Iron Inactivates the RNA Polymerase NS5B and Suppresses Subgenomic Replication of Hepatitis C Virus
2005; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês
10.1074/jbc.m412687200
ISSN1083-351X
AutoresCarine Fillebeen, Ana Rivas, Martin Bisaillon, Prem Ponka, Martina U. Muckenthaler, Matthias W. Hentze, Antonis E. Koromilas, Kostas Pantopoulos,
Tópico(s)Hepatitis B Virus Studies
ResumoClinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a well-established model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent Kd: 6 and 60 μm for Fe2+ and Fe3+, respectively) to the protein's Mg2+-binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication. Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a well-established model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent Kd: 6 and 60 μm for Fe2+ and Fe3+, respectively) to the protein's Mg2+-binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication. Infection with hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; DFO, desferrioxamine; SIH, salicylaldehyde isonicotinoyl hydrazone; Mt-2, metallothionein 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region; CAT, chloramphenicol acetyltransferase; NPT, neomycin phosphotransferase; IRES, internal ribosome entry site; EMCV, encephalomyocarditis virus; RdRp, RNA-dependent RNA polymerase. poses a serious health care problem worldwide and is the leading cause of blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1Seeff L.B. Hepatology. 2002; 36: S35-S46Crossref PubMed Google Scholar). HCV is a positive-polarity, single-stranded RNA virus, a member of the Hepacivirus genus of the Flaviviridae family (2Shi S.T. Lai M.M. Cell Mol. Life Sci. 2001; 58: 1276-1295Crossref PubMed Scopus (24) Google Scholar). There are at least six major HCV genotypes and a large number of subtypes (3Bukh J. Miller R.H. Purcell R.H. Semin. Liver Dis. 1995; 15: 41-63Crossref PubMed Scopus (754) Google Scholar). The viral genome comprises ∼9600 nucleotides and contains a single, large open reading frame, which encodes a precursor polypeptide of ∼3010 amino acids (4Tellinghuisen T.L. Rice C.M. Curr. Opin. Microbiol. 2002; 5: 419-427Crossref PubMed Scopus (154) Google Scholar). This is proteolytically cleaved to yield the functional proteins of the virus by combined actions of host-derived signal peptidase and viral-encoded protease activities. The course of infection is affected by various factors, including the body iron status (5Pietrangelo A. Gastroenterology. 2003; 124: 1509-1523Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Chronic hepatitis C is often associated with mild to moderate iron accumulation in the liver, primarily in sinusoidal/Kupffer cells. Clinical studies show a positive correlation between elevated iron indices, such as hepatic iron content, serum ferritin levels, or transferrin saturation, and liver damage in HCV infection (6Ioannou G.N. Tung B.Y. Kowdley K.V. Semin. Gastrointest. Dis. 2002; 13: 95-108PubMed Google Scholar, 7Sherrington C.A. Olynyk J.K. Liver. 2002; 22: 187-189Crossref PubMed Scopus (15) Google Scholar, 8Tung B.Y. Emond M.J. Bronner M.P. Raaka S.D. Cotler S.J. Kowdley K.V. Gastroenterology. 2003; 124: 318-326Abstract Full Text PDF PubMed Scopus (140) Google Scholar). Increased iron indices have also been associated with poor response to treatment with interferon-α (9Akiyoshi F. Sata M. Uchimura Y. Suzuki H. Tanikawa K. Am. J. 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Locarnini S. Am. J. Gastroenterol. 2002; 97: 982-987PubMed Google Scholar), and phlebotomy did not substantially improve the efficacy of antiviral therapies (15Sartori M. Andorno S. Rigamonti C. Boldorini R. Dig. Liver Dis. 2001; 33: 157-162Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 16Di Bisceglie A.M. Bonkovsky H.L. Chopra S. Flamm S. Reddy R.K. Grace N. Killenberg P. Hunt C. Tamburro C. Tavill A.S. Ferguson R. Krawitt E. Banner B. Bacon B.R. Hepatology. 2000; 32: 135-138Crossref PubMed Scopus (155) Google Scholar). Iron overload is, by itself, a risk factor for liver fibrosis, cirrhosis, and hepatocellular carcinoma (17Pietrangelo A. Semin. Liver Dis. 1996; 16: 13-30Crossref PubMed Scopus (151) Google Scholar), and it appears to aggravate the clinical picture of chronic hepatitis C. A pathogenic synergism is evident in the combination of HCV infection and the common disease of iron overload hereditary hemochromatosis, which is associated with accelerated liver damage (18Diwakaran H.H. Befeler A.S. Britton R.S. Brunt E.M. Bacon B.R. J. Hepatol. 