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The green tea polyphenol, epigallocatechin-3-gallate, inhibits hepatitis C virus entry
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Hepatology December 2011
Sandra Ciesek,1,2 Thomas von Hahn,1 Che C. Colpitts,3 Luis M. Schang,3 Martina Friesland,2 Jorg Steinmann,4 Michael P. Manns,1 Michael Ott,1 Heiner Wedemeyer,1 Philip Meuleman,5 Thomas Pietschmann,2 and Eike Steinmann2
From the 1Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; 2Division of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Center for Infection Research (HZI), Hannover, Germany; 3Departments of Biochemistry and Medical Microbiology and Immunology and Li Ka Shing Insitute of Virology, University of Alberta, Edmonton, Alberta, Canada; 4Institute of Medical Microbiology, University Hospital Essen, Essen, Germany; and 5Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium.
Abstract
Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin-3-gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell-culture-derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell-to-cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co-)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation. (HEPATOLOGY 2011)
Infection with the hepatitis C virus (HCV) is a major cause of chronic liver disease and the major indication of liver transplantations worldwide. Approximately 160 million people are chronically infected with HCV, representing approximately 2% of the world population, whereas in some countries up to 15%-20% of the population is infected.1 Chronic HCV infection causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Standard therapy currently consisting of a combination of pegylated interferon-alpha (IFN-α) with ribavirin will soon also include an HCV nonstructural protein (NS)3/4A protease inhibitor.2 This addition is highly expected to improve response rates, especially in HCV genotype 1-infected individuals. However, therapy will most likely still remain expensive, fraught with side effects, and may still fail to clear infection in a substantial proportion of cases. Individuals undergoing orthotopic liver transplantation (OLT) for complications of HCV infection pose a particular clinical problem: Graft reinfection with HCV occurs in nearly all cases, and long-term outcomes are unsatisfactory.3 The pharmacological repression of immune function after OLT results in enhanced viral replication. Moreover, some immunosuppressants have additional nonimmune-mediated proviral effects, as we have demonstrated for glucocorticoids.4 Prevention of graft reinfection, as routinely achieved in the case of hepatitis B, is a major clinical goal, but will likely require efficient pharmacological means of preventing viral entry into hepatocytes. Such compounds are, so far, not available.
HCV is a highly variable enveloped RNA virus of the Flaviviridae family that infects hepatocytes and establishes a chronic infection in the majority of cases. The HCV genome, 9.6 kilobases in size, encodes for a single polyprotein cleaved by cellular and viral proteases into 10 different proteins: core, E1, E2 (structural proteins), p7, and the nonstructural proteins (NSs), NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The single open reading frame (3,000 amino acids) encoded by the HCV genome is flanked by nontranslated regions (NTRs) at the 5'- and 3'-end.
Cell-culture-derived HCV particles (HCVcc), based on the Japanese fulminant hepatitis 1 (JFH1) strain of HCV genotype 2a, are infectious both in vitro and in vivo and, therefore, are a widely used model for the complete replication cycle of HCV.5-7 Unlike other HCV isolates, JFH1 replicates very efficiently in Huh-7.5 cells in the absence of cell-culture-adaptive mutations. Construction of chimeric virus genomes encoding JFH1-derived nonstructural proteins and structural protein to form the J6/CF strain (genotype 2a) has further improved the efficiency of the HCV infection model.8 This system allows the assessment of the impact of antiviral agents on HCV RNA replication, virus production, and infectivity.
Green tea catechins, such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been found to have antiviral and antioncogenic properties.9 Functional and structural differences are attributed to the number of hydroxyl groups on the B-ring and the presence or absence of a galloyl moiety. For EGCG, a major component of green tea, inhibition of herpes simplex virus type 1 and 2, enterovirus 71, human immunodeficiency virus, influenza A, and other viruses has been reported.10-14 The mechanism of the observed broad antiviral effect is not known, but has been suggested to differ between viruses. This study was conducted to evaluate the effects of green tea catechins on HCV and to explore the mechanism underlying their antiviral effects.
