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• W1969461545 abstract "Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. HCV genome replication occurs in the replication complex (RC) around the endoplasmic reticulum membrane. However, the mechanisms regulating the HCV RC remain widely unknown. Here, we used a chemical biology approach to show that estrogen receptor (ESR) is functionally associated with HCV replication. We found that tamoxifen suppressed HCV genome replication. Part of ESRα resided on the endoplasmic reticulum membranes and interacted with HCV RNA polymerase NS5B. RNA interference-mediated knockdown of endogenous ESRα reduced HCV replication. Mechanistic analysis suggested that ESRα promoted NS5B association with the RC and that tamoxifen abrogated NS5B-RC association. Thus, ESRα regulated the presence of NS5B in the RC and stimulated HCV replication. Moreover, the ability of ESRα to regulate NS5B was suggested to serve as a potential novel target for anti-HCV therapeutics. Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. HCV genome replication occurs in the replication complex (RC) around the endoplasmic reticulum membrane. However, the mechanisms regulating the HCV RC remain widely unknown. Here, we used a chemical biology approach to show that estrogen receptor (ESR) is functionally associated with HCV replication. We found that tamoxifen suppressed HCV genome replication. Part of ESRα resided on the endoplasmic reticulum membranes and interacted with HCV RNA polymerase NS5B. RNA interference-mediated knockdown of endogenous ESRα reduced HCV replication. Mechanistic analysis suggested that ESRα promoted NS5B association with the RC and that tamoxifen abrogated NS5B-RC association. Thus, ESRα regulated the presence of NS5B in the RC and stimulated HCV replication. Moreover, the ability of ESRα to regulate NS5B was suggested to serve as a potential novel target for anti-HCV therapeutics. Estrogen receptor (ESR) 2The abbreviations used are: ESR, estrogen receptor; HCV, hepatitis C virus; RC, replication complex; ER, endoplasmic reticulum; TAM, tamoxifen; ERE, ESR-responsive element(s); CAV, caveolin; NS, nonstructural protein; MM, microsomal membrane; siRNA, small interfering RNA; si-ESR, small interfering ESR; GST, glutathione S-transferase; aa, amino acid(s); RT, reverse transcription; NS3, NS4A, NS4B, NS5A, and NS5B, nonstructural protein 3, 4A, 4B, 5A, and 5B, respectively. belongs to the steroid hormone receptor family of the nuclear receptor superfamily (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6085) Google Scholar). ESR consists of two subtypes, ESRα and ESRβ. As a primary physiological function, ESR is involved in the transcription for downstream genes in response to stimulation by the ligand, estradiol. In the normal state, ESR is mainly located in the cytoplasm and nucleus. Upon binding of the ligand, ESR dimerizes and translocates into the nucleus, where it binds to the ESR-responsive elements (ERE) in the DNA promoter of downstream genes and drives transcription. In addition to this classical genomic action, a portion of ESR is located on the membrane, such as the plasma membrane, and involved in the nongenomic function of triggering signal transduction pathways, such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C (2Acconcia F. Kumar R. Cancer Lett. 2006; 238: 1-14Crossref PubMed Scopus (197) Google Scholar, 3Levin E.R. Mol. Endocrinol. 2005; 19: 1951-1959Crossref PubMed Scopus (605) Google Scholar, 4Song R.X. Zhang Z. Santen R.J. Trends Endocrinol. Metab. 2005; 16: 347-353Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Although the molecular basis of ESR membrane retention is not fully understood, one mechanism involves a membrane protein, caveolin (CAV); ESRα interacted with CAV, and this interaction facilitated ESRα localization to the membrane (5Razandi M. Alton G. Pedram A. Ghonshani S. Webb P. Levin E.R. Mol. Cell. Biol. 2003; 23: 1633-1646Crossref PubMed Scopus (287) Google Scholar, 6Razandi M. Oh P. Pedram A. Schnitzer J. Levin E.R. Mol. Endocrinol. 