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Iron inhibits replication of infectious hepatitis C virus in permissive Huh7.5.1 cells
Research Article
Iron inhibits replication of infectious hepatitis C virus in permissive Huh7.5.1 cells Carine Fillebeen1, Kostas Pantopoulos1,2,⇑ 1
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 3755 Cote-Ste-Catherine Road, Montreal, Quebec, Canada H3T 1E2; 2Department of Medicine, McGill University, Canada See Editorial, pages 990–992
Background & Aims: Chronic infection with hepatitis C virus (HCV) is often associated with elevated hepatic iron levels. Excess iron is known to promote oxidative stress and exacerbate liver disease. Nevertheless, biochemical studies in subgenomic HCV replicon systems showed that iron can also suppress the expression of viral RNA and proteins by inhibiting the enzymatic activity of the RNA polymerase NS5B. To explore the physiological relevance of this response, we evaluated the effects of iron during infection of permissive Huh7.5.1 hepatoma cells with HCV. Methods: We utilized Fe-SIH (iron complexed with salicylaldehyde isonicotinoyl hydrazone), a cell permeable and highly efficient iron donor. Results: Treatments of infected cells with Fe-SIH drastically reduced the expression of viral proteins (core and NS3) and RNA, in a dose-dependent manner. The inhibition was dramatic when Fe-SIH was administered simultaneously with the HCV inoculum or early afterwards, while pre-treatment of cells with Fe-SIH before infection failed to elicit antiviral responses. Iron chelation with SIH did not significantly alter the expression of viral proteins. Conclusions: Our data establish a critical role of hepatic iron concentration on the progression of HCV infection, and are consistent with iron-mediated inactivation of NS5B. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction The vast majority (85%) of individuals infected acutely with HCV develop chronic hepatitis C (CHC) that may progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma [32]. CHC is a leading cause of liver failure and poses a major health care challenge, with an estimated 170 million patients worldwide [15]. Keywords: Hepatitis C virus; Iron metabolism; NS5B; NS3; Ferritin; Transferrin receptor 1. DOI of original article: 10.1016/j.jhep.2010.08.003 ⇑ Corresponding author. Address: Lady Davis Institute for Medical Research Sir Mortimer B. Davis Jewish General Hospital 3755 Cote-Ste-Catherine Road Montreal, Quebec, Canada H3T 1E2. Tel.: +1 514 340 8260x5293; fax: +1 514 340 7502. E-mail address: [email protected] (K. Pantopoulos). Abbreviations: HCV, hepatitis C virus; SIH, salicylaldehyde isonicotinoyl hydrazone; NS3, non-structural protein 3; CHC, chronic hepatitis C; TfR1, transferrin receptor 1; PBS, phosphate-buffered saline; RT-PCR, reverse-transcription polymerase chain reaction; Hmox1, heme oxygenase 1.
The disease is often associated with mild to moderate hepatic iron overload with variable distribution among reticuloendothelial and parenchymal cells [1,25,31]. Excess hepatic iron is considered as a co-morbid factor that aggravates liver damage by promoting oxidative stress. Altered redox homeostasis disrupts organellar architecture and growth properties of hepatocytes and hepatic stellate cells [11,20,27]. Parenchymal iron accumulation in CHC patients has been linked to necroinflammation [31] and to misregulation of iron homeostasis by HCV-dependent inhibition in the expression of hepcidin [9,19,22]. A decrease in levels of this peptide hormone leads to unrestricted intestinal iron absorption and iron release from macrophages due to stabilization of the iron transporter ferroportin [21]. This phenotype is the hallmark of hereditary hemochromatosis, a disease caused by genetic defects in the hepcidin pathway [17,26]. The idea that excess iron exacerbates the clinical picture of CHC is concordant with the pathology of hereditary hemochromatosis that triggers in its own right liver fibrosis, cirrhosis, and hepatocellular carcinoma [14]. Nevertheless, biochemical experiments showed that iron can also exert antiviral effects. Thus, we previously reported that iron binds tightly to NS5B, the RNAdependent RNA polymerase of HCV and inhibits its catalytic activity by displacing Mg2+ from the enzyme’s active site [8]. The antiviral activity of iron was further validated in a subgenomic HCV replicon model, where the administration of exogenous iron blocked viral replication and attenuated the production of viral RNA and proteins [8]. Further data suggested that the expression of the subgenomic HCV replicon leads to an iron-poor phenotype in host Huh7 cells, possibly to bypass the iron-dependent block in viral replication [7]. Here, we employ an in vitro model for HCV infection based on permissive Huh7.5.1 hepatoma cells [40], and examine the effects of iron on this process. We demonstrate that exogenous iron diminishes HCV replication in these cells and inhibits the expression of viral proteins and RNA. Materials and methods Materials SIH was a kind gift of Dr. Prem Ponka (McGill University). Fe-SIH was prepared as described earlier [8]. The HCV genotype 2a consensus clone JFH-1 [35], derived from a Japanese patient with fulminant hepatitis, and permissive human Huh7.5.1 hepatoma cells [40] were kindly provided by Dr. Takaji Wakita (Tokyo Metropolitan Institute for Neuroscience).
Journal of Hepatology 2010 vol. 53 j 995–999
Research Article A
Cell culture Huh7.5.1 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat inactivated fetal bovine serum, 100 nM non-essential amino acids, 100 U/ml penicillin, and 100 lg/ml streptomycin.