2002; 36: 687-691Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 19Bonkovsky H.L. Troy N. McNeal K. Banner B.F. Sharma A. Obando J. Mehta S. Koff R.S. Liu Q. Hsieh C.C. J. Hepatol. 2002; 37: 848-854Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The most prevalent form of hereditary hemochromatosis is linked to mutations in the HFE gene, encoding an atypical major histocompatibility complex class I type molecule that appears to control dietary iron absorption and body iron reutilization (20Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1413) Google Scholar). Clinical studies suggest that HFE mutations exacerbate hepatic fibrogenesis in chronic hepatitis C (5Pietrangelo A. Gastroenterology. 2003; 124: 1509-1523Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), mostly at early stages (8Tung B.Y. Emond M.J. Bronner M.P. Raaka S.D. Cotler S.J. Kowdley K.V. Gastroenterology. 2003; 124: 318-326Abstract Full Text PDF PubMed Scopus (140) Google Scholar). A failure to find such a correlation in some reports may be related to the lack of control for confounding variables (5Pietrangelo A. Gastroenterology. 2003; 124: 1509-1523Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Whereas clinical data suggest that iron metabolism is tightly linked to HCV pathology, it is unknown whether and how iron interferes with viral replication and the expression of viral proteins. The molecular mechanisms by which HCV affects iron metabolism are also poorly understood. The development of subgenomic replicon systems has provided a powerful tool not only for basic studies on the biology of HCV but also for the design and evaluation of pharmacological interventions (21Randall G. Rice C.M. Curr. Opin. Infect. Dis. 2001; 14: 743-747Crossref PubMed Scopus (26) Google Scholar, 22Bartenschlager R. Nat. Rev. Drug Discov. 2002; 1: 911-916Crossref PubMed Scopus (136) Google Scholar). Here, we utilized HCV replicon cells to search for molecular links between iron metabolism and HCV replication. Materials—Hemin was purchased from Sigma, and desferrioxamine (DFO) was purchased from Novartis (Dorval, Quebec, Canada). Fe-SIH was prepared by mixing SIH with ferric citrate in 2:1 ratio (23Ponka P. Schulman H.M. J. Biol. Chem. 1985; 260: 14717-14721Abstract Full Text PDF PubMed Google Scholar). Cell Culture—Replicon and parent human Huh7 hepatoma cells (24Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2503) Google Scholar, 25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar) and human embryonic kidney 293 cells (26Ali S. Pellerin C. Lamarre D. Kukolj G. J. Virol. 2004; 78: 491-501Crossref PubMed Scopus (64) Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 1% non-essential amino acids, 100 units/ml penicillin, and 100 μg/ml streptomycin. The replicon cells were maintained in media containing 500 μg/ml G418 (Geneticin; Invitrogen) in addition to the above-mentioned supplements. For a typical experiment, 1 × 106 Huh7 or 5 × 106 293 cells were seeded into 10-cm plates and subjected to iron manipulations on the next day. Generation of Additional Replicon Huh7 Clones—Total RNA from the replicon Huh7 clone described in Ref. 25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar was transfected into parent Huh7 cells by the Lipofectamine reagent (Invitrogen), and stable clones were selected in the presence of 500 μg/ml G418 (27Kato T. Date T. Miyamoto M. Furusaka A. Tokushige K. Mizokami M. Wakita T. Gastroenterology. 2003; 125: 1808-1817Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Western Blotting—Cytoplasmic lysates were resolved by SDS-PAGE on 10% gels and transferred onto Immobilon™P polyvinylidene difluoride membranes (Millipore Corp.), as described in Ref. 25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar. The blots were saturated with 10% non-fat milk in phosphate-buffered saline and probed with 1:1000-diluted antibodies against NS5A (Biogenesis), NPT-II (Cortex Biochem), phosphorylated (at Ser51) eIF-2α (25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar), transferrin receptor 1 (Zymed Laboratories Inc.), or β-actin (Sigma). Dilutions were in phosphate-buffered saline containing 0.5% Tween 20 (PBST). After a wash with PBST, the blots with monoclonal NS5A antibodies were incubated