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Originally published In Press as doi:10.1074/jbc.M704418200 on August 17, 2007

J. Biol. Chem., Vol. 282, Issue 45, 32765-32772, November 9, 2007
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Anti-hepatitis C Virus Activity of Tamoxifen Reveals the Functional Association of Estrogen Receptor with Viral RNA Polymerase NS5B*

Koichi Watashi, Daisuke Inoue, Makoto Hijikata, Kaku Goto, Hussein H. Aly, and Kunitada Shimotohno1

From the Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan

Received for publication, May 30, 2007 , and in revised form, August 15, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ESR{alpha} resided on the endoplasmic reticulum membranes and interacted with HCV RNA polymerase NS5B. RNA interference-mediated knockdown of endogenous ESR{alpha} reduced HCV replication. Mechanistic analysis suggested that ESR{alpha} promoted NS5B association with the RC and that tamoxifen abrogated NS5B-RC association. Thus, ESR{alpha} regulated the presence of NS5B in the RC and stimulated HCV replication. Moreover, the ability of ESR{alpha} to regulate NS5B was suggested to serve as a potential novel target for anti-HCV therapeutics.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen receptor (ESR)2 belongs to the steroid hormone receptor family of the nuclear receptor superfamily (1). ESR consists of two subtypes, ESR{alpha} and ESRbeta. 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 (2-4). Although the molecular basis of ESR membrane retention is not fully understood, one mechanism involves a membrane protein, caveolin (CAV); ESR{alpha} interacted with CAV, and this interaction facilitated ESR{alpha} localization to the membrane (5, 6). It was also reported that ESR{alpha} localizes to the lipid rafts on the plasma membrane (7). 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 ESR{alpha} 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 (8). 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 (9, 10). NS5B is an RNA-dependent RNA polymerase, which plays a central role in viral genome replication (11, 12). HCV genome replication can be evaluated using a HCV subgenomic replicon system, which Lohmann et al. (13) 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 (14-17). It also has been reported that HCV genome replication associates with the lipid rafts on these intracellular membranes, such as the ER membrane (14, 18). These lipid rafts accumulate CAV2, and HCV proteins involved in viral genome replication cofractionate with CAV2 (18). 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 (19). 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 (20). We also reported that cyclophilin B regulated the RNA binding activity of NS5B (21). In the current study, this chemical screening approach linked ESR{alpha} to HCV replication. We showed that tamoxifen (TAM) suppressed HCV genome replication. Using TAM as a bioprobe, we found that ESR{alpha} interacted with NS5B and regulated the participation of NS5B in the RC.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—Huh-7 and cured MH-14 cells (21) 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 (16), and LucNeo#2 cells, carrying luciferase-containing subgenomic replicon (22), 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 (23), 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) (24). Plasmid transfection was performed with FuGENE 6 transfection reagent (Roche Applied Science), as described previously (25). RNA transfection was achieved using DMrie-C transfection reagent (Invitrogen), as described previously (21). siRNA was transfected by using siLentFect (Bio-Rad) (21).

Plasmid Construction—pCMV-FL-ESR{alpha}, encoding the whole open reading frame of ESR{alpha} 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 (21). 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 (21). Expression plasmids for HCV NS3, NS4B, NS5A, and NS5B (pcDNA-NS3, pcDNA-NS4B, pcDNA-NS5A, and pcDNA-NS5B, respectively) were described in Ref. 21. pGEX-ESR{alpha} A/B, C, D, and E/F, expressing the fusion protein of the domain A/B, C, D, and E/F of ESR{alpha} with GST, were prepared by the insertion of the PCR product with pCMV-FL-ESR{alpha} as a template and appropriate primers into the EcoRI-XhoI site of pGEX-6P1 vector (Clontech). The expression plasmids for the point mutants of ESR{alpha}, ESR{alpha}(L540Q), ESR{alpha}(255M), and ESR{alpha}(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 (21). The mammalian expression vector for the C domain of ESR{alpha} was generated by replacing the EcoRI-XhoI digestion of pCMV-FLAG vector (21) by that of pGEX-ESR{alpha} C. pLMH14 was described previously (26). pGL3-EREX3-TATA-Luc, pcDNA3-ER{alpha}, pcDNA3-hERbeta 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 (22, 26). In Fig. 1, A and F, we used LucNeo#2 cells, stably carrying luciferase-containing subgenomic replicon (22). 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 (26). A luciferase assay detecting the transcriptional activity driven from the ERE was performed as described previously (25).

