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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proofs
  8. References
  9. Supporting Information

Here, we identify (−)-epigallocatechin-3-gallate (EGCG) as a new inhibitor of hepatitis C virus (HCV) entry. EGCG is a flavonoid present in green tea extract belonging to the subclass of catechins, which has many properties. Particularly, EGCG possesses antiviral activity and impairs cellular lipid metabolism. Because of close links between HCV life cycle and lipid metabolism, we postulated that EGCG may interfere with HCV infection. We demonstrate that a concentration of 50 μM of EGCG inhibits HCV infectivity by more than 90% at an early step of the viral life cycle, most likely the entry step. This inhibition was not observed with other members of the Flaviviridae family tested. The antiviral activity of EGCG on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, using binding assays at 4°C, we demonstrate that EGCG prevents attachment of the virus to the cell surface, probably by acting directly on the particle. We also show that EGCG has no effect on viral replication and virion secretion. By inhibiting cell-free virus transmission using agarose or neutralizing antibodies, we show that EGCG inhibits HCV cell-to-cell spread. Finally, by successive inoculation of naïve cells with supernatant of HCV-infected cells in the presence of EGCG, we observed that EGCG leads to undetectable levels of infection after four passages. Conclusion: EGCG is a new, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals. Furthermore, it is a novel tool to further dissect the mechanisms of HCV entry into the hepatocyte. (HEPATOLOGY 2012;)

Hepatitis C virus (HCV) is a major cause of chronic liver disease. It is estimated that 3% of the world population is currently infected and thus is at high risk of developing cirrhosis and hepatocellular carcinoma. 1 No vaccine is available, and the current standard-of-care therapy with pegylated interferon-alpha (IFN-α) and ribavirin has a limited efficacy and significant side effects. 2 Very recently, an addition to the therapy of new direct-acting antivirals (DAAs) targeting HCV nonstructural protein (NS)3-4A protease, telaprevir, and boceprevir was shown to increase the sustained virological response in patients infected with HCV genotype 1 by up to 70%. 3 Efforts are currently being made to identify new DAAs with additive potency. The majority of these molecules target the replication step. 3 However, because of the high genetic heterogeneity of HCV and its rapid replication, monotherapy with DAA agents poses a high risk for selection of resistant variants. 4 Therefore, combinations of drugs targeting different steps of the viral life cycle, including virus entry, would likely improve viral response rates and therapeutic success.

HCV is a small enveloped virus with a positive stranded RNA genome belonging to the Hepacivirus genus in the Flaviviridae family. 5 Its genome encodes two envelope glycoproteins, E1 and E2, which play a key role in virus entry into the hepatocyte. HCV entry is a complex, multistep process involving sequential interactions with several cell-surface proteins. 6 The virus relies on glycosaminoglycans and perhaps on low-density lipoprotein receptor to attach to cells. Furthermore, four specific entry factors— scavenger receptor class B type 1, tetraspanin cluster of differentiation (CD)81, claudin-1, and occludin— are sequentially involved after initial virus binding. Finally, HCV enters cells via clathrin-mediated endocytosis. 7 The viral particle also has the peculiar feature of being associated with low- or very-low-density lipoproteins. 7 Importantly, two modes of infection have been observed in vitro: either cell-free or cell-to-cell transmission, with the latter being refractory to neutralization by anti-E2 antibodies, thus representing an alternative mode of transmission, which may be important in vivo. 8

Green tea has a potential to effect a variety of human diseases, in particular, cancers. 9 Most of the properties of green tea are mediated by (−)-epigallocatechin-3-gallate (EGCG), the most abundant polyphenol catechin present in green tea extracts. EGCG administration is safe in healthy individuals. 10 EGCG also displays some antiviral activity against human immunodeficiency virus (HIV), human herpes simplex virus (HSV), and influenza virus. 11-13 In all cases, EGCG was shown to inhibit entry, either by targeting cellular proteins, CD4 receptor for HIV, 11 and nuclear factor erythroid 2–related factor 2 for influenza, 14 or by direct action on the particle for HSV and influenza. 11, 13 Furthermore, EGCG was shown to increase lipid-droplet formation and to impair very-low-density lipoprotein secretion in hepatocytes. 15 Thus, because of the link between lipid metabolism and HCV life cycle, we hypothesized that this molecule might interfere with HCV. Here, we investigated the potential antiviral effect of EGCG on HCV. Importantly, our data show that EGCG is a new anti-HCV agent that blocks an early step of the entry process, the attachment step, probably by targeting HCV particle.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proofs
  8. References
  9. Supporting Information

Chemicals.

Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), glutaMAX-I, and goat, horse, and fetal calf sera were purchased from Invitrogen (Carlsbad, CA). 4′,6-Diamidino-2-phenylindole (DAPI) was from Molecular Probes (Invitrogen). EGCG was from Calbiochem (Merck Chemicals, Darmstadt, Germany), except when a set of green tea catechins, (+)-catechin, (−)-epicatechin (EC), (−)-epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC), and EGCG, was used, which was purchased from Extrasynthèse (Lyon, France). Stocks were resuspended in dimethyl sulfoxide (DMSO) at 0.5 M. Other chemicals were from Sigma-Aldrich (St. Louis, MO).

Antibodies.

Mouse anti-E1 A4, 16 rat anti-E2 3/11, 17 mouse anti–yellow fever virus (YFV) envelope protein 2D12 (ATCC CRL-1689), and mouse anti–bovine viral diarrhea virus (BVDV) NS3 Osc-23 18 monoclonal antibodies (mAbs) were produced in vitro. Cyanin 3 (Cy3)-conjugated goat antimouse immunoglobulin G (IgG) was from Jackson Immunoresearch (West Grove, PA).

Cells and Culture Conditions.

Huh-7, 19 HEK 293T (ATCC number CRL-11268), Vero (ATCC CCL-81), and Madin-Darby Bovine Kidney (MDBK; ATCC number CCL-22) cells were grown in DMEM, supplemented with glutaMAX-I and either 10% fetal calf serum (Huh-7, HEK 293T, and Vero) or 10% horse serum (MDBK).

HCV Grown in Cell Culture.

We used a modified Japanese fulminant hepatitis (JFH)1 virus containing titer-enhancing mutations, 20 in which the A4 epitope of HCV glycoprotein E1 of genotype 1a was reconstituted. 21 The JFH1-Luc plasmid, containing a Renilla Luciferase reporter gene, the JFH1-ΔE1/E2-Luc or JFH1-ΔE1/E2 plasmids, which contain an in-frame deletion in the E1/E2 region, and the JFH1/GND-Luc replication mutant, have been described previously. 21, 22 Infections were scored by measuring luciferase activity in cell lysates, using a Renilla luciferase assay system from Promega (Madison, WI), or by measuring infectivity by indirect immunofluorescence (IF) with anti-E1 mAb. For quantitative binding experiments, purified virus was obtained by the precipitation of HCV grown in cell culture (HCVcc)-infected Huh-7 cell supernatants with 8% polyethylene glycol 6000. Pelleted virus was then loaded onto a continuous 10%-40% iodixanol gradient. One-milliliter fractions were collected and the most infectious were pooled. The titer of the stock was 5 × 106 focus-forming units/mL.

HCV Pseudotyped Particles.

The luciferase-based HCV pseudotyped retroviral particle (HCVpp) infection assay was used as previously described. 23

Other Viruses.

HSV type 1 strain HF (HSV-1; ATCC VR-260) was used to infect Vero cells, then seeded in 96-well plates for 1 hour at 37°C. Five days after infection, titers were calculated by quantifying the cytopathic effect. BVDV strain NADL and YFV strain 17D were used to infect MDBK or Huh-7 cells at a multiplicity of infection (MOI) of 1.5 or 1, respectively, seeded on coverslips for 1 hour at 37°C, and cultured for either 15 or 23 hours. MOIs were determined based on BVDV and YFV infectious titers, determined on MDBK and Huh-7 cells, respectively, and on the number of cells at the inoculation step. Stocks of Toto1101/Luc, 24 a Sindbis virus (SINV) expressing the Firefly luciferase (kindly provided by M. MacDonald, Rockefeller University, New York, NY), were generated as previously described. 24 The inoculation period was 1 hour, and cells were lysed at 23 hours postinfection.