Our results reveal that EGCG, but not other green tea catechins, is an inhibitor of HCVcc that does not interfere with genome replication. Instead, EGCG specifically targets viral cell entry (i.e., the NS3/4A-independent initial stage of the viral replication cycle) into both hepatoma cell lines and primary human hepatocytes. Furthermore, we showed that EGCG inhibits viral attachment to the target cell as well as cell-to-cell transmission between adjacent cells.
INN, boceprevir; CD, cluster of differentiation; CLDN1, claudin-1; cpm, counts per minute; CyA, cyclosporine A; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; EC, epicatechin; ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate; EGF-R, epidermal growth factor receptor; FACS, fluorescence-activated cell sorting; FBS, fetal bovine saerum; ffu, focus-forming units; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HCVcc, cell-culture-derived HCV; HCVpp, HCV pseudoparticles; HPM, hepatocyte plating media; IC50, half-maximal inhibitory concentration; IFN-α, interferon-alpha; JFH1, Japanese fulminant hepatitis 1; MLV, murine leukemia virus; MOI, multiplicity of infection; NS, nonstructural protein; NTRs, nontranslated regions; OCLN, occluding; OLT, orthotopic liver transplantation; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PHHs, primary human hepatocytes; RFP, red fluorescent protein; SR-BI, scavenger receptor class B type 1; VSV-G, vesicular stomatitis virus glycoprotein.
Discussion
In this study, we identified EGCG, a natural compound contained in green tea, as an inhibitor of HCV entry into target cells and cell-to-cell spread between neighboring cells. The effect is unique to EGCG and not shared by other green tea catechins. It was readily detectable for all HCV genotypes tested and in hepatoma cell lines as well as PHH. Moreover, we demonstrated that EGCG acts by blocking viral attachment to target cells (i.e., the initial step of the cell entry process), whereas it does not affect other replication cycle stages. The biophysical properties of the virions and the receptor expression levels on the target cells also remained unaltered by EGCG.
HCV entry is essential for initiation, spread, and maintenance of virus infection and represents an interesting target for antiviral therapy. With the NS3/4A protease inhibitors, INN and telaprevir, the first anti-HCV drugs beyond IFN and ribavirin are reaching the market in 2011. Yet, like all other anti-HCV compounds in late-stage clinical development, they target the inhibition of viral RNA replication. In the setting of liver transplantation for HCV-associated end-stage liver disease, moreover, the ability to block cell entry would help in minimizing the currently universal reinfection of the donor liver by virions in the blood.
Other agents have previously been reported to inhibit HCV cell entry through various mechanisms. The lectin, cyanovirin-N, interacts with high-mannose oligosaccharides on viral envelope glycoproteins and prevents its interaction with cellular receptor molecules, presumably CD81.21 Oxidized low-density lipoproteins (oxLDL), a physiologically occurring subfraction of LDL, are thought to inhibit an SR-BI-mediated step in the HCV entry process.22 Antibodies targeting the glycoproteins or cellular receptors, such as CD81, SR-BI, and CLDN1, have also been shown to block viral entry and control spread in vitro and in vivo.23 The small molecule compound, ITX-5061, was described to disrupt the interaction of E2 and SR-BI and is currently entering a phase Ib study in humans.24 Baldick et al. have recently identified a compound (El-1) that inhibits a postattachment step of the cell entry process, but which appears less active against non-1 genotypes.20 A recent study by Lupberger et al. identified the epidermal growth factor receptor (EGF-R) as a host factor required for HCV entry and suggested the EGF-R inhibitor, erlotinib, as a possible anti-HCV agent.25 Also, small molecule inhibitors of viral entry, which act on envelope lipids to prevent the formation of the negative curvature required for fusion, inhibit HCV JFH-1 infectivity to Huh-7.5 cells in culture.26 However, compared to these other inhibitors of HCV entry, EGCG is of particular interest, because it is active in vitro against all HCV genotypes tested, known to be innocuous in humans, readily available, and cheap. In fact, EGCG and various green tea preparations are available as an over-the-counter remedy in many countries.