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core In vitro transcribed JFH-1 RNA was transfected into Huh7.5.1 cells by electroporation [13]. Culture media were collected 14 days after transfection, cleared using low speed centrifugation and filtered [13]. Naïve Huh7.5.1 cells were infected by inoculation with this material for 24 h, then washed and incubated with fresh media for 1–4 days.
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The cells were washed twice in phosphate-buffered saline (PBS) and lysed in RIPA buffer (50 mM Tris–Cl, pH 7.4, 150 mM NaCl, 1% SDS, 0.5% Na deoxycholate, 1% Triton X-100). Lysates were resolved by SDS–PAGE on 13% or 7% gels and transferred onto nitrocellulose filters. The blots were saturated with 10% non-fat milk in PBS and probed with 1:1000 diluted antibodies against NS3 (Abcam), core protein (Affinity BioReagents), TfR1 (Zymed), ferritin (Novus) or b-actin (Sigma). Dilutions were in PBS containing 0.5% Tween-20 (PBST). Following wash with PBST, the blots with monoclonal NS3, core protein, and TfR1 antibodies were incubated with peroxidase-coupled rabbit anti-mouse IgG (1:5000 dilution), and the blots with polyclonal ferritin and b-actin antibodies were incubated with peroxidase-coupled goat anti-rabbit IgG (1:10,000 dilution). Peroxidase-coupled antibodies were detected with the enhanced chemiluminescence method (Amersham), according to the manufacturer’s instructions.
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Results To generate infectious HCV particles, Huh7.5.1 cells were initially transfected with JFH-1 RNA. Lysates generated at different time intervals were analyzed for expression of the virally encoded proteins NS3 and core (Fig. 1A). Both NS3 and core proteins were readily detectable by Western blotting within 5–7 days (lanes 2–3) and their expression peaked between 11 and 15 days post-transfection (lanes 5–7), in line with previous findings [35,40]. The peak in viral protein expression was accompanied by a robust increase in viral RNA levels (Fig. 1B). Under these conditions, the culture supernatant is expected to contain infectious HCV particles [35,40]. Indeed, the inoculation of naïve Huh7.5.1 cells with culture supernatant of JFH-1-transfected counterparts resulted in infection of these cells with HCV, as judged by the expression of NS3 and core proteins (Fig. 1C), as well as the expression of viral RNA (Fig. 1D). We employed Fe-SIH, a lipophilic iron delivery vehicle [28], to address the effects of iron on the progression of HCV infection. Naïve Huh7.5.1 cells were inoculated with culture supernatant from JFH-1-transfectants containing HCV particles. The cells received increasing doses of Fe-SIH either concurrently with the inoculation (day 0) or on the following days 1–3, and the incuba996
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Fig. 1. Infection of permissive Huh7.5.1 cells with HCV. The cells were initially electroporated with JFH-1 RNA (A and B) and the culture supernatant was used to inoculate naïve counterparts (C and D). The expression of the viral proteins NS3 and core and of cellular b-actin was analyzed by Western blotting, and the levels of HCV RNA were determined by RT-PCR. Viral RNA from four independent experiments was quantified by RT-PCR (mean ± SD).
tion was continued until day 4. The addition of 25 or 50 lM FeSIH together with the inoculum, or 1–2 days post-infection, dramatically inhibited the expression of the viral proteins NS3 and
Journal of Hepatology 2010 vol. 53 j 995–999
JOURNAL OF HEPATOLOGY expression of NS3 was monitored over 4 days (Fig. 4A). Fe-SIH efficiently blocked the early accumulation of NS3 on the second day post-infection, while longer treatments with iron (3–4 days) yielded a similar outcome (lanes 6–9). As in previous experimental settings, SIH did not significantly affect NS3 (lanes 10–13). As early as one day post-infection, Fe-SIH decreased the expression of viral RNA by 60% (p <0.01); this response was enhanced up to 80% (p <0.01) on days 3–4 (Fig. 4B).
core (Fig. 2A, lanes 14–16 and 18–20). Moreover, at this concentration range (25–50 lM), Fe-SIH profoundly reduced the expression of viral RNA (Fig. 2B). The inhibitory capacity of Fe-SIH was attenuated at lower concentrations (Fig. 2A and B). The effectiveness of Fe-SIH as an iron donor is demonstrated by the induction of ferritin (Fig. 2A, 4th panel), the iron storage protein. In addition, high doses of Fe-SIH promoted a decrease in transferrin receptor 1 (TfR1) levels (Fig. 2A, 3rd panel), in line with the coordinate iron-dependent regulation of ferritin and TfR1 [24]. Notably, a pre-treatment of Huh7.5.1 cells with 50 lM Fe-SIH two days before the infection with HCV failed to reduce the expression of the viral protein NS3 (Fig. 2C). While the co-administration of Fe-SIH together with the HCV inoculum diminished the levels of virally encoded proteins in infected cells, its precursor SIH did not affect the NS3 content (Fig. 3A), demonstrating the iron specificity. As a known chelator of intracellular iron [4], SIH potently suppressed ferritin (3rd panel). At a higher dose, SIH appeared to slightly stimulate the expression of HCV RNA (Fig. 3B); a similar response was previously observed in a subgenomic HCV replicon system and was attributed to stabilization of HCV RNA by iron deficiency [8]. To evaluate the kinetics of iron-mediated inhibition in the replication of infectious HCV, 50 lM Fe-SIH was added to Huh7.5.1 cells simultaneously with the HCV inoculum, and the
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Discussion Here, we show that the administration of exogenous iron drastically inhibits the progression of HCV infection of permissive Huh7.5.1 cells. This finding is fully consistent with the previously reported iron-mediated block of subgenomic HCV replication in Huh7 and 293Rep cells [8]. Considering that the previous experiments were performed on subgenomic HCV replicon systems of genotype 1b, while the infectious model utilized here is based on HCV 2a, we conclude that the inhibitory effects of iron are independent of the HCV genotype. Biochemical experiments with purified NS5B, the HCV RNAdependent polymerase, revealed that iron binds with high affinity and specificity to this enzyme. Moreover, the binding site of
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Fig. 2. Iron inhibits the expression of HCV proteins and RNA in infected cells, in a dose-dependent manner. Huh7.5.1 cells were infected with HCV and left untreated, or treated with various doses of Fe-SIH at the indicated time intervals. All cells were harvested and lysed on day 4 post-infection. (A and C) The expression of the HCV proteins NS3 and core, and of cellular TfR1, ferritin and b-actin was analyzed by Western blotting. (B) HCV RNA from three independent experiments was quantified by RTPCR (mean ± SD).