Quantification of HCV Core Protein.

Huh-7 cells were inoculated for 2 hours with HCVcc in 35-mm wells of six-well cell-culture plates or were electroporated with JFH1-ΔE1/E2 RNA. HCV core antigen, expressed within cells or secreted into the supernatant, was quantified using chemiluminescent microparticle technology (Architect HCV Ag Test; Abbott SA, Rungis, France), as previously described. 25 In parallel, total amounts of proteins in cell lysates were quantified using the bicinchoninic acid assay (Sigma-Aldrich).

Indirect IF Microscopy.

Infected cells grown onto glass coverslips were processed for IF detection of viral proteins, as previously described. 26 Nuclei were stained with 1 μg/mL of DAPI. Coverslips were observed with a Zeiss Axiophot microscope equipped with either 10× or 20× magnification objectives (Carl Zeiss AG, Oberkochen, Germany). Fluorescent signals were collected with a Coolsnap ES camera (Photometrix, Kew, Australia). For quantification, images of randomly picked areas from each coverslip were recorded.

Viability Assay.

Subconfluent cell cultures grown in 96-well plates were incubated in culture medium. An MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based viability assay (CellTiter 96 aqueous nonradioactive cell proliferation assay from Promega) was conducted as recommended by the manufacturer.

HCVcc Cell-to-Cell Transmission Assays.

Huh-7 cells seeded on coverslips in 24-well plates were infected with HCVcc for 2 hours. The inoculum was removed and replaced with culture medium containing 1% Seaplaque low-melting-temperature agarose (Lonza, Walkersville, MD) or 3/11 anti-E2 neutralizing mAb at 50 μg/mL. At 3 days postinfection, the foci were detected using indirect IF.

Entry Assay.

Huh-7 cells were infected with JFH1-Luc in 24-well plates for 1 hour at 4°C (attachment/binding period), washed with serum-free medium, and incubated for another 1 hour at 4°C (postattachment/-binding period). Then, cells were washed and incubated for 1 hour at 37°C (endocytosis/fusion period). Finally, cells were washed and incubated in complete culture medium at 37°C for 45 hours. Infections were scored by measuring luciferase activity.

Quantitative Binding Assay.

Huh-7 cells were infected with purified HCVcc in 24-well plates for 1 hour at 4°C in the presence of either DMSO, or 50 μM of EGCG, or 500 μg/mL of porcine intestinal heparin. Cells were washed with PBS, and total RNA was extracted using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions. HCV RNA was quantified by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assay as described previously. 27

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proofs
  8. References
  9. Supporting Information

EGCG Inhibits HCV Infection.

Before analyzing the potential antiviral effect of EGCG on HCV, we first determined the toxicity of EGCG on Huh-7 cells (Fig. 1A). Although some toxicity began to be observed at 100 μM, a concentration of 50 μM was shown to have no toxic effect, even after 72 hours. The half lethal dose was between 150 and 175 μM in Huh-7 cells, depending on the exposition time. To test the effect of EGCG on the HCV life cycle, the molecule was added to the medium during HCVcc infection. Interestingly, more than 1 log10 decrease of HCVcc infectious titers was observed in cells treated with 50 μM of EGCG (Fig. 1B). To confirm our results on HCV, we used a recombinant HCV expressing the Renilla luciferase (i.e., JFH1-Luc) to infect cells in the presence of increasing concentrations of EGCG. A dose-dependent decrease of JFH1-Luc infection was observed (Fig. 1C). The half-maximal inhibitory concentration (IC50) was estimated to approximately 5 μM, and the 90% inhibitory concentration (IC90) was close to 50 μM. Thus, the therapeutic index of EGCG is approximately 30. Together, these results show that EGCG has an antiviral activity against HCV.