Real Time RT-PCR Analysis—Real time RT-PCR analysis was performed as previously described (20).

Immunoblot Analysis—Immunoblot analysis was performed as previously described (25). 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-I{kappa}B{alpha} (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 (25). The antibodies used were anti-NS5A and anti-protein-disulfide isomerase antibodies (StressGen).

siRNA—siRNA duplexes (5'-GUGUGCAAUGACUAUGCUUCA-3' for si-ESR{alpha} and 5'-CGCAUCGGGAUAUCACUAUGG-3' for si-ESRbeta) 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 (29) 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 (20) by using the following primer sets: 5'-CCTACTACCTGGAGAACG-3' and 5'-GCTGGACACATATAGTCG-3' for the detection of ESR{alpha} and 5'-AGCCATGACATTCTATAGC-3' and 5'-CCACTTCGTAACACTTCC-3' for ESRbeta.

GST Pull-down Assay—The GST pull-down assay was conducted as described previously (25).

Immunoprecipitation Analysis—Immunoprecipitation analysis was performed as described previously (25). 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.


Figure 1
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  		FIGURE 1.
TAM suppressed the replication of the HCV genome. A, luciferase activities were measured using the LucNeo#2 cells, which carried a luciferase-containing replicon RNA, upon treatment with TAM at the indicated doses for 7 days. Relative luciferase activities are plotted against the concentrations of TAM. The data show the means of three independent experiments. The error bars are indicated. B, HCV RNA was quantified by real time RT-PCR analysis using the lysates from MH-14 cells, harboring the HCV subgenomic replicon, treated with the indicated doses of TAM for 7 days. Relative amounts of HCV RNA are shown. C, HCV NS5A and NS5B proteins as well as tubulin as an internal control were detected by immunoblot analysis in the lysates from MH-14 cells (replicon) treated without (control) or with 500 nM TAM or 100 IU/ml interferon-{alpha} as a positive control for 7 days and Huh-7 cells. D, HCV NS5A and protein-disulfide isomerase (PDI) as an internal control were detected by indirect immunofluorescence analysis in the cells treated without (control) or with 500 nM TAM for 7 days. 4',6-Diamidino-2-phenylindole (DAPI) shows a nuclear staining. E, cell number was counted after 5 days upon treatment with various concentrations of TAM. Relative cell numbers are shown. F, luciferase activities with LucNeo#2 cells treated with various concentrations of ICI182780 were measured as described in A. G, cell number was counted under treatment with ICI182780 at the indicated concentrations. H, core in the culture medium of JFH1 RNA-transfected cells upon treatment with TAM was quantified as described under "Experimental Procedures."

 
Fractionation of Cell Extracts—MH-14 cells transfected with the expression plasmid for FLAG-tagged ESR{alpha} were fractionated essentially as described previously (25).

HCV Replication Complex Assay—Isolation of HCV RC was done as described previously (16, 21).

In Vitro HCV Infection Experiment—In vitro HCV infection was conducted essentially as described (23). Briefly, HCV-infected serum (~2 x 105 copies) was inoculated into HuS-E7/DN24 cells (5 x 104 cells) for 24 h. After washes, cells were cultured in the medium supplemented with 10 µM PD98059 to stimulate HCV translation (27) (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 (23).

