand the pathophysiology of the HBVassociated liver diseases, the role of HBx and Nur77 in the process of immune evasion and apoptotic cell death is guaranteed for investigation in future. ACKNOWLEDGMENTS We thank Dr. Jacques Drouin for the reporter construct NurREpomc-Luc. We also thank Dr. Thomas Perlmann for the antibody specific to Nur77. This work was supported by a grant of the Korea Science and Engineering Foundation (2000-2-20900-011-3 to M.O.L.).

REFERENCES 1. Cromlish, J. A. (1996) Trends Microbiol. 4, 270 –274. 2. Yen, T. S. B. (1996) J. Biomed. Sci. 3, 20 –30. 3. Lee, H., Lee, Y.-H., Huh, Y.-S., and Yun, Y. (1995) J. Biol. Chem. 270, 31405–31412. 4. Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 143–150. 5. Haviv, I., Shamay, M., Doitsh, G., and Shaul, Y. (1998) Mol. Cell. Biol. 18, 1562–1569. 6. Qadri, I., Maguire, H. F., and Siddiqui, A. (1995) Proc. Natl. Acad. Sci. USA 92, 1003–1007. 7. Benn, J., and Schneider, R. J. (1994) Proc. Natl. Acad. Sci. USA 91, 10350 –10354. 8. Benn, J., Su, F., Doria, M., and Schneider, R. J. (1996) J. Virol. 70, 4978 – 4985. 9. Klein, N. P., Bouchard, M. J., Wang, L. H., Kobarg, C., and Schneider, R. J. (1999) EMBO J. 18, 5019 –5027. 10. Lee, Y.-H., and Yun, Y. (1998) J. Biol. Chem. 273, 25510 –25515. 11. Chirillo, P., Falco, M., Puri, P. L., Artini, M., Balsano, C., Levrero, M., and Natoli, G. (1996) J. Virol. 70, 641– 646. 12. Walker, P. R., Saas, P., and Dietrich, P. Y. (1998) Curr. Opin. Immunol. 110, 564 –572. 13. Hahne, M., Rimoldi, D., Schroter, M., Romero, P., Schreier, M., French, L. E., Schneider, P., Bornand, T., Fontana, A., Lienard, D., Cerottini, J., and Tschopp, J. (1996) Science 274, 1363–1366. 14. Strand, S., Hofmann, W. J., Hug, H., Muller, M., Otto, G., Strand, D., Mariani, S. M., Stremmel, W., Krammer, P. H., and Galle, P. R. (1996) Nat. Med. 2, 1361–1366. 15. Shin, E.-C., Shin, J.-S., Park, J.-H., Kim, H., and Kim, S.-J. (1999) Int. J. Cancer 82, 587–591. 16. Zhang, D., Zhang, D., and Tao, X. (1999) Chung Hwa Kan Tsang Ping Tsa Chih 7, 67– 68. 17. Liu, Z.-G., Smith, S. W., McLaughlin, K. A., Schwartz, L. M., and Osborne, B. A. (1994) Nature 367, 281–284. 18. Uemura, H., and Chang, C. (1998) Endocrinology 129, 2329 – 2334. 19. Woronicz, J. D., Calnan, B., Ngo, V., and Winoto, A. (1995) Mol. Cell. Biol. 15, 6364 – 6376. 20. Woronicz, J. D., Lina, A., Calnan, B. J., Szychowski, S., Cheng, L., and Winoto, A. (1994) Nature 367, 277–281. 21. Yun, C., Lee, J. H., Park, H., Jin, Y. M., Park, S., Park, K., and Cho, H. (2000) Oncogene 19, 5163–5172. 22. Park, J. G., Lee, J. H., Kang, M. S., Park, K. J., Jeon, Y. M., Lee, H. J., Kwon, H. S., Park, K. S., Yeo, K. U., Lee, S. T., Kim, J. K., Chung, Y. J., Hwang, H. S., Lee, C. Y., Kim, Y. I., Lee, T. R., Chen, R. J., Hay, S. Y., Song, W. H., Kim, C. W., and Kim, Y. I. (1995) Int. J. Cancer 62, 276 –282. 23. Kang, M. S., Lee, H. J., Lee, J. H., Ku, J. L., Lee, K. P., Kelley, M. J., Won, Y. J., Kim, S. T., and Park, J. G. (1996) Int. J. Cancer 67, 898 –902. 24. Park, O. Y., Jin, Y. H., Lee, M., Shin, H. J., Kim, H. I., Cho, H, Yun, C. W., Youn, J. K., and Park, S. (2000) Hybridoma 19, 73– 80. 25. Kang, H.-J., Song, M.-R., Lee, S.-K., Shin, E.-C., Choi, Y.-H., Kim, S. J., Lee, J., and Lee, M.-O. (2000) Exp. Cell Res. 256, 545–554. 26. Wu, Q, Li, Y., Agadir, A., Lee, M.-O., Liu, Y., and Zhang, X.-k. (1997) EMBO J. 16, 1656 –1669. 27. Perlmann, T., and Jansson, L. (1995) Genes Dev. 9, 769 –782. 28. Wilson, T. E., Fahrner, T. J., Johnston, M., and Milbrandt, J. (1991) Science 252, 1296 –1300.