2002; 16: 100-115Crossref PubMed Scopus (298) Google Scholar). It was also reported that ESRα localizes to the lipid rafts on the plasma membrane (7Marquez D.C. Chen H.W. Curran E.M. Welshons W.V. Pietras R.J. Mol. Cell. Endocrinol. 2006; 246: 91-100Crossref PubMed Scopus (94) Google Scholar). The lipid rafts are microdomains of the membrane that form platforms enriched in cholesterol and glycosphingolipids. However, the characteristics and relevance of membrane-associated ESR have not been fully disclosed. Here, we report the novel role of ESRα in the regulation of viral replication. Hepatitis C virus (HCV), a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, constitutes a serious health problem worldwide (8Liang T.J. Jeffers L.J. Reddy K.R. Medina DeM. Parker I.T. Cheinquer H. Idrovo V. Rabassa A. Schiff E.R. Hepatology. 1993; 18: 1326-1333PubMed Google Scholar). HCV has a positive strand RNA genome that produces at least 10 functional viral proteins: core, envelope 1, envelope 2, p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (9Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar, 10Hijikata M. Kato N. Ootsuyama Y. Nakagawa M. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5547-5551Crossref PubMed Scopus (581) Google Scholar). NS5B is an RNA-dependent RNA polymerase, which plays a central role in viral genome replication (11Bartenschlager R. Lohmann V. Antiviral Res. 2001; 52: 1-17Crossref PubMed Scopus (157) Google Scholar, 12Tellinghuisen T.L. Rice C.M. Curr. Opin. Microbiol. 2002; 5: 419-427Crossref PubMed Scopus (154) Google Scholar). HCV genome replication can be evaluated using a HCV subgenomic replicon system, which Lohmann et al. (13Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2498) Google Scholar) first established. In this system, cells carry an HCV subgenome RNA encoding NS3 to NS5B. Using this system, it has been proposed that HCV genome replication occurs in the replication complex (RC), which contains the viral genome RNA and HCV NS proteins. The RC forms on the surface of the intracellular membranes, including the endoplasmic reticulum (ER) membrane, and is surrounded by a membrane structure (14Aizaki H. Lee K.J. Sung V.M. Ishiko H. Lai M.M. Virology. 2004; 324: 450-461Crossref PubMed Scopus (232) Google Scholar, 15Egger D. Wolk B. Gosert R. Bianchi L. Blum H.E. Moradpour D. Bienz K. J. Virol. 2002; 76: 5974-5984Crossref PubMed Scopus (696) Google Scholar, 16Miyanari Y. Hijikata M. Yamaji M. Hosaka M. Takahashi H. Shimotohno K. J. Biol. Chem. 2003; 278: 50301-50308Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 17Moradpour D. Gosert R. Egger D. Penin F. Blum H.E. Bienz K. Antiviral Res. 2003; 60: 103-109Crossref PubMed Scopus (132) Google Scholar). It also has been reported that HCV genome replication associates with the lipid rafts on these intracellular membranes, such as the ER membrane (14Aizaki H. Lee K.J. Sung V.M. Ishiko H. Lai M.M. Virology. 2004; 324: 450-461Crossref PubMed Scopus (232) Google Scholar, 18Shi S.T. Lee K.J. Aizaki H. Hwang S.B. Lai M.M. J. Virol. 2003; 77: 4160-4168Crossref PubMed Scopus (225) Google Scholar). These lipid rafts accumulate CAV2, and HCV proteins involved in viral genome replication cofractionate with CAV2 (18Shi S.T. Lee K.J. Aizaki H. Hwang S.B. Lai M.M. J. Virol. 2003; 77: 4160-4168Crossref PubMed Scopus (225) Google Scholar). However, it is largely unknown how the RC is formed and under what mechanism the HCV proteins participate in the RC. A chemical biology approach is a useful method to analyze the molecular mechanism of viral life cycles as well as cellular physiological processes (19Watashi K. Shimotohno K. Rev. Med. Virol. 