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Research Article treatment (μM)
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Fig. 3. The antiviral effect of Fe-SIH depends on its iron moiety. Huh7.5.1 cells were infected with HCV and left untreated (control), or treated with the indicated concentrations of Fe-SIH or SIH for 4 days. (A) The expression of the viral protein NS3 and of cellular TfR1, ferritin, and b-actin was analyzed by Western blotting. (B) HCV RNA from three independent experiments was quantified by RT-PCR (mean ± SD). *p <0.05 vs control and **p <0.01 vs control (Student’s t-test).
iron overlaps with that of Mg2+ at the catalytic center of NS5B. Since iron binds approximately 5–50 times more tightly than Mg2+ to NS5B (apparent Kd values are: 6 lM for Fe2+, 60 lM for Fe3+, and 3.1 mM for Mg2+), it efficiently outcompetes and displaces Mg2+ from the NS5B active site, and thereby inactivates the enzyme [8]. Thus, a direct inactivation of NS5B by iron would offer a plausible mechanism for the observed resistance of ironloaded Huh7.5.1 cells to the progression of HCV infection. A treatment of cells with exogenous iron is thought to increase the labile iron pool (LIP) that triggers homeostatic adaptations, culminating in the storage of excess iron into ferritin (Figs. 2–4). The failure of preloading of Huh7.5.1 cells with iron, to protect them against HCV infection (Fig. 2C), may suggest that iron deposited into ferritin is unavailable for binding to NS5B. Here, we exclusively used Fe-SIH as iron donor and not hemin, that likewise blocks subgenomic HCV replication [8,23], to avoid potential confounding effects of heme oxygenase 1 (Hmox1). This heme-metabolizing enzyme and anti-inflammatory factor is induced by various stress stimuli including heme [29] and inhibits HCV replicons [33,41], possibly by protecting host cells against oxidative stress [41]. It was recently shown that Hmox1 induces antiviral interferon responses via its metabolic product biliverdin and it was also argued that exogenous iron administration does not suffice to block subgenomic HCV replication [18]. Nevertheless, inasmuch as the cells were exposed to relatively poor iron donors (10 lM FeCl3 or iron-loaded lactoferrin), these negative results do not contradict the dose-dependent inhibition of HCV replication by iron shown in Fig. 2. 998
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Fig. 4. Kinetics of iron-mediated inhibition in the infectious genomic HCV replicon. Huh7.5.1 cells were infected with HCV and left untreated, or treated with 50 lM Fe-SIH or SIH. The cells were harvested at the indicated time intervals. (A) The expression of the viral protein NS3 and of cellular TfR1, ferritin and b-actin was analyzed by Western blotting. (B) HCV RNA from three independent experiments was quantified by RT-PCR (mean ± SD).