with peroxidase-coupled rabbit anti-mouse IgG (1:4000 dilution), and the blots with all other polyclonal antibodies were incubated with peroxidase-coupled goat anti-rabbit IgG (1:5000 dilution). Detection of peroxidase-coupled antibodies was performed with the ECL method (Amersham Biosciences). Northern Blotting—Cells were lysed with TRizol reagent (Invitrogen), and RNA was prepared according to the manufacturer's recommendations. Total cellular RNA (10 μg) was electrophoretically resolved on denaturing agarose gels, transferred onto nylon membranes, and hybridized to radiolabeled cDNA probes against replicon RNA (25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar), human Mt-2, or rat GAPDH. Autoradiograms were quantified by phosphorimaging. Reporter Gene Assays—Huh7 cells expressing the HCV IRES/3′-UTR (25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar) or the EMCV IRES (28Wilson J.E. Powell M.J. Hoover S.E. Sarnow P. Mol. Cell. Biol. 2000; 20: 4990-4999Crossref PubMed Scopus (251) Google Scholar) bicistronic construct (Fig. 2A) were subjected to iron manipulations, and cell extracts were prepared for reporter gene assays. The expression of HCV IRES/3′-UTR or EMCV IRES was analyzed by CAT/luciferase (25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar) or dual luciferase (Promega) assays, respectively. The luciferase/CAT and firefly/Renilla luciferase ratios were used to estimate the activities of the HCV and EMCV IRES, respectively. Assessment of Replicon RNA Stability—Replicon and parent Huh7 cells were pretreated for 2 h with 4 μg/ml actinomycin D and metabolically labeled (in the presence of actinomycin D) for 16 h in phosphatefree media with 200 μCi of [α-32P]UTP (ICN), as described in Ref. 29Bost A.G. Venable D. Liu L. Heinz B.A. J. Virol. 2003; 77: 4401-4408Crossref PubMed Scopus (66) Google Scholar. Subsequently, the cells were washed and chased in cold media. Total RNA (0.5 μg) was extracted from each plate and resolved on a 1% formaldehyde-agarose gel. The gel was dried, and RNA was visualized by autoradiography and quantified by phosphorimaging. Expression and Purification of NS5BΔ21—NS5BΔ21, a truncated form of HCV NS5B protein lacking the 21 C-terminal amino acids, was expressed and purified as described previously (30Bougie I. Charpentier S. Bisaillon M. J. Biol. Chem. 2003; 278: 3868-3875Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Fluorescence Measurements—Fluorescence was measured using a Hitachi F-2500 fluorescence spectrophotometer. Background emission was eliminated by subtracting the signal from either buffer alone or buffer containing the appropriate quantity of substrate. The extent to which ligands bind to the NS5B protein was determined by monitoring the fluorescence emission of a fixed concentration of proteins and titrating with a given ligand in binding buffer (50 mm Tris-Cl, pH 7.5, 50 mm KOAc). In experiments with ferrous iron (provided by FeSO4) as a ligand, the redox state of Fe2+ was maintained by the addition of 1 mm ascorbate to the binding buffer. The binding can be described by Eq. 1:Kd=[NS5B][ligand][NS5B⋅ligand](Eq. 1) where Kd is the apparent dissociation constant, [NS5B] is the concentration of the protein, [NS5B·ligand] is the concentration of complexed protein, and [ligand] is the concentration of unbound ligand. The proportion of ligand-bound protein as described by Eq. 1 is related to measured fluorescence emission intensity by Eq. 2:ΔF/ΔFmax=[NS5B⋅ligand]/[NS5B]tot(Eq. 2) where ΔF is the magnitude of the difference between the observed fluorescence intensity at a given concentration of ligand and the fluorescence intensity in the absence of ligand, ΔFmax is the difference at infinite [ligand], and [NS5B]tot is the total protein concentration. If the total ligand concentration, [ligand]tot, is in large molar excess relative to [NS5B]tot, then it can be assumed that [ligand] is approximately equal to [ligand]tot. Eqs. 1 and 2 can then be combined to give Eq. 3.