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Tamoxifen Suppressed HCV Genome Replication—We screened for agents that suppressed HCV genome replication using a HCV subgenomic replicon system (13, 16). Among the compounds tested, we observed that TAM inhibited HCV genome replication. HCV replication activity, monitored by luciferase activity (22), 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) (28-30). 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 (31), 2) P-glycoprotein (32, 33), 3) calmodulin (34), 4) protein kinase C (35, 36), 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 ESR{alpha} and ESRbeta (Fig. 2A) reduced HCV RNA in replicon-containing cells to ~20-40% and 60-70%, respectively (Fig. 2B). Transient transfection with ESR{alpha} and ESRbeta expression plasmids, which activated ERE-driven transcription 4-5-fold (Fig. 2C), showed that ectopically expressed ESR{alpha} augmented HCV replication activity in a dose-dependent manner, whereas ESRbeta did not (Fig. 2D). ESR{alpha}-induced augmentation of the replication was reversed upon TAM treatment (Fig. 2D). These results suggested a significant role of ESR, especially ESR{alpha}, in HCV genome replication. ESR{alpha}(L540Q), carrying a leucine to glutamine point mutation at aa 540 within the LXXLL motif (aa 536-540) of ESR{alpha} (37), had much lower transactivation activity driven from ERE (Fig. 2C). However, ESR{alpha}(L540Q) stimulated HCV replication activity ~5-fold, although the stimulation was less than that by wild-type ESR{alpha} (Fig. 2E). Thus, ESR{alpha} having lower transactivating capacity could still facilitate HCV replication.


Figure 2
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  		FIGURE 2.
ESR was involved in HCV genome replication. A, specific knockdown of endogenous ESR{alpha} and ESRbeta. RT-PCR analysis was performed to detect the expression of ESR{alpha}, ESRbeta, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control in the cells transfected with siRNA recognizing ESR{alpha} (si-ESR{alpha}, si-ESR{alpha}2), ESRbeta (si-ESRbeta, si-ESRbeta2), 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-ESR{alpha}, si-ESR{alpha}2, si-ESRbeta, and si-ESRbeta2 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-ER{alpha} (ESR{alpha}), pcDNA3-hERbeta (ESRbeta), pcDNA-ESR{alpha}(L540Q), or the empty vector (control) (left) or varying amounts (ng) of pcDNA3-ER{alpha} (ESR{alpha}) or pcDNA-ESR{alpha}(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 ESR{alpha} or ESRbeta (D) or 30 ng of ESR{alpha}, ER{alpha}(L540Q), or the empty vector (control) (E) together with 0.125 µg of LMH14 RNA without or with 1 µM TAM for 4 days.

 
ESR{alpha} Interacted with HCV NS5B—Thus, the chemical biology approach revealed the involvement of ESR in HCV genome replication. Then we investigated the molecular mechanism of ESR-induced HCV replication. A binding assay between ESR{alpha} and HCV proteins expressed in the HCV subgenomic replicon showed that the C domain of ESR{alpha} coprecipitated with NS5B but not NS3, NS4B, and NS5A (Fig. 3A). Other ESR{alpha} domains, A/B, D, and E/F, did not bind to any HCV proteins. A coimmunoprecipitation assay also indicated the presence of ESR{alpha} in the immunoprecipitate by anti-NS5B antibody (Fig. 3, B and D), and vice versa (Fig. 3C). Thus, ESR{alpha} specifically interacted with NS5B. Deletion analysis indicated that the region of 71-591 and 1-520 but not 521-591 of NS5B coprecipitated with the recombinant C domain of ESR{alpha} (Fig. 3E). This binding profile is different from that between cyclophilin B and NS5B, which we previously reported (21).


Figure 3
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  		FIGURE 3.
ESR{alpha} interacted with HCV NS5B. A, top, schematic representation of the primary structure of ESR{alpha}. ESR{alpha} consists of domains A-F. The amino acid numbers are also shown. Bottom, GST pull-down assays were performed using the recombinant proteins of the A/B, C, D, and E/F domain of ESR{alpha} fused with GST and in vitro translated HCV NS3, NS4B, NS5A, and NS5B protein. Input, the one-fifth amount of protein used for the pull-down assay. The Coomassie Brilliant Blue staining pattern of the precipitated fraction is also shown in the bottom panel. B-D, the lysates from the cells ectopically expressing NS5B (B and C) or the whole open reading frame of the HCV JFH1 strain (D) and/or FLAG-tagged ESR{alpha} were immunoprecipitated (IP) with anti-NS5B (B; NS5B), anti-FLAG antibody (C and D; FL), or mouse normal IgG as a negative control followed by the detection of ESR{alpha} and NS5B by immunoblot analysis (IB). E, deletion mutants of NS5B were subjected to a GST pull-down assay with GST-fused C domain of ESR{alpha} as described in A. The left panel shows a schematic representation of the full-length and truncated mutants of NS5B. The numbers indicate the amino acid numbers in NS5B.