Our findings may deserve particular attention in light of clinical data where CHC patients with hereditary iron overload due to mutations in the HFE gene exhibit paradoxically better responses to antiviral therapy [3,6,16]. Even though an immunological function of HFE cannot be excluded [5], it is tempting to speculate that an increased hepatic iron content may contribute to viral RNA clearance and antagonize the relapse of HCV infection following therapy. It should, however, be noted that iron overload secondary to CHC or other chronic liver diseases does not improve, but rather worsens the clinical outcome. Iron-induced oxidative stress very likely plays a major role in this process [11]. Moreover, iron is known to affect immune responses and cytokine production [30,37]. In a cohort of 55 CHC patients, increased transferrin saturation correlated with more advanced liver disease and a shift from pro-inflammatory (Th-1) to anti-inflammatory (Th-2) responses of T-helper cells that do not favor viral clearance [38]. The iron content of macrophages is critical for their immune effector functions. High iron levels inhibit NO biosynthesis [39], while iron deficiency impairs TLR4 signaling [36]. Interestingly, the distribution of hepatic iron varies considerably between primary and secondary iron overload states. Thus, in hereditary hemochromatosis, iron is almost exclusively deposited in parenchymal cells, and macrophages remain relatively iron-deficient [26]. By contrast, in secondary iron overload, including transfusional siderosis, macrophages contain an excess of iron [2,31]. Iron-dependent variability in immune effector functions of mac-
Journal of Hepatology 2010 vol. 53 j 995–999
JOURNAL OF HEPATOLOGY rophages may underlie clinical differences in HCV-infected patients with primary or secondary iron overload. Thus, while excess iron appeared to improve antiviral therapy in a background of hereditary hemochromatosis [3,6,16], it did not offer any apparent benefits to HCV-infected patients with b-thalassemia [10]. Likewise, intravenous iron administration did not improve the responses of hemodialyzed HCV-infected patients to antiviral therapy [12]. Consequently, the inhibitory effects of iron on HCV replication are unlikely to be exploitable for the pharmacological treatment of CHC. Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Financial support K.P. holds a Chercheur National career award from the Fonds de la Recherche en Santé du Quebéc (FRSQ). Supported by a grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN 288283-06). References [1] Alla V, Bonkovsky HL. Iron in nonhemochromatotic liver disorders. Semin Liver Dis 2005;25:461–472. [2] Andrews NC. Disorders of iron metabolism. N Engl J Med 1999;341: 1986–1995. [3] Bonkovsky HL, Naishadham D, Lambrecht RW, Chung RT, Hoefs JC, Nash SR, et al. Roles of iron and HFE mutations on severity and response to therapy during retreatment of advanced chronic hepatitis C. Gastroenterology 2006;131:1440–1451. [4] Buss JL, Hermes-Lima M, Ponka P. Pyridoxal isonicotinoyl hydrazone and its analogues. In: Hershko C, editors. Progress in iron research, vol. 509; 2002. p. 205–29. [5] de Almeida SF, Carvalho IF, Cardoso CS, Cordeiro JV, Azevedo JE, Neefjes J, et al. HFE cross-talks with the MHC class I antigen presentation pathway. Blood 2005;106:971–977. [6] Distante S, Bjoro K, Hellum KB, Myrvang B, Berg JP, Skaug K, et al. Raised serum ferritin predicts non-response to interferon and ribavirin treatment in patients with chronic hepatitis C infection. Liver 2002;22:269–275. [7] Fillebeen C, Muckenthaler M, Andriopoulos B, Bisaillon M, Mounir Z, Hentze MW, et al. Expression of the subgenomic hepatitis C virus replicon alters iron homeostasis in Huh7 cells. J Hepatol 2007;47:12–22. [8] Fillebeen C, Rivas-Estilla AM, Bisaillon M, Ponka P, Muckenthaler M, Hentze MW, et al. Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus. J Biol Chem 2005;280: 9049–9057. [9] Fujita N, Sugimoto R, Takeo M, Urawa N, Mifuji R, Tanaka H, et al. Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C. Mol Med 2007;13:97–104. [10] Harmatz P, Jonas MM, Kwiatkowski JL, Wright EC, Fischer R, Vichinsky E, et al. Safety and efficacy of pegylated interferon alpha-2a and ribavirin for the treatment of hepatitis C in patients with thalassemia. Haematologica 2008;93:1247–1251. [11] Isom HC, McDevitt EI, Moon MS. Elevated hepatic iron: a confounding factor in chronic hepatitis C. Biochim Biophys Acta 2009;1790:650–662. [12] Kahraman S, Yilmaz R, Genctoy G, Arici M, Altun B, Erdem Y, et al. Efficacy and safety of intravenous iron therapy for HCV-positive haemodialysis patients. Nephron Clin Pract 2005;100:c78–c85. [13] Kato T, Date T, Murayama A, Morikawa K, Akazawa D, Wakita T. Cell culture and infection system for hepatitis C virus. Nat Protoc 2006;1:2334–2339.
[14] Kowdley KV. Iron, hemochromatosis, and hepatocellular carcinoma. Gastroenterology 2004;127:S79–S86. [15] Lavanchy D. The global burden of hepatitis C. Liver Int 2009;29:74–81. [16] Lebray P, Zylberberg H, Hue S, Poulet B, Carnot F, Martin S, et al. Influence of HFE gene polymorphism on the progression and treatment of chronic hepatitis C. J Viral Hepat 2004;11:175–182. [17] Lee PL, Beutler E. Regulation of hepcidin and iron-overload disease. Annu Rev Pathol 2009;4:489–515. [18] Lehmann E, El-Tantawy WH, Ocker M, Bartenschlager R, Lohmann V, Hashemolhosseini S, et al. The heme oxygenase 1 product biliverdin interferes with hepatitis C virus replication by increasing antiviral interferon response. Hepatology 2010;51:398–404. [19] Miura K, Taura K, Kodama Y, Schnabl B, Brenner DA. Hepatitis C virusinduced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity. Hepatology 2008;48:1420–1429. [20] Mueller S, Afdhal NH, Schuppan D. Iron, HCV, and liver cancer: hard metal setting the pace? Gastroenterology 2006;130:2229–2234. [21] Nemeth E, Ganz T. The role of hepcidin in iron metabolism. Acta Haematol 2009;122:78–86. [22] Nishina S, Hino K, Korenaga M, Vecchi C, Pietrangelo A, Mizukami Y, et al. Hepatitis C virus-induced reactive oxygen species raise hepatic iron level in mice by reducing hepcidin transcription. Gastroenterology 2008;134: 226–238. [23] Paeshuyse J, Coelmont L, Vliegen I, Hemel JV, Vandenkerckhove J, Peys E, et al. Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin. Biochem Biophys Res Commun 2006;348:139–144. [24] Pantopoulos K. Iron metabolism and the IRE/IRP regulatory system: an update. Ann NY Acad Sci 2004;1012:1–13. [25] Pietrangelo A. Hemochromatosis gene modifies course of hepatitis C viral infection. Gastroenterology 2003;124:1509–1523. [26] Pietrangelo A. Hereditary hemochromatosis – a new look at an old disease. N Engl J Med 2004;350:2383–2397. [27] Pietrangelo A. Metals, oxidative stress, and hepatic fibrogenesis. Semin Liver Dis 1996;16:13–30. [28] Ponka P, Schulman HM. Acquisition of iron from transferrin regulates reticulocyte heme synthesis. J Biol Chem 1985;260:14717–14721. [29] Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev 2006;86:583–650. [30] Schaible UE, Kaufmann SH. Iron and microbial infection. Nat Rev Microbiol 2004;2:946–953. [31] Sebastiani G, Walker AP. HFE gene in primary and secondary hepatic iron overload. World J Gastroenterol 2007;13:4673–4689. [32] Seeff LB. The history of the ‘‘natural history” of hepatitis C (1968–2009). Liver Int 2009;29:89–99. [33] Shan Y, Zheng J, Lambrecht RW, Bonkovsky HL. Reciprocal effects of microRNA-122 on expression of heme oxygenase-1 and hepatitis C virus genes in human hepatocytes. Gastroenterology 2007;133:1166–1174. [34] Takeuchi T, Katsume A, Tanaka T, Abe A, Inoue K, Tsukiyama-Kohara K, et al. Real-time detection system for quantification of hepatitis C virus genome. Gastroenterology 1999;116:636–642. [35] Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11:791–796. [36] Wang L, Harrington L, Trebicka E, Shi HN, Kagan JC, Hong CC, et al. Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice. J Clin Invest 2009;119:3322–3328. [37] Weiss G. Iron and immunity: a double-edged sword. Eur J Clin Invest 2002;32:70–78. [38] Weiss G, Umlauft F, Urbanek M, Herold M, Loyevsky M, Offner F, et al. Associations between cellular immune effector function, iron metabolism, and disease activity in patients with chronic hepatitis C virus infection. J Infect Dis 1999;180:1452–1458. [39] Weiss G, Werner-Felmayer G, Werner ER, Grünewald K, Wachter H, Hentze MW. Iron regulates nitric oxide synthase activity by controlling nuclear transcription. J Exp Med 1994;180:969–976. [40] Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton DR, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 2005;102: 9294–9299. [41] Zhu Z, Wilson AT, Mathahs MM, Wen F, Brown KE, Luxon BA, et al. Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury. Hepatology 2008;48:1430–1439.
Journal of Hepatology 2010 vol. 53 j 995–999
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Iron inhibits replication of infectious hepatitis C virus in permissive Huh7.5.1 cells Carine Fillebeen1, Kostas Pantopoulos1,2,⇑ 1
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 3755 Cote-Ste-Catherine Road, Montreal, Quebec, Canada H3T 1E2; 2Department of Medicine, McGill University, Canada See Editorial, pages 990–992
Background & Aims: Chronic infection with hepatitis C virus (HCV) is often associated with elevated hepatic iron levels. Excess iron is known to promote oxidative stress and exacerbate liver disease. Nevertheless, biochemical studies in subgenomic HCV replicon systems showed that iron can also suppress the expression of viral RNA and proteins by inhibiting the enzymatic activity of the RNA polymerase NS5B. To explore the physiological relevance of this response, we evaluated the effects of iron during infection of permissive Huh7.5.1 hepatoma cells with HCV. Methods: We utilized Fe-SIH (iron complexed with salicylaldehyde isonicotinoyl hydrazone), a cell permeable and highly efficient iron donor. Results: Treatments of infected cells with Fe-SIH drastically reduced the expression of viral proteins (core and NS3) and RNA, in a dose-dependent manner. The inhibition was dramatic when Fe-SIH was administered simultaneously with the HCV inoculum or early afterwards, while pre-treatment of cells with Fe-SIH before infection failed to elicit antiviral responses. Iron chelation with SIH did not significantly alter the expression of viral proteins. Conclusions: Our data establish a critical role of hepatic iron concentration on the progression of HCV infection, and are consistent with iron-mediated inactivation of NS5B. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction The vast majority (85%) of individuals infected acutely with HCV develop chronic hepatitis C (CHC) that may progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma [32]. CHC is a leading cause of liver failure and poses a major health care challenge, with an estimated 170 million patients worldwide [15]. Keywords: Hepatitis C virus; Iron metabolism; NS5B; NS3; Ferritin; Transferrin receptor 1. DOI of original article: 10.1016/j.jhep.2010.08.003 ⇑ Corresponding author. Address: Lady Davis Institute for Medical Research Sir Mortimer B. Davis Jewish General Hospital 3755 Cote-Ste-Catherine Road Montreal, Quebec, Canada H3T 1E2. Tel.: +1 514 340 8260x5293; fax: +1 514 340 7502. E-mail address: [email protected] (K. Pantopoulos). Abbreviations: HCV, hepatitis C virus; SIH, salicylaldehyde isonicotinoyl hydrazone; NS3, non-structural protein 3; CHC, chronic hepatitis C; TfR1, transferrin receptor 1; PBS, phosphate-buffered saline; RT-PCR, reverse-transcription polymerase chain reaction; Hmox1, heme oxygenase 1.