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Figure 1. EGCG impairs HCV infection. (A) EGCG toxicity was tested on Huh-7 cells. Cells were cultured in the presence of given concentrations of EGCG. After removal of the EGCG-containing culture medium, their viability was monitored using an MTS-based viability assay at 24, 48, and 72 hours by determining OD at 490 nm. Results are means ± SD (error bars) from three independent experiments, expressed as relative values compared to untreated cells, for which a value of 1 was attributed. (B) Huh-7 cells were infected with HCVcc in the presence or absence of 50 μM of EGCG for 2 hours, then incubated with or without 50 μM of EGCG for an additional 46 hours. Supernatants were collected, and infectivity titers were determined. (C) Huh-7 cells were infected with JFH1-Luc for 2 hours in the presence of given concentrations of EGCG and were further incubated with the same concentrations of EGCG for 48 hours. Cells were lysed, and luciferase activity was quantified. Error bars represent SD from three experiments. (D) Schematic representation of the five different catechins extracted from green tea. (E) Huh-7 cells were infected with JFH1-Luc for 2 hours in the presence of 50 μM of each catechin separately. Cells were further incubated in catechin-free medium for 46 hours and lysed to quantify luciferase activity. Infectivity is expressed as the percentage relative to the luciferase activity measured without catechin. Error bars represent SD from three experiments. EGCG (1) was from Calbiochem (La Jolla, CA) and EGCG (2) was from Extrasynthèse (Lyon, France). OD, optical density; SD, standard deviation.

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Other catechins extracted from green tea include (+)-catechin, EC, ECG, and EGC (Fig. 1D). The toxicity of each catechin was determined individually (Supporting Fig. 1). Then, each catechin was tested for its antiviral activity. (+)-Catechin and EC did not display any anti-HCV activity (Fig. 1E). In contrast, both ECG and EGC exhibited an inhibition of HCV infection of approximately 40% and 80%, respectively. Furthermore, we confirmed the antiviral activity of EGCG by using EGCG provided by another manufacturer.

To test whether EGCG would be a general viral inhibitor, experiments were performed with two other members of the Flaviviridae family (BVDV and YFV) and another unrelated virus (SINV) (Fig. 2A). HSV-1 was used as a positive control, because EGCG has an antiviral activity against this virus 12 (Fig. 2B). In contrast to HCV and HSV-1, EGCG treatment at 50 μM has no antiviral effect on BVDV, YFV, and SINV, indicating that the effect of EGCG could not be generalized to the Flaviviridae family in our experimental conditions.

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Figure 2. The antiviral effect of EGCG is not observed for YFV or BVDV. (A) MDBK cells were infected with BVDV at a MOI of 1.5 for 1 hour in the presence of 50 μM of EGCG and were further incubated in culture medium for 15 hours. Cells were fixed, and infection was quantified by IF using an anti-NS3 (Osc-23) antibody and Cy3-conjugated goat antimouse IgG secondary antibody. For BVDV, the MOI of 1.5 led to approximately 28% of infected cells. Huh-7 cells were infected in the presence of 50 μM of EGCG for 1 hour with YFV at a MOI of 1 or with SINV or for 2 hours with JFH1-Luc (HCV). At 23 hours postinfection with YFV, cells were fixed and infection was quantified by IF using anti-E antibody (2D12) and Cy3-conjugated goat antimouse IgG secondary antibody. For YFV, the MOI of 1 led to approximately 32% of infection in the control. For SINV and HCVcc quantification, cells were lysed at 23 and 46 hours postinfection, respectively, and luciferase activities were quantified. The graph represents, for BVDV and YFV, the number of infected cells and, for SINV and HCV, the relative luciferase activity. For each virus, infectivity is expressed as the percentage of the control (DMSO) for which the 100% value was arbitrarily attributed. (B) HSV-1 (TCID 50/mL = 106.7) was incubated with DMSO or 100 μM of EGCG for 1 hour at 37°C before 1-hour inoculation of Vero cells. Five days after infection, titers were calculated by quantifying the cytopathic effect of the virus. Median TCID (50/mL) is presented. Mean values ± SD (error bars) of three different experiments are given. TCID, tissue-culture infective dose. SD, standard deviation.