 
The ESR{alpha}-NS5B Interaction Was Important for the Regulation of HCV Genome Replication—To examine whether the interaction between ESR{alpha} and NS5B was essential for the ESR{alpha}-mediated regulation of HCV replication or not, we searched for a point mutant of ESR{alpha} that could not bind to NS5B by alanine-scanning mutation analysis. ESR{alpha} mutants, ESR{alpha}(255M) and ESR{alpha}(258M), in which IRK at aa 255-257 and DRR at aa 258-260 was replaced by TGT and AQT, respectively, had little affinity with NS5B (Fig. 4A) but still possessed the ERE-mediated transactivation capacity (Fig. 4B). However, both ESR{alpha}(255M) and ESR{alpha}(258M) caused only weak activations of HCV replication, compared with wild type ESR{alpha} (Fig. 4C). The data suggest that the interaction of ESR{alpha} with NS5B is critical for ESR{alpha}-mediated regulation of HCV genome replication.


Figure 4
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  		FIGURE 4.
The interaction of NS5B mediated the regulation of HCV genome replication by ESR{alpha}. A, GST pull-down assays were performed as described in Fig. 3A using the wild type ESR{alpha} or point mutant of ESR{alpha}, ESR{alpha}(255M), and ESR{alpha}(258M). B, the mutation within ESR{alpha}(255M) and ESR{alpha}(258M) did not reduce the activation capacity of ERE-mediated transcription. Huh-7 cells were transfected with the expression plasmids for ESR{alpha}, ESR{alpha}(255M), or ESR{alpha}(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 ESR{alpha}, ESR{alpha}(255M), or ESR{alpha}(258M). D, the cells were fractionated into the nucleus (N), MM, and cytoplasm (C). Each fraction was detected for FLAG-tagged ESR{alpha}, SC-35, calnexin, and I{kappa}B{alpha}, 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 ESR{alpha}.

 
Thus, ESR{alpha} interaction with NS5B regulates HCV replication. NS5B is mainly located on the cytoplasmic surface of the ER membrane (21, 38). On the other hand, ESR{alpha} as a nuclear hormone receptor is normally distributed in the cytoplasm and translocates into the nucleus upon ligand stimulation. In addition, a part of ESR{alpha} localizes on the membrane fraction. In our experiment, NS5B was mainly located around the ER, colocalized with the ER marker, protein-disulfide isomerase (data not shown) (21). Ectopically expressed ESR{alpha} showed diffuse distribution in the cells (data not shown). We fractionated cell homogenates and observed that a part of the ESR{alpha} resided in the microsomal membrane (MM) fraction (Fig. 4D). Moreover, ESR{alpha} in the MM fraction was coprecipitated with NS5B (Fig. 4E). It suggests the possibility that the interaction between NS5B and ESR{alpha}, at least in part of them, occurs on the ER membrane.