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29. Philips, A., Lesage, S., Gingras, R., Maira, M.-H., Gauthier, Y., Hugo, P., and Drouin, J. (1997) Mol. Cell. Biol. 17, 5946 –5951. 30. Lara-Pezzi, E., Armesilla, A. L., Majano, P. L., Redondo, J. M., and Lo´pez-Cabrera, M. (1999) EMBO J. 17, 7066 –7077. 31. Weih, F., Ryseck, R.-P., Chen, L., and Bravo, R. (1996) Proc. Natl. Acad. Sci. USA 93, 5533–5538. 32. Mages, H. W., Rilke, O., Bravo, R., Senger, G., and Kroczek, R. A. (1994) Mol. Endocrinol. 8, 1583–1591. 33. Nakai, A., Kartha, S., Sakurai, A., Toback, F. G., and DeGroot, L. J. (1990) Mol. Endocrinol. 4, 1438 –1443. 34. Ryseck, R.-P., Macdonald-Bravo, H., Matte´i, M.-G., Ruppert, S., and Bravo, R. (1989) EMBO J. 8, 3327–3335. 35. Winoto, A. (1997) Curr. Opin. Immunol. 9, 365–370. 36. Furuke, K., Shiraishi, M., Mostowski, H. S., and Bloom, E. T. (1999) J. Immunol. 162, 1988 –1993.

37. Latinis, K. M., Norian, L. A., Eliason, S. L., and Koretzky, G. A. (1997) J. Biol. Chem. 50, 31427–31434. 38. Chen, X., Zachar, V., Zdravkovic, M., Guo, M., Ebbesen, P., and Liu, X. (1997) J. Gen. Virol. 78, 3277–3285. 39. Chen, X., Zachar, V., Chang, C., Ebbesen, P., and Liu, X. (1998) J. Virol. 72, 6902– 6906. 40. Hodge, S., Novembre, F. J., Whetter, L., Gelbard, H. A., and Dewhurst, S. (1998) Virology 252, 354 –363. 41. Chirillo, P., Pagano, S., Natoli, G., Puri, P. L., Burgio, V. L., Balsano, C., and Levrero, M. (1997) Proc. Natl. Acad. Sci. USA 94, 8162– 8167. 42. Kim, H., Lee, H., and Yun, Y. (1998) J. Biol. Chem. 273, 381–385. 43. Shintani, Y., Yotsuyanagi, H., Moriya, K., Fujie, H., Tsutsumi, T., Kanegae, Y., Kimura, S., Saito, I., and Koike, K. J. (1999) Gen. Virol. 80, 3257–3265.

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