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EGCG Inhibits HCV Entry.

To determine whether it had any effect on HCV entry, EGCG was added at different time points before, during, and after inoculation of Huh-7 cells with JFH1-Luc. The duration of each step was determined experimentally using specific controls (Supporting Fig. 2). The results clearly show that a decrease in HCVcc infection was only observed when EGCG was present during virus infection (Fig. 3A, second, third, fourth, and sixth bars in the bar-graph), and that there was no effect of EGCG if added as a pretreatment of the cells (Fig. 3A, first bar) or postinfection (Fig. 3A, fifth bar). These results suggest that EGCG inhibits an early step of the HCV life cycle, most likely the entry step.

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Figure 3. EGCG inhibits HCV entry independently of the genotype. (A) EGCG (+), or DMSO (−) as a control, was added to the culture medium of Huh-7 cells for 2 hours before infection (cell pretreatment), for 2 hours during infection with the JFH1-Luc virus (infection), and/or for 2 hours after infection (postinfection). Forty-six hours after infection, cells were lysed and luciferase activity was quantified. Infectivity is expressed as a percentage relative to the luciferase activity measured in the control without EGCG in any step (DMSO, darker bar on the right). Mean values ± SD (error bars) of three different experiments are presented. (B) Huh-7 cells were infected with HCVpp of the indicated genotypes or with VSVpp for 2 hours in the presence of DMSO or EGCG (5 or 50 μM). At 46 hours postinfection, cells were lysed and luciferase activity was quantified. Infectivity is expressed as the percentage of the control (DMSO) for which the 100% value was arbitrarily attributed. Mean values ± SD (error bars) of three different experiments are presented. SD, standard deviation.

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To confirm the effect of EGCG on HCV entry, HCVpp harboring E1 and E2 of different genotypes were produced. HCVpp infectivity was reduced by approximately 10-fold with a concentration of 50 μM, confirming the effect of EGCG on HCV entry, whatever the genotype used (Fig. 3B). However, some differences between genotypes could be observed at a lower EGCG concentration (5 μM). In contrast, vesicular stomatitis virus (VSV)pp entry was much less inhibited. These results suggest that the antiviral activity of EGCG is directed against HCV envelope glycoproteins and is genotype independent.

Together, these data indicate that EGCG inhibits HCV entry in a genotype-independent manner.

EGCG Does Not Inhibit HCV Replication and Egress.

Although the above data indicate that EGCG has a strong effect on HCV entry, we cannot exclude additional effects on other steps of the HCV life cycle. To analyze the effect of EGCG on HCV genome replication, Huh-7 cells were electroporated with in vitro transcribed assembly-defective JFH1-ΔE1/E2-Luc RNA, to bypass the entry step, and avoid any interference with late steps of the HCV life cycle. EGCG had no major effect on HCV replication, even after a longer period of treatment (96 hours postelectroporation) (Fig. 4A). In contrast, IFN-α, at 2 IU/mL, approximately twice the IC50 calculated for HCVcc in Huh-7 cells (1.15 IU/ml), 28 induced 1 log10 decrease of luciferase activity.

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Figure 4. EGCG does not inhibit HCV replication and secretion. (A) JFH1-ΔE1/E2-Luc and JFH1-GND-Luc RNA (10 μg) were electroporated in Huh-7 cells, and, at 4 hours postelectroporation, 50 μM of EGCG or IFN-α (2 IU/mL) were added or not to the culture medium of cells electroporated with JFH1-ΔE1/E2-Luc. Cells were lysed at given time points postelectroporation and luciferase activity was quantified. Luciferase activity relative to activity quantified at 4 hours postelectroporation is presented. The data presented are representative of three independent experiments. Error bars represent SD of values collected in three different samples. The JFH1-GND-Luc mutant deficient in replication (GND) was added as a negative control. (B) Cells were either inoculated with HCVcc for 2 hours and cultured in the presence of DMSO or 50 μM of EGCG for 70 hours, or electroporated with JFH1-ΔE1/E2, and cultured in the presence of DMSO or 50 μM of EGCG for 44 hours, 4 hours postelectroporation. Supernatants were collected, cells were lysed, and quantities of intra- and extracellular core protein were measured. The data are expressed as femtomoles of core protein relative to the amount of total proteins quantified in cell lysates. The data presented are representative of three independent experiments. Error bars represent SD of values collected in three different samples. Statistical analyses were performed using the Mann-Whitney nonparametric test. Calculated two-sided exact P values showed no significant differences between DMSO and EGCG values (P > 0.05) in all conditions. SD, standard deviation.