The disease is often associated with mild to moderate hepatic iron overload with variable distribution among reticuloendothelial and parenchymal cells [1,25,31]. Excess hepatic iron is considered as a co-morbid factor that aggravates liver damage by promoting oxidative stress. Altered redox homeostasis disrupts organellar architecture and growth properties of hepatocytes and hepatic stellate cells [11,20,27]. Parenchymal iron accumulation in CHC patients has been linked to necroinflammation [31] and to misregulation of iron homeostasis by HCV-dependent inhibition in the expression of hepcidin [9,19,22]. A decrease in levels of this peptide hormone leads to unrestricted intestinal iron absorption and iron release from macrophages due to stabilization of the iron transporter ferroportin [21]. This phenotype is the hallmark of hereditary hemochromatosis, a disease caused by genetic defects in the hepcidin pathway [17,26]. The idea that excess iron exacerbates the clinical picture of CHC is concordant with the pathology of hereditary hemochromatosis that triggers in its own right liver fibrosis, cirrhosis, and hepatocellular carcinoma [14]. Nevertheless, biochemical experiments showed that iron can also exert antiviral effects. Thus, we previously reported that iron binds tightly to NS5B, the RNAdependent RNA polymerase of HCV and inhibits its catalytic activity by displacing Mg2+ from the enzyme’s active site [8]. The antiviral activity of iron was further validated in a subgenomic HCV replicon model, where the administration of exogenous iron blocked viral replication and attenuated the production of viral RNA and proteins [8]. Further data suggested that the expression of the subgenomic HCV replicon leads to an iron-poor phenotype in host Huh7 cells, possibly to bypass the iron-dependent block in viral replication [7]. Here, we employ an in vitro model for HCV infection based on permissive Huh7.5.1 hepatoma cells [40], and examine the effects of iron on this process. We demonstrate that exogenous iron diminishes HCV replication in these cells and inhibits the expression of viral proteins and RNA. Materials and methods Materials SIH was a kind gift of Dr. Prem Ponka (McGill University). Fe-SIH was prepared as described earlier [8]. The HCV genotype 2a consensus clone JFH-1 [35], derived from a Japanese patient with fulminant hepatitis, and permissive human Huh7.5.1 hepatoma cells [40] were kindly provided by Dr. Takaji Wakita (Tokyo Metropolitan Institute for Neuroscience).
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Research Article A
Cell culture Huh7.5.1 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat inactivated fetal bovine serum, 100 nM non-essential amino acids, 100 U/ml penicillin, and 100 lg/ml streptomycin.
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core In vitro transcribed JFH-1 RNA was transfected into Huh7.5.1 cells by electroporation [13]. Culture media were collected 14 days after transfection, cleared using low speed centrifugation and filtered [13]. Naïve Huh7.5.1 cells were infected by inoculation with this material for 24 h, then washed and incubated with fresh media for 1–4 days.
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The cells were washed twice in phosphate-buffered saline (PBS) and lysed in RIPA buffer (50 mM Tris–Cl, pH 7.4, 150 mM NaCl, 1% SDS, 0.5% Na deoxycholate, 1% Triton X-100). Lysates were resolved by SDS–PAGE on 13% or 7% gels and transferred onto nitrocellulose filters. The blots were saturated with 10% non-fat milk in PBS and probed with 1:1000 diluted antibodies against NS3 (Abcam), core protein (Affinity BioReagents), TfR1 (Zymed), ferritin (Novus) or b-actin (Sigma). Dilutions were in PBS containing 0.5% Tween-20 (PBST). Following wash with PBST, the blots with monoclonal NS3, core protein, and TfR1 antibodies were incubated with peroxidase-coupled rabbit anti-mouse IgG (1:5000 dilution), and the blots with polyclonal ferritin and b-actin antibodies were incubated with peroxidase-coupled goat anti-rabbit IgG (1:10,000 dilution). Peroxidase-coupled antibodies were detected with the enhanced chemiluminescence method (Amersham), according to the manufacturer’s instructions.
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Results To generate infectious HCV particles, Huh7.5.1 cells were initially transfected with JFH-1 RNA. Lysates generated at different time intervals were analyzed for expression of the virally encoded proteins NS3 and core (Fig. 1A). Both NS3 and core proteins were readily detectable by Western blotting within 5–7 days (lanes 2–3) and their expression peaked between 11 and 15 days post-transfection (lanes 5–7), in line with previous findings [35,40]. The peak in viral protein expression was accompanied by a robust increase in viral RNA levels (Fig. 1B). Under these conditions, the culture supernatant is expected to contain infectious HCV particles [35,40]. Indeed, the inoculation of naïve Huh7.5.1 cells with culture supernatant of JFH-1-transfected counterparts resulted in infection of these cells with HCV, as judged by the expression of NS3 and core proteins (Fig. 1C), as well as the expression of viral RNA (Fig. 1D). We employed Fe-SIH, a lipophilic iron delivery vehicle [28], to address the effects of iron on the progression of HCV infection. Naïve Huh7.5.1 cells were inoculated with culture supernatant from JFH-1-transfectants containing HCV particles. The cells received increasing doses of Fe-SIH either concurrently with the inoculation (day 0) or on the following days 1–3, and the incuba996
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Fig. 1. Infection of permissive Huh7.5.1 cells with HCV. The cells were initially electroporated with JFH-1 RNA (A and B) and the culture supernatant was used to inoculate naïve counterparts (C and D). The expression of the viral proteins NS3 and core and of cellular b-actin was analyzed by Western blotting, and the levels of HCV RNA were determined by RT-PCR. Viral RNA from four independent experiments was quantified by RT-PCR (mean ± SD).