ΔF/ΔFmax=[ligand]tot/(Kd+[ligand]tot)(Eq. 3) The Kd values were determined from a nonlinear least square regression analysis of titration data by using Eq. 3. Analysis of Competitive Metal Ion Binding—Analysis of the effect of a fixed concentration of one metal ion ligand (iona) on the binding of a second ion ligand (ionb) was performed in a manner analogous to that reported previously for analyzing the kinetics of a system in which two alternative substrates compete for the same enzyme binding site (31Painter G.R. Wright L.L. Hopkins S. Furman P.A. J. Biol. Chem. 1991; 266: 19362-19368Abstract Full Text PDF PubMed Google Scholar). The change in fluorescence (ΔF) observed upon titration of NS5B with iona in the presence of a fixed concentration of competing substrate (ionb) can be described by Eq. 4:ΔF=ΔFmax(a)([iona]/Ka)+ΔFmax(b)([ionb]/Kb)+([iona]/Ka+[ionb]/Kb)(Eq. 4) where ΔFmax(a) and ΔFmax(b) are the changes in fluorescence produced at infinite concentrations of iona and ionb, respectively. Ka and Kb are the apparent dissociation constants for iona and ionb, respectively. Eq. 4 was fit to the simple ligand saturation isotherms for both iona and ionb. Primer-independent RNA Polymerase Assay—Polymerization assays with purified NS5BΔ21 (5 nm) were performed in the presence of 50 nm HCV-specific 3′-UTR RNA template (32Pellerin C. Lefebvre S. Little M.J. McKercher G. Lamarre D. Kukolj G. Biochem. Biophys. Res. Commun. 2002; 295: 682-688Crossref PubMed Scopus (10) Google Scholar), 20 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 1 mm dithiothreitol, 5 μCi of [α-32P]UTP, 10 μm UTP, and 500 μm of the three other NTPs. The reactions were performed in standard buffer supplemented (or not) with various concentrations of Fe2+ and 1 mm ascorbate. The reactions were incubated at 22 °C for 2.5 h. The RNA products were analyzed on denaturing 8 m urea, 5% polyacrylamide gels; visualized by autoradiography; and quantified by phosphorimaging. The IC50 value for Fe2+ was determined by nonlinear least square regression analysis of titration data. RNA Polymerase Assay in Extracts of Replicon Huh7 Cells—Cytoplasmic extracts of replicon and parent Huh7 cells were prepared by a modified protocol of Ref. 33Ali N. Tardif K.D. Siddiqui A. J. Virol. 2002; 76: 12001-12007Crossref PubMed Scopus (77) Google Scholar. Briefly, after washing with wash buffer (150 mm sucrose, 30 mm HEPES, pH 7.4, 33 mm NH4Cl, and 7 mm KCl), the cells were treated with 250 μg/ml lysolecithin for 1 min and washed again. The cells were collected by scraping in 120 μl of lysis buffer (100 mm HEPES, pH 7.4, 50 mm NH4Cl, 7 mm KCl, and 10% glycerol) and lysed gently by pipetting up and down at least 15 times. The cell suspension was centrifuged at 1600 rpm for 5 min at 4 °C. The cytoplasmic fraction was aliquoted and stored at –80 °C until use. The cytoplasmic extract (50 μl for each reaction) was incubated at 34 °C with 50 μl of replication buffer (50 μCi of [α-32P]UTP, 20 μm UTP, 2 mm of the three other NTPs, 2 mm spermidine, 2 mm dithiothreitol, 4 μg/ml actinomycin D, 1000 units/ml RNasin, 10 mm creatine phosphate, and 80 units/ml creatine phosphokinase) in the presence or absence of Mg2+ and Fe2+/1 mm ascorbate. After 60 min, alkaline phosphatase (5 units; Sigma) was added, and incubation continued for another 20 min. Reactions were terminated by the addition of 100 μl of stop buffer (10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 150 mm NaCl, and 1% SDS). RNA products isolated by phenol/chloroform extraction and ethanol precipitation were analyzed on a denaturing formaldehyde-agarose gel, visualized by autoradiography, and quantified by phosphorimaging. The IC50 value for Fe2+ was determined by nonlinear least square regression analysis of titration data. Loading of Replicon Huh7 Cells with Iron Decreases the Expression of NS5A and NPT-II—The prototype replicon consists of a subgenomic HCV RNA, which is sufficient for replication in Huh7 hepatoma cells (24Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2503) Google Scholar). In this system, the HCV structural region has been replaced by the NPT-II gene that is translated under the control of the HCV IRES. The translation of the viral proteins NS3 to NS5 is directed by the EMCV IRES (Fig. 1A). To explore the effects of iron on HCV replication, replicon Huh7 cells were exposed for 24 h to increasing concentrations of hemin, an iron donor, or DFO, an iron chelator, and the expression of the viral protein NS5A and the marker NPT-II was assessed by Western blotting (Fig. 1B). Quantitative conditions for the analysis of these proteins were established previously (25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar). The treatment with hemin inhibited the expression of both NS5A and NPT-II (Fig. 1B, first and second panels, respectively, lanes 5–7), whereas DFO appeared to elicit opposite responses (lanes 1–4), and levels of control β-actin remained unchanged (bottom panel). The inhibitory effect of hemin was consistently much stronger on NS5A compared with NPT-II, possibly reflecting differences in the stability of these proteins (25Rivas-Estilla A.M. Svitkin Y. Lopez Lastra M. Hatzoglou M. Sherker A. Koromilas A.E. J. Virol. 