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To determine whether EGCG could have any effect on HCV assembly or secretion, intra- and extracellular core protein was quantified in infected cells treated postinfection with 50 μM of EGCG for 70 hours. The amount of core in the culture supernatant reflects the quantity of secreted viral particles. A slight, but not significant (P = 0.10), decrease in intracellular core was observed in the presence of EGCG (Fig. 4B). This cannot be explained by a decrease in RNA replication, because it has been shown above that EGCG has no effect on HCV replication (Fig. 4A). However, the quantification of extracellular core showed a small, but not significant (P = 0.10), increase of secreted core in the presence of EGCG, as compared to the nontreated control (Fig. 4B), showing that EGCG does not impair viral secretion. Similar experiments were performed with JFH1-ΔE1/E2 to avoid reinfection of the cells and to quantify the levels of extracellular core resulting from cell lysis. As expected, no difference in intra- or extracellular core was observed in the presence of EGCG, confirming that EGCG does not inhibit HCV replication and translation and does not induce any cell lysis. Similar results were obtained for NS5A by western blotting (Supporting Fig. 3). All together, these data show that EGCG does not inhibit viral replication or virion assembly and egress.

EGCG Blocks an Early Step of HCV Entry.

Because EGCG has an antiviral activity at an early step of the HCV life cycle, we further investigated its mode of action on HCV entry. To test whether EGCG would act on the viral particle or on the target cells, HCVcc was preincubated with EGCG before contact with target cells. In these conditions, EGCG should have a higher inhibitory effect, especially if it acts on the viral particle. In contrast, the antiviral action of EGCG should not be modified if EGCG acts on the cell. Preincubation of the virus with 50 μM of EGCG, followed by a dilution to 5 μM during infection, led to a stronger inhibition of infection than the one observed with the same concentration of EGCG during the inoculation, with no preincubation (Fig. 5A). Together with the inhibition of HCVpp infectivity, these data strongly suggest that EGCG inhibits HCV entry by affecting the HCV envelope.

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Figure 5. EGCG blocks an early step of HCV entry. (A) Huh-7 cells were inoculated 2 hours with JFH1-Luc (diluted 10 times) in the presence of 5 μM of EGCG, or with JFH1-Luc previously treated with 50 μM of EGCG and then diluted 10 times, leading to a concentration of 5 μM of EGCG for the inoculation period. The virus titers were kept constant in the three different conditions. At 46 hours postinfection, cells were lysed and luciferase activity was quantified. Infectivity is expressed as the percentage of the control (DMSO) for which the 100% value was arbitrarily attributed. Mean values ± SD (error bars) of three independent experiments are presented. (B) As represented at the top of the right panel, infection of Huh-7 cells with JFH1-Luc was divided into three steps. A first step of 1 hour at 4°C in the presence of the virus, allowing attachment of the particle to the cell, a second step of 1 hour at 4°C after removal of viral inoculum, permitting the attached virion to further bind to its receptors, and a third step of 1 hour at 37°C allowing endocytosis and fusion of virus with cellular membranes. EGCG (50 μM) or heparin (500 μg/mL) were added, as represented by bolded bars in the diagram. Cells were further incubated for 45 hours at 37°C without EGCG or heparin and were lysed to quantify luciferase activity. Infectivity is expressed as the percentage relative to the luciferase activity measured with DMSO. Mean values ± SD (error bars) of three different experiments are presented. (C) Huh-7 cells were inoculated with purified HCVcc at a MOI of 10 for 1 hour at 4°C in presence of DMSO, 50 μM of EGCG, or 500 μg/mL of heparin. Cells were washed thrice with ice-cold PBS, and total RNA was extracted. Bound HCV virions were detected by quantification of HCV gRNA by qRT-PCR. Relative binding is expressed as the percentage of the control (DMSO) for which the 100% value was arbitrarily attributed. Mean values ± SD (error bars) of three different experiments are presented. SD, standard deviation.