2007; 17: 245-252Crossref PubMed Scopus (45) Google Scholar). We employed forward chemical genetics in which we analyzed HCV replication activity as a phenotypic indicator of a cell-based assay to screen chemical compounds that inhibited HCV replication. Using this system, we previously identified an immunosuppressant, cyclosporin A, as an anti-HCV compound (20Watashi K. Hijikata M. Hosaka M. Yamaji M. Shimotohno K. Hepatology. 2003; 38: 1282-1288Crossref PubMed Scopus (463) Google Scholar). We also reported that cyclophilin B regulated the RNA binding activity of NS5B (21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). In the current study, this chemical screening approach linked ESRα to HCV replication. We showed that tamoxifen (TAM) suppressed HCV genome replication. Using TAM as a bioprobe, we found that ESRα interacted with NS5B and regulated the participation of NS5B in the RC. Cell Culture and Transfection—Huh-7 and cured MH-14 cells (21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, minimal essential medium nonessential amino acid (Invitrogen), and kanamycin (Meiji). MH-14 cells, carrying HCV subgenomic replicon (16Miyanari Y. Hijikata M. Yamaji M. Hosaka M. Takahashi H. Shimotohno K. J. Biol. Chem. 2003; 278: 50301-50308Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and LucNeo#2 cells, carrying luciferase-containing subgenomic replicon (22Goto K. Watashi K. Murata T. Hishiki T. Hijikata M. Shimotohno K. Biochem. Biophys. Res. Commun. 2006; 343: 879-884Crossref PubMed Scopus (124) Google Scholar), were cultured in the same medium supplemented with 300 μg/ml G418 (Invitrogen). Hus-E7/DN24 cells, a human hepatocyte cell line established by immortalization with HPV E6E7 and hTERT from human primary hepatocytes and introduction with a dominant negative form of interferon regulatory factor-7 (23Aly H.H. Watashi K. Hijikata M. Kaneko H. Takada Y. Egawa H. Uemoto S. Shimotohno K. J. Hepatol. 2007; 46: 26-36Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), were cultured with Dulbecco's modified Eagle's medium with 20 mm Hepes (Invitrogen), 15 g/ml l-proline, 0.25 g/ml insulin (Sigma), 50 nm dexamethasone (Sigma), 44 mm NaHCO3, 10 mm nicotinamide, 5 ng/ml epidermal growth factor, 0.1 mm Asc-2P, 100 IU/ml penicillin G (Invitrogen), 100 μg/ml streptomycin (Invitrogen), 5% fetal bovine serum, 1% Dulbecco's modified Eagle's medium, and 2 UG/ml Fungizone (Invitrogen) (24Hino H. Tateno C. Sato H. Yamasaki C. Katayama S. Kohashi T. Aratani A. Asahara T. Dohi K. Yoshizato K. Biochem. Biophys. Res. Commun. 1999; 256: 184-191Crossref PubMed Scopus (54) Google Scholar). Plasmid transfection was performed with FuGENE 6 transfection reagent (Roche Applied Science), as described previously (25Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar). RNA transfection was achieved using DMrie-C transfection reagent (Invitrogen), as described previously (21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). siRNA was transfected by using siLentFect (Bio-Rad) (21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Plasmid Construction—pCMV-FL-ESRα, encoding the whole open reading frame of ESRα fused with a FLAG tag, was generated by inserting the PCR product using 5′-GTTGAATTCATGACCATGACCCTCCAC-3′ and 5′-GTTGATCTCGAGTCAGACTGTGGCAGGGAAAC-3′ as primer set and human lymphocyte cDNA library (Clontech) as a template into the EcoRI-XhoI site of pCMV-FLAG vector (21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). pCAG-HA-NS5B, encoding the NS5B protein fused with a hemagglutinin tag, was made by subcloning the PCR product with 5′-GTTGCGGCCGCTATGTCAATGTCCTACTCA-3′ and 5′-GTTCTCGAGTCACCGGTTGGGGAGCAGGTA-3′ as primers and pMH14 as a template into NotI-XhoI digestion of PCAG-HA vector (21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Expression plasmids for HCV NS3, NS4B, NS5A, and NS5B (pcDNA-NS3, pcDNA-NS4B, pcDNA-NS5A, and pcDNA-NS5B, respectively) were described in Ref. 21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar. pGEX-ESRα A/B, C, D, and E/F, expressing the fusion protein of the domain A/B, C, D, and E/F of ESRα with GST, were prepared by the insertion of the PCR product with pCMV-FL-ESRα as a template and appropriate primers into the EcoRI-XhoI site of pGEX-6P1 vector (Clontech). The expression plasmids for the point mutants of ESRα, ESRα(L540Q), ESRα(255M), and ESRα(258M), of which Leu at aa 540, IRK at aa 255-257, and DRR at aa 258-260 were replaced by Gln, TGT, and ANT, respectively, was generated by oligonucleotide-directed mutagenesis. pCMV-FL-CAV2, encoding FLAG-tagged CAV2, was prepared by inserting the PCR product amplified with 5′-GTTGTCGACTATGGGGCTGGAGAC-3′ and 5′-GTTAAGCTTTCAATCCTGGCTC-3′ as primers and human liver cDNA library (Clontech) as a template into the SalI-HindIII site of pCMV-FLAG vector (21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). The mammalian expression vector for the C domain of ESRα was generated by replacing the EcoRI-XhoI digestion of pCMV-FLAG vector (21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar) by that of pGEX-ESRα C. pLMH14 was described previously (26Murata T. Ohshima T. Yamaji M. Hosaka M. Miyanari Y. Hijikata M. Shimotohno K. Virology. 2005; 331: 407-417Crossref PubMed Scopus (57) Google Scholar). pGL3-EREX3-TATA-Luc, pcDNA3-ERα, pcDNA3-hERβ were kindly provided by Dr. Kato (Institute of Molecular and Cellular Biosciences, University of Tokyo). JFH1 expression plasmid was provided by Dr. Wakita (National Institute of Infectious Diseases). Luciferase Assay—A luciferase assay monitoring HCV replication activity was performed as described previously (22Goto K. Watashi K. Murata T. Hishiki T. Hijikata M. Shimotohno K. Biochem. Biophys. Res. Commun. 2006; 343: 879-884Crossref PubMed Scopus (124) Google Scholar, 26Murata T. Ohshima T. Yamaji M. Hosaka M. Miyanari Y. Hijikata M. Shimotohno K. Virology. 2005; 331: 407-417Crossref PubMed Scopus (57) Google Scholar). In Fig. 1, A and F, we used LucNeo#2 cells, stably carrying luciferase-containing subgenomic replicon (22Goto K. Watashi K. Murata T. Hishiki T. Hijikata M. Shimotohno K. Biochem. Biophys. Res. Commun. 2006; 343: 879-884Crossref PubMed Scopus (124) Google Scholar). In Figs. 2 (D and E), 4C, and 6A, we transiently transduced luciferase-containing replicon LMH14 RNA together with each expression plasmid into cured MH-14 cells (26Murata T. Ohshima T. Yamaji M. Hosaka M. Miyanari Y. Hijikata M. Shimotohno K. Virology. 2005; 331: 407-417Crossref PubMed Scopus (57) Google Scholar). A luciferase assay detecting the transcriptional activity driven from the ERE was performed as described previously (25Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar).FIGURE 2ESR was involved in HCV genome replication. A, specific knockdown of endogenous ESRα and ESRβ. RT-PCR analysis was performed to detect the expression of ESRα, ESRβ, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control in the cells transfected with siRNA recognizing ESRα (si-ESRα, si-ESRα2), ESRβ (si-ESRβ, si-ESRβ2), or randomized siRNA (si-control, si-control2). B, HCV RNA was quantified as shown in Fig. 1B, using the cells transfected with si-control, si-control2, si-ESRα, si-ESRα2, si-ESRβ, and si-ESRβ2 for 5 days. C, the ERE-mediated transcriptional activities were measured by a luciferase assay using the lysates from the cells transfected with pGL3-EREX3-TATA-Luc reporter plasmid together with pcDNA3-ERα (ESRα), pcDNA3-hERβ (ESRβ), pcDNA-ESRα(L540Q), or the empty vector (control) (left) or varying amounts (ng) of pcDNA3-ERα (ESRα) or pcDNA-ESRα(L540Q) (right) and treated with 100 nm estradiol for 36 h. D and E, HCV replication activities were examined by quantifying the luciferase activities using cured MH-14 cells transfected with the indicated doses (ng) of ESRα or ESRβ (D) or 30 ng of ESRα, ERα(L540Q), or the empty vector (control) (E) together with 0.125 μg of LMH14 RNA without or with 1 μm TAM for 4 days.