EGCG is water soluble in green tea produced from the leaves of the plant, Camellia sinensis. The major active ingredients of green tea are polyphenolic compounds, known as catechins. The catechines include EGCG, EGC, ECG, and EC, of which EGCG accounts for approximately 50% of the total green tea catechins. Interestingly, EC is inactive against HCV and lacks the gallic acid ester moiety of EGCG, as well as one additional phenolic hydroxyl group. Thus, these functional groups, therefore, contribute to the ability of EGCG to inhibit HCV attachment and infectivity.
In clinical studies with healthy human volunteers, it could be shown that EGCG is safe and very well tolerated with oral doses of 800 mg of EGCG per day over 4 weeks, which equals approximately 8-16 cups of green tea once a day.27 Plasma concentration ranged from 0.13 to 3.4 μg/mL, which reaches the IC50 value of EGCG that we determined here (2.5 μg/mL), but would probably not be high enough to eliminate HCV completely. However, the bioavailability of EGCG can be increased by peracetylation, and further studies are required to determine the tissue distribution and the in vivo potency of the molecule against HCV.
Using radioactively labeled HCV, we showed that EGCG targets the very first step of the HCV cell entry process (i.e., attachment). Later entry steps, such as viral receptor interactions, endocytosis, or membrane fusion, appear not to be directly affected. Accordingly, viral association with lipoproteins and the HCV (co-)receptor expression levels on the host cell were not altered by EGCG treatment. Importantly, cell-to-cell HCV spread, which may be critical in vivo, was also inhibited by EGCG treatment.
Based on work in a cell-free system, it was previously suggested that EGCG also inhibits the essential HCV NS3/4A serine protease, but this inhibitory effect has not been validated in an HCV replication setting.28 In our hands, HCV replication and assembly of full-length HCV genomes were unaffected by EGCG.
EGCG and the other green tea catechins have been reported to be active against viruses other than HCV, and different mechanisms of action appear to be involved. For influenza A virus, Song et al. proposed that EGCG and ECG are potent inhibitors of multiple steps of the viral life cycle, including hemagglutination, neuraminidase activity, and viral RNA synthesis.12 In the case of hepatitis B virus, an inhibition of viral DNA synthesis has been proposed.14
In summary, the green tea molecule, EGCG, potently inhibits HCV entry independent of the genotype and in primary human hepatocytes by blocking viral attachment. This novel inhibitor may provide a new approach to prevent HCV infection, especially in the setting of liver transplantation of chronically infected HCV patients.
Materials and Methods
Compounds.
EGCG, ECG, EC, and EGC were purchased from Sigma-Aldrich (Seelze, Germany). Cyclosprine A (CyA) was provided by Novartis (Basel, Switzerland), and boceprevir (INN) was provided by the Institute Pasteur Korea (Seongnam, Korea).
Plasmids and Viruses.
Plasmids pFK-Jc1, pFK-JFH1, and H77/JFH1 have been described recently.8, 15 The construct, Luc-Jc1, is a bicistronic firefly luciferase reporter virus that encodes a chimeric HCV polyprotein consisting of codons 1-846 derived from J6/CF combined with codons 847-3033 of JFH1.16
Cell Culture.
Huh-7.5 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS), 1x nonessential amino acids (Invitrogen), 100 μg/mL of streptomycin (Invitrogen), and 100 IU/mL of penicillin (DMEMcomplete; Invitrogen).