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The mechanism of HCV entry is a complex multistep process involving binding of the viral particle to the cell surface, interaction with several entry factors followed by endocytosis, and fusion of the viral envelope with an internal membrane. To determine which step of HCV entry is impaired by EGCG, virus binding was performed at 4°C. Then, the cells were shifted to 37°C to allow endocytosis and fusion, with EGCG being added at different times (Fig. 5B). Heparin, a known inhibitor of HCV binding, was used as a positive control. 29 A strong decrease in HCV infection was observed when EGCG was added at 4°C during the first hour (i.e., binding step), whereas no inhibition was observed when EGCG was added after virus binding, even before endocytosis. As expected, similar results were obtained with heparin. These results are consistent with a direct action of EGCG on the viral particle, as suggested before, for the antiviral effect to take place.

To determine whether EGCG impairs directly the binding of particles to the cell surface or a later step of virus entry, we analyzed virus binding in the presence of EGCG. Cells were inoculated with purified HCVcc at 4°C in the presence of EGCG or heparin, and the amount of bound viruses was determined by quantifying HCV genomic RNA (gRNA). As expected, heparin strongly reduced HCV attachment to the cell surface (Fig. 5C). In the presence of EGCG, a strong decrease in virus binding was also observed. Together, these results show that EGCG, likely by acting on the virion, inhibits virus entry by impairing virus binding to the cell surface.

EGCG Inhibits HCV Cell-to-Cell Spread and Clears Cell Culture From HCV.

After infection of Huh-7 cells with HCVcc, progeny viruses are transmitted to adjacent cells, resulting in focal areas of spreading infection (i.e., foci). This mode of transmission is refractory to neutralization by anti-E2 antibodies. 8 To determine whether EGCG could block cell-to-cell spread, HCV-infected Huh-7 cells were either overlaid with agarose-containing medium or incubated with neutralizing mAb 3/11 in the presence or absence of 50 μM of EGCG. Both methods are known to prevent reinfection of cells by newly secreted HCV particles, but allow cell-to-cell spreading. 30, 31 Three days after infection, foci were visualized by IF (Fig. 6A) and sizes of foci were measured by counting the number of cells per focus (Fig. 6B). The two methods, even if they led to differences in average size of foci in the control condition (approximately 47 and 55 cells), showed a strong reduction in the number of cells per focus in the presence of EGCG (4 and 16 cells). These data clearly indicate that EGCG blocks HCV cell-to-cell transmission.

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Figure 6. EGCG blocks HCV cell-to-cell transmission and clears virus from cell culture. (A) Huh-7 cells were inoculated with HCVcc for 2 hours, and cells were either overlaid with 1% agarose dissolved in culture medium or incubated, with 3/11 anti-E2 neutralizing antibodies (without agarose), in the presence or absence of 50 μM of EGCG. At 70 hours postinfection, cells were fixed and processed for IF detection of HCV E1 envelope glycoprotein using A4 mAb and Cy3-conjugated goat antimouse IgG secondary antibody. Nuclei were stained with DAPI in parallel. (B) Numbers of cells per focus were quantified in 16 foci of each condition. Bar indicates the mean value. (C) Huh-7 cells were infected with HCVcc (P0), and 48 hours later, cellular supernatant was collected and used to inoculate naïve Huh-7 cells in the presence of DMSO or 50 μM of EGCG for 2 hours (P1). This manipulation was repeated thrice (P2, P3, and P4). Infected cells were quantified at each step by IF staining of E1 protein using A4 mAb and Cy3-conjugated goat antimouse IgG secondary antibody and compared to the total number of cells determined by staining nuclei with DAPI. (D) Infectious titers were determined from supernatants collected at P0, P1, and P4.