2002; 76: 10637-10653Crossref PubMed Scopus (41) Google Scholar). Similar results were also obtained following iron perturbations for 12 h, but the range of regulation was lower (data not shown). As a control for the hemin treatment, we evaluated the phosphorylation status of the translation initiation factor eIF-2α by probing with a phospho-specific antibody (Fig. 1B, third panel). Considering that many cells express the heme-regulated inhibitor, which is an eIF-2α kinase (34Chen J.-J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 529-546Google Scholar), a treatment with hemin is expected to decrease eIF-2α phosphorylation. The observed decrease (Fig. 1B, lanes 5–7) is in line with the notion that in addition to serving as an iron donor, heme also modulates multiple biochemical pathways. We therefore examined whether the inhibition on NS5A and NPT-II expression shown in Fig. 1B is an iron-dependent or a hemin-specific phenomenon. Treatment with the lipophilic, cell-permeable iron donor Fe-SIH (23Ponka P. Schulman H.M. J. Biol. Chem. 1985; 260: 14717-14721Abstract Full Text PDF PubMed Google Scholar) recapitulated the inhibitory effects of hemin (Fig. 1C, lanes 4 and 5) and clearly suggested that inorganic iron negatively regulates the expression of NS5A and NPT-II. Further evidence that inorganic iron is the critical component was provided by the fact that two iron chelators, DFO and SIH, efficiently antagonized the hemin-mediated decrease in NS5A expression (Fig. 1D). We noticed that in some experiments, DFO and SIH slightly stimulated the expression of replicon proteins (for example, in Fig. 1D, lanes 1–5); however, this was not consistent (for example Fig. 1C, lanes 1–3). To analyze whether the inhibitory effects of iron are also shared by other metals, the replicon cells were exposed for 24 h to either 100 μm hemin or 100 μm copper, manganese, zinc, or cobalt salts. Note that only hemin significantly inhibited (p < 0.01, Student's t test) the expression of NS5A (Fig. 2A). As a control for the cellular response to the metal treatments, we analyzed Mt-2 mRNA levels by Northern blotting (Fig. 2B). As expected (35Haq F. Mahoney M. Koropatnick J. Mutat. Res. 2003; 533: 211-226Crossref PubMed Scopus (339) Google Scholar), copper and zinc strongly induced Mt-2 mRNA expression. Taken together, the above results establish a molecular link between iron metabolism and HCV gene expression by showing that pharmacological modulation of cellular iron levels affects the expression of proteins of the subgenomic HCV replicon. Translation via the HCV and EMCV IRES Is Not Affected by Iron—The iron-dependent inhibition of NS5A and NPT-II expression could result from changes in viral RNA translation, possibly via the HCV or EMCV IRES. To address this scenario, we employed a bicistronic CAT/firefly luciferase indicator containing the HCV IRES and 3′-UTR sequences of viral RNA and a bicistronic Renilla/firefly luciferase indicator containing the EMCV IRES (Fig. 3A). These constructs were transfected into parent Huh7 cells. The cells were subjected to iron manipulations, and lysates were prepared for the analysis of luciferase and CAT activities. No significant iron-dependent variations were observed in the activity of the firefly luciferase indicator after normalization with the respective CAT or Renilla luciferase values (Fig. 3B), suggesting that altered translation via the HCV or EMCV IRES cannot explain the observed effects on HCV gene expression. As expected (20Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1413) Google Scholar), treatment with the iron chelator DFO stimulates the expression of endogenous transferrin receptor 1 by ∼2.5-fold, whereas the iron donors hemin and Fe-SIH decrease transferrin receptor 1 steady-state levels by ∼90% (Fig. 3C). Iron Inhibits the Expression of Replicon RNA without Affecting Its Stability—We next analyzed whether iron alters the expression of subgenomic HCV RNA. The Northern blotting