ESR{alpha} Promoted the Participation of NS5B in the HCV Replication Complex—It was reported that HCV proteins involved in the replication machinery was associated with the lipid raft on the ER and cofractionated with CAV2. A coimmunoprecipitation assay showed that NS5B associated with CAV2 (Fig. 5A). In the experiment investigating the role of ESR{alpha} in NS5B-CAV2 association, the coprecipitation of NS5B with CAV2 was decreased upon the knocking down of ESR{alpha} (Fig. 5B). Treatment with TAM abrogated the association of NS5B with CAV2 (Fig. 5C), although the total amount of NS5B in the cells is similar in the presence and absence of TAM for 24 h in this experiment (data not shown). Thus, ESR{alpha} was suggested to promote the association between NS5B and CAV2. Since a part of CAV2 resided on the lipid raft on the ER (18), ESR{alpha}-mediated binding between NS5B and CAV2 was possible to affect the localization of NS5B to the HCV RC. To see the consequential relevance of ESR{alpha} on NS5B function, we analyzed the HCV RC by treatment with digitonin/protease as described previously (16). HCV proteins involved in the RC and surrounded by the membrane structure are resistant to the treatment with digitonin followed by protease, whereas those unrelated to the replication outside the RC are digested by the treatment. By using this technique measuring the sensitivity to protease, HCV RC can be distinguished from the ER that is not related to the replication, although the RC and the nucleus cannot be separated. The experimental condition for fractionation was confirmed with the detection with I{kappa}B{alpha} and calnexin; a cytosolic protein I{kappa}B{alpha} was washed out following the treatment with digotinin (Fig. 5D, lanes 1 and 2), and ER protein calnexin, which did not accumulate in the RC, was digested by treatment with digotinin/protease (Fig. 5D, lanes 2-4). An ER lipid raft component, CAV2, was still detected under the digitonin/protease treatment (the RC-containing fraction) (Fig. 5D, lanes 3 and 4). Under this condition, a part of NS5B was detected in the digitonin/protease-resistant fraction, as described previously (16) (Fig. 5D, lanes 3 and 4). However, NS5B in this fraction was decreased upon treatment with TAM (Fig. 5D, lanes 3, 4, 7, and 8). On the other hand, the amount of NS5A was not significantly changed by TAM treatment. Knocking down of ESR{alpha} also disrupted the association of NS5B with the RC-containing fraction (Fig. 5E). From the above results, it was suggested that ESR{alpha} promoted the participation of NS5B in the RC (also see "Discussion").

ESR{alpha} Could Serve as a Molecular Target of Anti-HCV Agents—Finally, we assessed the possibility that the association of ESR{alpha} with NS5B could serve as a target of anti-HCV agents. By introducing a decoy peptide against ESR{alpha}-NS5B interaction, consisting of the C domain of ESR{alpha} into replicon-bearing cells, HCV replication activity was reduced in a dose-dependent manner (Fig. 6A). To further observe the significance of ESR{alpha} in a physiological condition, we performed an in vitro infection experiment using serum from an HCV-infected patient as a nascent virus inoculum and nonneoplastic human hepatocytes as highly infection-permissive cells (Fig. 6B). Treatment with 1 µM TAM did not show a cytotoxic effect on these cells in any time course examined (Fig. 6C). However, treatment with TAM as well as cyclosporin A as a positive control inhibited the multiplication of viral genome RNA in the cells along with the time course (Fig. 6D). Thus, ESR{alpha} could serve as a potent molecular target of anti-HCV agents.


Figure 5
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  		FIGURE 5.
ESR{alpha} promoted the participation of NS5B in HCV RC. A-C, a coimmunoprecipitation assay (IP) was performed with anti-FLAG (A), anti-CAV2 (B and C) antibody, or mouse normal IgG from the lysates of the cells transfected without or with FLAG-tagged CAV2 (A), transfected with si-control or si-ESR{alpha} (B), or treated without or with 1 µM TAM (C). NS5B (top) and CAV2 (bottom) were detected by immunoblot analysis. D, detection of the amount of NS5B in the digitonin/protease-resistant fraction. MH-14 cells were treated without (lanes 1-4) or with 1 µM TAM (lanes 5-8) for 24 h. Cells were then treated without (lanes 1 and 5) or with digitonin (lanes 2-4 and 6-8), followed by digestion with proteinase K (0 µg/ml for lanes 2 and 6, 0.3 µg/ml for lanes 3 and 7, and 1 µg/ml for lanes 4 and 8). NS5B, NS5A, I{kappa}B{alpha}, calnexin, and CAV2 were detected by immunoblot analysis. E, HCV RC was isolated as described in D using the cells transfected with si-control or si-ESR{alpha}, and NS5B was detected. A similar result was obtained by using si-ESR{alpha}2.

 


Figure 6
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  		FIGURE 6.
ESR{alpha} 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 ESR{alpha}. 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In general, viruses take advantage of host cell factors for their replication. So far, some factors have been shown to relevantly regulate HCV replication, including hVAP33 (39, 40), FBL2 (41), and cyclophilin B (21). Among these, FBL2 and cyclophilin B were identified by a chemical biological approach; FBL2 from the observation of an anti-HCV activity of lovastatin and an inhibitor of geranylgeranyl transferase (41-43); cyclophilin B from the inhibitory effect of cyclosporin A on HCV replication (20, 21). In this study, we found a suppressive capacity of TAM to HCV genome replication. Through further examination using TAM, we revealed ESR{alpha} as a host cell factor regulating HCV replication and suggested its regulation mechanism.