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4The interaction of NS5B mediated the regulation of HCV genome replication by ESRα. A, GST pull-down assays were performed as described in Fig. 3A using the wild type ESRα or point mutant of ESRα, ESRα(255M), and ESRα(258M). B, the mutation within ESRα(255M) and ESRα(258M) did not reduce the activation capacity of ERE-mediated transcription. Huh-7 cells were transfected with the expression plasmids for ESRα, ESRα(255M), or ESRα(258M) at doses of 10, 30, and 100 ng each together with pGL3-EREX3-TATA-Luc reporter plasmid and treated without (white bar) or with 100 nm estradiol (black bar) to quantify the luciferase activity. C, HCV replication activities were examined by quantifying the luciferase activities as described in the legend to Fig. 2D in the cells upon transfection with the expression plasmids for wild type ESRα, ESRα(255M), or ESRα(258M). D, the cells were fractionated into the nucleus (N), MM, and cytoplasm (C). Each fraction was detected for FLAG-tagged ESRα, SC-35, calnexin, and IκBα, respectively, by immunoblot analysis. Calnexin, an ER marker protein, was detected in the nucleus as well as MM, probably because of the existence of the nuclear membrane in the nuclear fraction. E, the MM fraction obtained in D was subjected to a coimmunoprecipitation assay using anti-NS5B or IgG followed by immunoblot analysis for the detection for ESRα.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6ESRα could serve as a molecular target for anti-HCV agents. A, HCV replication activity was measured by quantifying the luciferase activity as described in the legend to Fig. 2D in the cells overexpressing a decoy peptide consisting of the C domain of ESRα. B, experimental scheme of in vitro HCV infection experiment. After seeding the HuS-E7/DN24 cells, HCV-positive serum was inoculated for 24 h. After extensive washes, the cells were cultured with the medium supplemented without (control) or with 1 μm TAM or 3 μg/ml cyclosporin A. HCV genome RNA was quantified along with the time course (days 1, 3, and 5 postinoculation) by real time RT-PCR analysis.C, the treatment with 1 μm TAM did not show any cytotoxic effect on HuS-E7/DN24 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed as described under “Experimental Procedures” to examine the viability of the cells at days 2, 3, and 5 postinoculation. D, HCV genome RNA was quantified as described in B and plotted against the time course.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Real Time RT-PCR Analysis—Real time RT-PCR analysis was performed as previously described (20Watashi K. Hijikata M. Hosaka M. Yamaji M. Shimotohno K. Hepatology. 2003; 38: 1282-1288Crossref PubMed Scopus (463) Google Scholar). Immunoblot Analysis—Immunoblot analysis was performed as previously described (25Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar). The antibodies used in this study are anti-NS5A (kindly provided by Dr. Takamizawa (Osaka University)), anti-NS5B (anti-NS5B#14; a generous gift from Dr. Kohara (Tokyo Metropolitan Institute of Medical Science)), anti-NS5B (NS5B#6; a kind gift from Dr. Fukuya (Osaka University)), anti-tubulin (Oncogene), anti-FLAG (Sigma), anti-IκBα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-calnexin (StressGen), and anti-caveolin-2 antibodies (BD Biosciences Pharmingen). Indirect Immunofluorescence Analysis—Indirect immunofluorescence analysis was performed as described previously (25Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar). The antibodies used were anti-NS5A and anti-protein-disulfide isomerase antibodies (StressGen). siRNA—siRNA duplexes (5′-GUGUGCAAUGACUAUGCUUCA-3′ for si-ESRα and 5′-CGCAUCGGGAUAUCACUAUGG-3′ for si-ESRβ) were synthesized (Proligo). A randomized siRNA, si-control, was purchased from Dharmacon (nonspecific control duplex IX). Enzyme-linked Immunosorbent Assay—HCV core was quantified in the culture medium of the cells transfected with JFH1 RNA (29Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Nat. Med. 2005; 11: 791-796Crossref PubMed Scopus (2417) Google Scholar) with an enzyme-linked immunosorbent assay according to the manufacturer's protocol (HCV antigen enzyme-linked immunosorbent assay test; Ortho-Clinical Diagnostics). RT-PCR Analysis—RT-PCR analysis was performed as described (20Watashi K. Hijikata M. Hosaka M. Yamaji M. Shimotohno K. Hepatology. 2003; 38: 1282-1288Crossref PubMed Scopus (463) Google Scholar) by using the following primer sets: 5′-CCTACTACCTGGAGAACG-3′ and 5′-GCTGGACACATATAGTCG-3′ for the detection of ESRα and 5′-AGCCATGACATTCTATAGC-3′ and 5′-CCACTTCGTAACACTTCC-3′ for ESRβ. GST Pull-down Assay—The GST pull-down assay was conducted as described previously (25Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar). Immunoprecipitation Analysis—Immunoprecipitation analysis was performed as described previously (25Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar). The antibodies used in this study were mouse normal IgG as a negative control (Zymed Laboratories), anti-NS5B (anti-NS5B#10; a generous gift from Dr. Kohara at the Tokyo Metropolitan Institute of Medical Science), anti-FLAG, and anti-caveolin-2 antibodies. Fractionation of Cell Extracts—MH-14 cells transfected with the expression plasmid for FLAG-tagged ESRα were fractionated essentially as described previously (25Watashi K. Hijikata M. Tagawa A. Doi T. Marusawa H. Shimotohno K. Mol. Cell. Biol. 2003; 23: 7498-7509Crossref PubMed Scopus (52) Google Scholar). HCV Replication Complex Assay—Isolation of HCV RC was done as described previously (16Miyanari Y. Hijikata M. Yamaji M. Hosaka M. Takahashi H. Shimotohno K. J. Biol. Chem. 2003; 278: 50301-50308Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 21Watashi K. Ishii N. Hijikata M. Inoue D. Murata T. Miyanari Y. Shimotohno K. Mol. Cell. 2005; 19: 111-122Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). In Vitro HCV Infection Experiment—In vitro HCV infection was conducted essentially as described (23Aly H.H. Watashi K. Hijikata M. Kaneko H. Takada Y. Egawa H. Uemoto S. Shimotohno K. J. Hepatol. 2007; 46: 26-36Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Briefly, HCV-infected serum (∼2 × 105 copies) was inoculated into HuS-E7/DN24 cells (5 × 104 cells) for 24 h. After washes, cells were cultured in the medium supplemented with 10 μm PD98059 to stimulate HCV translation (27Murata T. Hijikata M. Shimotohno K. Virology. 2005; 340: 105-115Crossref PubMed Scopus (23) Google Scholar) (scheme in Fig. 6B). To observe HCV amplification, HCV RNA in the cells was quantified, since HCV RNA was hardly detected significantly in the culture medium (23Aly H.H. Watashi K. Hijikata M. Kaneko H. Takada Y. Egawa H. Uemoto S. Shimotohno K. J. Hepatol. 2007; 46: 26-36Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Assay—The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to examine the cell viability using Cell Proliferation kit II, XTT (Roche Applied Science) according to the manufacturer's protocol. Tamoxifen Suppressed HCV Genome Replication—We screened for agents that suppressed HCV genome replication using a HCV subgenomic replicon system (13Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2498) Google Scholar, 16Miyanari Y. Hijikata M. Yamaji M. Hosaka M. Takahashi H. Shimotohno K. J. Biol. Chem. 2003; 278: 50301-50308Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Among the compounds tested, we observed that TAM inhibited HCV genome replication. HCV replication activity, monitored by luciferase activity (22Goto K. Watashi K. Murata T. Hishiki T. Hijikata M. Shimotohno K. Biochem. Biophys. Res. Commun. 