experiment depicted in Fig. 4A demonstrates that treatment of replicon cells with hemin for 24 h decreased the replicon RNA levels in a dose-dependent manner (top panel, lanes 1 and 5–7), whereas DFO had a slight stimulatory effect (top panel, lanes1–4). Probing with the cDNA of cellular GAPDH serves as control (Fig. 4A, bottom panel). To elucidate whether the iron-dependent decrease in the steady-state levels of replicon RNA is a result of alterations in its stability, a pulse-chase experiment was performed (Fig. 4B). Parent and replicon Huh7 cells were incubated for 16 h with [α-32P]UTP in the presence of 4 μg/ml actinomycin D to block cellular mRNA transcription by RNA polymerase II. Subsequently, the replicon cells were chased for different time intervals with cold media alone or with cold media containing 100 μm hemin or DFO, and RNA was prepared for analysis by agarose gel electrophoresis. Iron chelation seems to partially (∼20%) stabilize the replicon RNA, which may contribute to the DFO-dependent increase shown in Fig. 4A. The treatment with hemin appeared to accelerate the decay of replicon RNA rather modestly (by ∼20%) within 6 h and did not have any effects afterward. On the basis of this finding, we conclude that the profound iron-induced decrease in replicon RNA expression cannot be sufficiently explained by alterations in its half-life. Iron Inhibits Subgenomic HCV Replication in Multiple Replicon Systems—The data presented thus far collectively suggest that iron may inhibit HCV replication. However, because these data were obtained with a single clone of replicon Huh7 cells (24Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2503) Google Scholar), it was important to exclude that the inhibitory effects of iron may represent a clonal phenomenon. To address this, we established further replicon Huh7 clones and evaluated their response to iron. Three new clones were isolated, which express different amounts of replicon RNA. The cells were treated with hemin or DFO, and the expression of replicon RNA was analyzed by Northern blotting (Fig. 5A). Whereas no stimulatory effects of DFO can be observed, a profound iron-induced inhibition of replicon RNA expression is evident in all clones, confirming the previous data. To further validate this interpretation, we utilized the recently described 293Rep replicon system, consisting of human embryonic kidney 293 cells fused with the S22.3 clone of replicon Huh7 cells into a heterokaryon (26Ali S. Pellerin C. Lamarre D. Kukolj G. J. Virol. 2004; 78: 491-501Crossref PubMed Scopus (64) Google Scholar). Parent 293 and replicon 293Rep cells were subjected to iron manipulations, and the expression of NS5A protein and replicon RNA was analyzed by Western and Northern blotting, respectively. Treatment of 293Rep cells with hemin dramatically decreased the steady-state levels of both NS5A (Fig. 5B) and replicon RNA (Fig. 5C), in agreement with the data obtained in replicon Huh7 cells. Iron chelation with DFO did not stimulate the expression of the replicon (Fig. 5, B and C), as in the newly established replicon Huh7 clones (Fig. 5A). Iron Binds Specifically to Purified HCV RNA Polymerase and Inhibits Its Activity—The results in Figs. 1, 2, 3, 4, 5 could be best explained if iron inhibited viral RNA transcription, which is mediated by the RNA-dependent RNA polymerase (RdRp) activity of the NS5B protein (36Behrens S.E. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar, 37Lohmann V. Korner F. Herian U. Bartenschlager R. J. Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar). To investigate this directly, we utilized the soluble, catalytically active (38Ferrari E. Wright-Minogue J. Fang J.W. Baroudy B.M. Lau J.Y. Hong Z. J. Virol. 1999; 73: 1649-1654Crossref PubMed Google Scholar) NS5BΔ21 protein and tested whether iron affects its catalytic properties. The NS5BΔ21 protein was highly purified (Fig. 6A) and analyzed by endogenous tryptophan fluorescence emission spectroscopy (30Bougie I. Charpentier S. Bisaillon M. J. Biol. Chem. 2003; 278: 3868-3875Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) for the binding of ferrous (Fe2+) iron. Fluorescence spectra were quenched in the presence of increasing micromolar concentrations of Fe2+ without affecting the emission maximum and the spectra bandwidth (Fig. 6B), suggesting that iron direc
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