Primary human hepatocytes were purchased from Invitrogen and resuspended in hepatocyte plating media (HPM; 500 mL of DMEM high glucose and 10% FBS) and plated at a concentration of at 2.5 x 105 cells/cm2. We used diluted collagen (type 1, rat tail; BD, Heidelberg, Germany) (50 μg/mL in 0.02 N of acetic acid) for coating coverslips and plates in approximately 10 mL at room temperature for 1 hour. The collagen solution was then removed, and the plastic surface was rinsed twice with phosphate-buffered saline (PBS). After the cells attached (>3 hours), the HPM was replaced by hepatocyte basal medium media+hepatocyte culture medium Single Quots (Clonetics; Lonza, Walkersville, MD).
Preparation of retroviral pseudoparticles.
Murine leukemia virus (MLV)-based pseudotypes bearing vesicular stomatitis virus glycoproteins (VSV-Gs) or HCV E1-E2 proteins of the Con1, H77, or J6CF isolates were generated as described recently.4
HCV Pseudoparticle Infection of Primary Human Hepatocytes.
Primary human hepatocyte cells infected with HCV pseudoparticles (HCVpp) were treated with increasing amounts of EGCG for 4 hours. After 48 hours, cells were analyzed on a fluorescence-activated cell sorter (FACS) (FACScalibur; BD). Data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
HCV Luciferase Replication and Infection Assay.
Huh-7.5 cells were electroporated with 5 μg of the reporter virus genome, as previously described.16 After 4 hours, different concentrations of EGCG were added to the cell-culture medium. HCV RNA replication was quantified by measuring luciferase activity. After 48 hours, supernatants were collected, filtered through 0.45-μm pore-size filters, and used to infect naïve Huh-7.5 target cells.
Detection of HCV Receptor Expression by Flow Cytometry and Western Blotting.
Huh-7.5 cells were treated with increasing amounts of EGCG for 4 hours. After incubation with EGCG, cells were stained with antibodies for flow cytometry and western blotting, as previously described.4
Iodixanol Density-Gradient Fractionation.
Density-gradient centrifugation was performed as described recently.17
Cell-to-cell spread assay.
To measure HCV infection by direct cell-to-cell transmission between adjacent cells, we performed an agarose overlay assay in the presence or absence of EGCG, as described recently.18
35S Labeling.
Huh-7.5 cells seeded in 100-mm dishes were infected with HCV JFH-1 (multiplicity of infection [MOI]: 0.13 focus-forming units [ffu]/cell). Inocula were removed after 4 hours at 37°C, and the infected cells were washed twice with PBS before the addition of DMEM/10% FBS. Cells were methionine-starved 3 hours later by replacing the media with methionine-free DMEM/10% FBS. After 2 hours of methionine starvation, cells were washed twice with warm PBS and overlaid with 4 mL of methionine-free DMEM/10% FBS, supplemented with 42 μCi/mL of 35S-methionine (PerkinElmer, Waltham, MA). Supernatants were recovered approximately 48 hours later and centrifuged at 1,200 rpm at 4°C for 5 minutes to pellet cell debris. The supernatant was filtered through a 0.22-μm filter and concentrated using Amicon 100-K molecular-weight cut-off filters (Millipore, Bedford, MA). The resulting virus stock was titrated using the focus-forming assay and stored at -80°C. 35S incorporation was tested using a Beckman Coulter LS 6500 scintillation counter (Beckman Coulter, Inc., Brea, CA). HCV virions were labeled to a calculated 199 cpm/ffu.
Binding Assay.
[35S]-methionine-labeled HCV virions were exposed to EGCG, EC, or dimethyl sulfoxide (DMSO) vehicle for 10 minutes at 37°C. Next, 6 x 105 cpm (3 x 103 ffu) of 35S-Met-HCV was adsorbed onto nearly confluent monolayers of Huh-7.5 cells for 1 hour at 4°C before washing four times with ice-cold PBS. Radioactivity attached to cells after the washes was measured using a Beckman Coulter LS 6500 scintillation counter. Binding was calculated as counts per minute (cpm) bound to cells divided by total cpm, adjusted by background and expressed as a percentage. Percent binding was then expressed relative to binding of virions exposed to vehicle control.
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