2006; 343: 879-884Crossref PubMed Scopus (124) Google Scholar), and the amount of HCV RNA were decreased with TAM treatment in a dose-dependent manner (Fig. 1, A and B). The expression of HCV proteins, NS5A and NS5B, detected by immunoblot (Fig. 1C) and indirect immuofluorescence analyses (Fig. 1D), also drastically decreased by treatment with TAM. A high concentration of TAM decreased cell proliferation (Fig. 1E). However, TAM suppressed HCV replication without any cytotoxicity in another cell line, HuS-E7/DN24 cells (Fig. 6, C and D). In addition, a pure anti-estrogen compound ICI182780, which had little cytotoxic effect, reduced HCV RNA (Fig. 1, F and G). Moreover, TAM inhibited the production of core in the culture medium of HCV JFH1-transfected cells, in a recently developed system of the production of infectious HCV particles (Fig. 1H) (28Lindenbach B.D. Evans M.J. Syder A.J. Wolk B. Tellinghuisen T.L. Liu C.C. Maruyama T. Hynes R.O. Burton D.R. McKeating J.A. Rice C.M. Science. 2005; 309: 623-626Crossref PubMed Scopus (1954) Google Scholar, 29Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Nat. Med. 2005; 11: 791-796Crossref PubMed Scopus (2417) Google Scholar, 30Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. Wieland S.F. Uprichard S.L. Wakita T. Chisari F.V. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9294-9299Crossref PubMed Scopus (1519) Google Scholar). The above data indicate that TAM suppresses HCV genome replication. ESR Was Involved in HCV Genome Replication—Next, we investigated which cellular protein TAM targets to suppress HCV replication. It has been reported that TAM targets 1) ESR (31Shang Y. Nat. Rev. Cancer. 2006; 6: 360-368Crossref PubMed Scopus (209) Google Scholar), 2) P-glycoprotein (32Callaghan R. Higgins C.F. Br. J. Cancer. 1995; 71: 294-299Crossref PubMed Scopus (102) Google Scholar, 33Raderer M. Scheithauer W. Cancer. 1993; 72: 3553-3563Crossref PubMed Scopus (265) Google Scholar), 3) calmodulin (34Lopes M.C. Vale M.G. Carvalho A.P. Cancer Res. 1990; 50: 2753-2758PubMed Google Scholar), 4) protein kinase C (35O'Brian C.A. Liskamp R.M. Solomon D.H. Weinstein I.B. Cancer Res. 1985; 45: 2462-2465PubMed Google Scholar, 36O'Brian C.A. Ward N.E. Anderson B.W. J. Natl. Cancer Inst. 1988; 80: 1628-1633Crossref PubMed Scopus (63) Google Scholar), etc. Although other compounds targeting P-glycoprotein, calmodulin, and protein kinase C did not affect HCV replication in our screening (data not shown), ESR was suggested to play a role in HCV replication as shown below. RNAi-mediated specific knockdown of endogenous ESRα and ESRβ (Fig. 2A) reduced HCV RNA in replicon-containing cells to ∼20-40% and 60-70%, respectively (Fig. 2B). Transient transfection with ESRα and ESRβ expression plasmids, which activated ERE-driven transcription 4-5-fold (Fig. 2C), showed that ectopically expressed ESRα augmented HCV replication activity in a dose-dependent manner, whereas ESRβ did not (Fig. 2D). ESRα-induced augmentation of the replication was reversed upon TAM treatment (Fig. 2D). These results suggested a significant role of ESR, especially ESRα, in HCV genome replication. ESRα(L540Q), carrying a leucine to glutamine point mutation at aa 540 within the LXXLL motif (aa 536-540) of ESRα (37Leers J. Treuter E. Gustafsson J.A. Mol. Cell. Biol. 1998; 18: 6001-6013Crossref PubMed Scopus (93) Google Scholar), had much lower transactivation activity driven from" @default.
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