Currently, it is proposed that HCV RC that replicates the HCV genome is formed on the intracellular membrane, including the ER membrane (14-17). It was also reported that HCV genome replication was associated with the lipid raft on the intracellular membrane (18). Most HCV proteins are not related to the RC, whereas only a minor portion of HCV proteins take part in the RC to drive the viral replication (16). It has remained widely unknown, however, how HCV proteins are regulated to participate in the RC. It was reported that hVAP-33 binds to NS5A and NS5B, and this protein is related to the amount of NS5B in the lipid raft (40). hVAP-33 was speculated to recruit NS5B to the lipid raft, although its molecular mechanism has not been analyzed. This study suggested the interaction between ESR{alpha} and NS5B in the ER fraction, although we did not show the existence of ESR{alpha} in the RC, since the RC and the nucleus cannot be separated in the digitonin/protease treatment experiment. ESR{alpha} promoted the interaction of NS5B with CAV2. Previous papers reported that ESR{alpha} bound to CAV1 and CAV2 (6). From these observations, ESR{alpha} is likely to function as a bridging factor that connects NS5B to CAV2, although we cannot fully neglect the possibility that ESR{alpha} augments NS5B-CAV2 binding via another function, such as transcriptional activity. Because CAV2 resided on the lipid raft of the intracellular membrane (18), this action of ESR{alpha} may recruit NS5B to the lipid raft and the HCV RC. In fact, ESR{alpha} promoted the participation of NS5B in the HCV RC. Thus, ESR{alpha} is suggested to escort NS5B to the HCV RC, although it is also possible that ESR{alpha} augments the number of the RC itself. However, ESR{alpha} at least augments the amount of NS5B involved in HCV replication machinery to stimulate the replication. It was reported that the membrane-associated ESR{alpha} served as a platform where signalsomes, including receptor tyrosine kinase, nonreceptor tyrosine kinase Src, and G proteins, assembled and activated downstream signaling pathways (44-46). HCV may also take advantage of such platform characteristics of ESR{alpha} to form the RC for their efficient replication. Although the mechanisms of the nuclear receptor function of ESR{alpha} have been extensively elucidated, the functions of membrane-associated ESR{alpha} have not been widely characterized so far. This study suggested a novel physiological relevance of membrane-associated ESR{alpha} as a regulator of the viral replication.

Until now, there are no clinical studies that report a direct interaction of TAM treatment with HCV replication in patients infected with HCV. Given our results, examinations on the effect of TAM or other anti-estrogen drugs may be one of the useful approaches to develop a new anti-HCV strategy. On the other hand, we disclosed the mechanism of ESR-mediated regulation of HCV genome replication. Screening for compounds that inhibit this mechanism expectedly led to novel types of anti-HCV agents. Further analyses on ESR are needed to develop anti-HCV therapeutics as well as reveal the regulation mechanism of HCV replication.


   FOOTNOTES
 
* This work was supported by grants-in-aid for cancer research and for the second term comprehensive 10-year strategies for cancer control from the Ministry of Health, Labor, and Welfare; by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology; by grants-in-aid for the Research for the Future Program from the Japanese Society for the Promotion of Science; and by grants-in-aid for the Program for Promotion of Fundamental Studies in Health Science from the Organization for Pharmaceutical Safety. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: 81-75-751-4000; Fax: 81-75-751-3998; E-mail: kshimoto{at}virus.kyoto-u.ac.jp.

2 The 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. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. T. Murata, T. Hishiki, and M. Hosaka for establishing the replicon-containing cells. We thank Dr. Aratake (Asahi Kasei Pharma) for helpful discussions. We also thank Dr. Kato, Dr. Takamizawa, Dr. Kohara, Dr. Fukuya, and Dr. Wakita for kindly providing the plasmids: pGL3-EREX3-TATA-Luc, pcDNA3-ER{alpha}, and pcDNA3-hERbeta; anti-NS5A antibody; anti-NS5B antibody; anti-NS5B antibody; and JFH1 expression plasmid, respectively.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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