tion was continued until day 4. The addition of 25 or 50 lM FeSIH together with the inoculum, or 1–2 days post-infection, dramatically inhibited the expression of the viral proteins NS3 and
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JOURNAL OF HEPATOLOGY expression of NS3 was monitored over 4 days (Fig. 4A). Fe-SIH efficiently blocked the early accumulation of NS3 on the second day post-infection, while longer treatments with iron (3–4 days) yielded a similar outcome (lanes 6–9). As in previous experimental settings, SIH did not significantly affect NS3 (lanes 10–13). As early as one day post-infection, Fe-SIH decreased the expression of viral RNA by 60% (p <0.01); this response was enhanced up to 80% (p <0.01) on days 3–4 (Fig. 4B).
core (Fig. 2A, lanes 14–16 and 18–20). Moreover, at this concentration range (25–50 lM), Fe-SIH profoundly reduced the expression of viral RNA (Fig. 2B). The inhibitory capacity of Fe-SIH was attenuated at lower concentrations (Fig. 2A and B). The effectiveness of Fe-SIH as an iron donor is demonstrated by the induction of ferritin (Fig. 2A, 4th panel), the iron storage protein. In addition, high doses of Fe-SIH promoted a decrease in transferrin receptor 1 (TfR1) levels (Fig. 2A, 3rd panel), in line with the coordinate iron-dependent regulation of ferritin and TfR1 [24]. Notably, a pre-treatment of Huh7.5.1 cells with 50 lM Fe-SIH two days before the infection with HCV failed to reduce the expression of the viral protein NS3 (Fig. 2C). While the co-administration of Fe-SIH together with the HCV inoculum diminished the levels of virally encoded proteins in infected cells, its precursor SIH did not affect the NS3 content (Fig. 3A), demonstrating the iron specificity. As a known chelator of intracellular iron [4], SIH potently suppressed ferritin (3rd panel). At a higher dose, SIH appeared to slightly stimulate the expression of HCV RNA (Fig. 3B); a similar response was previously observed in a subgenomic HCV replicon system and was attributed to stabilization of HCV RNA by iron deficiency [8]. To evaluate the kinetics of iron-mediated inhibition in the replication of infectious HCV, 50 lM Fe-SIH was added to Huh7.5.1 cells simultaneously with the HCV inoculum, and the
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Discussion Here, we show that the administration of exogenous iron drastically inhibits the progression of HCV infection of permissive Huh7.5.1 cells. This finding is fully consistent with the previously reported iron-mediated block of subgenomic HCV replication in Huh7 and 293Rep cells [8]. Considering that the previous experiments were performed on subgenomic HCV replicon systems of genotype 1b, while the infectious model utilized here is based on HCV 2a, we conclude that the inhibitory effects of iron are independent of the HCV genotype. Biochemical experiments with purified NS5B, the HCV RNAdependent polymerase, revealed that iron binds with high affinity and specificity to this enzyme. Moreover, the binding site of
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Fig. 2. Iron inhibits the expression of HCV proteins and RNA in infected cells, in a dose-dependent manner. Huh7.5.1 cells were infected with HCV and left untreated, or treated with various doses of Fe-SIH at the indicated time intervals. All cells were harvested and lysed on day 4 post-infection. (A and C) The expression of the HCV proteins NS3 and core, and of cellular TfR1, ferritin and b-actin was analyzed by Western blotting. (B) HCV RNA from three independent experiments was quantified by RTPCR (mean ± SD).
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Fig. 3. The antiviral effect of Fe-SIH depends on its iron moiety. Huh7.5.1 cells were infected with HCV and left untreated (control), or treated with the indicated concentrations of Fe-SIH or SIH for 4 days. (A) The expression of the viral protein NS3 and of cellular TfR1, ferritin, and b-actin was analyzed by Western blotting. (B) HCV RNA from three independent experiments was quantified by RT-PCR (mean ± SD). *p <0.05 vs control and **p <0.01 vs control (Student’s t-test).
iron overlaps with that of Mg2+ at the catalytic center of NS5B. Since iron binds approximately 5–50 times more tightly than Mg2+ to NS5B (apparent Kd values are: 6 lM for Fe2+, 60 lM for Fe3+, and 3.1 mM for Mg2+), it efficiently outcompetes and displaces Mg2+ from the NS5B active site, and thereby inactivates the enzyme [8]. Thus, a direct inactivation of NS5B by iron would offer a plausible mechanism for the observed resistance of ironloaded Huh7.5.1 cells to the progression of HCV infection. A treatment of cells with exogenous iron is thought to increase the labile iron pool (LIP) that triggers homeostatic adaptations, culminating in the storage of excess iron into ferritin (Figs. 2–4). The failure of preloading of Huh7.5.1 cells with iron, to protect them against HCV infection (Fig. 2C), may suggest that iron deposited into ferritin is unavailable for binding to NS5B. Here, we exclusively used Fe-SIH as iron donor and not hemin, that likewise blocks subgenomic HCV replication [8,23], to avoid potential confounding effects of heme oxygenase 1 (Hmox1). This heme-metabolizing enzyme and anti-inflammatory factor is induced by various stress stimuli including heme [29] and inhibits HCV replicons [33,41], possibly by protecting host cells against oxidative stress [41]. It was recently shown that Hmox1 induces antiviral interferon responses via its metabolic product biliverdin and it was also argued that exogenous iron administration does not suffice to block subgenomic HCV replication [18]. Nevertheless, inasmuch as the cells were exposed to relatively poor iron donors (10 lM FeCl3 or iron-loaded lactoferrin), these negative results do not contradict the dose-dependent inhibition of HCV replication by iron shown in Fig. 2. 998
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Fig. 4. Kinetics of iron-mediated inhibition in the infectious genomic HCV replicon. Huh7.5.1 cells were infected with HCV and left untreated, or treated with 50 lM Fe-SIH or SIH. The cells were harvested at the indicated time intervals. (A) The expression of the viral protein NS3 and of cellular TfR1, ferritin and b-actin was analyzed by Western blotting. (B) HCV RNA from three independent experiments was quantified by RT-PCR (mean ± SD).
Our findings may deserve particular attention in light of clinical data where CHC patients with hereditary iron overload due to mutations in the HFE gene exhibit paradoxically better responses to antiviral therapy [3,6,16]. Even though an immunological function of HFE cannot be excluded [5], it is tempting to speculate that an increased hepatic iron content may contribute to viral RNA clearance and antagonize the relapse of HCV infection following therapy. It should, however, be noted that iron overload secondary to CHC or other chronic liver diseases does not improve, but rather worsens the clinical outcome. Iron-induced oxidative stress very likely plays a major role in this process [11]. Moreover, iron is known to affect immune responses and cytokine production [30,37]. In a cohort of 55 CHC patients, increased transferrin saturation correlated with more advanced liver disease and a shift from pro-inflammatory (Th-1) to anti-inflammatory (Th-2) responses of T-helper cells that do not favor viral clearance [38]. The iron content of macrophages is critical for their immune effector functions. High iron levels inhibit NO biosynthesis [39], while iron deficiency impairs TLR4 signaling [36]. Interestingly, the distribution of hepatic iron varies considerably between primary and secondary iron overload states. Thus, in hereditary hemochromatosis, iron is almost exclusively deposited in parenchymal cells, and macrophages remain relatively iron-deficient [26]. By contrast, in secondary iron overload, including transfusional siderosis, macrophages contain an excess of iron [2,31]. Iron-dependent variability in immune effector functions of mac-
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JOURNAL OF HEPATOLOGY rophages may underlie clinical differences in HCV-infected patients with primary or secondary iron overload. Thus, while excess iron appeared to improve antiviral therapy in a background of hereditary hemochromatosis [3,6,16], it did not offer any apparent benefits to HCV-infected patients with b-thalassemia [10]. Likewise, intravenous iron administration did not improve the responses of hemodialyzed HCV-infected patients to antiviral therapy [12]. Consequently, the inhibitory effects of iron on HCV replication are unlikely to be exploitable for the pharmacological treatment of CHC. Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Financial support K.P. holds a Chercheur National career award from the Fonds de la Recherche en Santé du Quebéc (FRSQ). Supported by a grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN 288283-06). References [1] Alla V, Bonkovsky HL. Iron in nonhemochromatotic liver disorders. Semin Liver Dis 2005;25:461–472. [2] Andrews NC. Disorders of iron metabolism. N Engl J Med 1999;341: 1986–1995. [3] Bonkovsky HL, Naishadham D, Lambrecht RW, Chung RT, Hoefs JC, Nash SR, et al. Roles of iron and HFE mutations on severity and response to therapy during retreatment of advanced chronic hepatitis C. Gastroenterology 2006;131:1440–1451. [4] Buss JL, Hermes-Lima M, Ponka P. Pyridoxal isonicotinoyl hydrazone and its analogues. In: Hershko C, editors. Progress in iron research, vol. 509; 2002. p. 205–29. [5] de Almeida SF, Carvalho IF, Cardoso CS, Cordeiro JV, Azevedo JE, Neefjes J, et al. HFE cross-talks with the MHC class I antigen presentation pathway. Blood 2005;106:971–977. [6] Distante S, Bjoro K, Hellum KB, Myrvang B, Berg JP, Skaug K, et al. Raised serum ferritin predicts non-response to interferon and ribavirin treatment in patients with chronic hepatitis C infection. Liver 2002;22:269–275. [7] Fillebeen C, Muckenthaler M, Andriopoulos B, Bisaillon M, Mounir Z, Hentze MW, et al. Expression of the subgenomic hepatitis C virus replicon alters iron homeostasis in Huh7 cells. J Hepatol 2007;47:12–22. [8] Fillebeen C, Rivas-Estilla AM, Bisaillon M, Ponka P, Muckenthaler M, Hentze MW, et al. Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus. J Biol Chem 2005;280: 9049–9057. [9] Fujita N, Sugimoto R, Takeo M, Urawa N, Mifuji R, Tanaka H, et al. Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C. Mol Med 2007;13:97–104. [10] Harmatz P, Jonas MM, Kwiatkowski JL, Wright EC, Fischer R, Vichinsky E, et al. Safety and efficacy of pegylated interferon alpha-2a and ribavirin for the treatment of hepatitis C in patients with thalassemia. Haematologica 2008;93:1247–1251. [11] Isom HC, McDevitt EI, Moon MS. Elevated hepatic iron: a confounding factor in chronic hepatitis C. Biochim Biophys Acta 2009;1790:650–662. [12] Kahraman S, Yilmaz R, Genctoy G, Arici M, Altun B, Erdem Y, et al. Efficacy and safety of intravenous iron therapy for HCV-positive haemodialysis patients. Nephron Clin Pract 2005;100:c78–c85. [13] Kato T, Date T, Murayama A, Morikawa K, Akazawa D, Wakita T. Cell culture and infection system for hepatitis C virus. Nat Protoc 2006;1:2334–2339.
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