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In parallel, experiments were performed to determine whether EGCG could lead to the elimination of HCV from infected cell supernatants after successive passages on naïve cells. The number of infected cells was quantified at each step, and virus titers were calculated at P0, P1, and P4 (Fig. 6C,D). Interestingly, a rapid, strong decrease in the number of infected cells was observed in the presence of EGCG, leading to almost undetectable levels of infected cells after four passages. In contrast, a slight decrease in the number of infected cells was observed in the absence of drug. These results were correlated with the measured virus titers (Fig. 6D). Moreover, we did not detect any change in antiviral activity of EGCG, whatever the titer (Supporting Fig. 4). These results show that the anti-HCV effect of EGCG can lead to undetectable levels of virions in the supernatant of infected cells.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proofs
  8. References
  9. Supporting Information

In this article, we identify a new inhibitor of HCV entry (EGCG) that might have some applications in HCV therapy. We demonstrate that this major component of green tea extract inhibits HCVcc as well as HCVpp entry, regardless of the genotype. Furthermore, EGCG inhibits viral cell-to-cell spread and is able to cure HCV from cell-culture supernatants after a few passages. We also demonstrate that EGCG acts at a very early step of entry, probably by inhibiting the docking of the virus to the cell surface.

A few polyphenol molecules have been reported to impair HCV infection. Among them, silymarin 32 and naringenin 33 inhibit HCV replication and/or secretion. Furthermore, silymarin has been recently shown to inhibit HCV entry, probably at the fusion step. 32 Here, we observed that EGCG is efficient in blocking HCV entry at a concentration of 50 μM (i.e., IC90), which is similar to active concentrations reported for other flavonoids (10-200 μM). Our results show that both the galloyl group in R3 and the hydroxyl group in R5′ are necessary to confer its anti-HCV activity to EGCG. EGC, which is not very toxic in vitro, might be used in combination with EGCG because it displays quite an interesting antiviral activity. Pharmacokinetics and safety studies of EGCG on healthy individuals show that (1) consumption of up to 800 mg of EGCG is safe, and (2) that it increases the concentration of EGCG detected in the plasma. 10 However, EGCG plasma concentration rises up to only 0.39 μg/mL, which is approximately 6 times lower than the IC50 observed in our study (5 μM corresponds to 2.3 μg/mL). Thus, other routes of EGCG delivery should be tested to increase its plasma concentration. Alternatively, modifying the molecule to improve its bioavailability and antiviral activity might be necessary to establish EGCG as a potent antiviral drug in human therapy.

We demonstrate here that EGCG acts at an early step of virus entry, probably at the binding step. This step of the virus life cycle is still poorly defined, and EGCG might be a new tool to go further in its characterization. The first step of virus entry involves an association of the virus with heparan sulfate proteoglycans present at the surface of many different cell types. 29 Moreover, the low-density lipoprotein receptor has also been implicated in the binding of HCV-associated low-density lipoproteins. 34 More experiments will be necessary to determine whether EGCG interferes with these processes or if it acts at a different level. Because EGCG acts on the viral particle, and inhibits both HCVpp and HCVcc infection, it is reasonable to think that EGCG interferes with E1/E2 function. It seems unlikely that it has any effect on the phospholipidic bilayer of the viral envelope because of its absence of inhibition for other viruses from the same family.

EGCG has already been reported to have an antiviral activity against several other viruses. In the case of HIV, it was demonstrated that EGCG inhibits HIV-1 infection by blocking interaction between T cell CD4 and viral glycoprotein 120 by binding to CD4. 35 Much like what we observed with HCV, EGCG inhibits influenza A and B and HSV at the entry step by affecting the viral particle. 12, 13, 36 EGCG was shown to agglutinate influenza viruses, leading to hemagglutination inhibition, and thus impairing virus adsorption on cells. In the case of HSV, EGCG seems to alter the viral particle, probably by interacting with the envelope glycoproteins, gB and gD, leading to a blockade in virus entry. The inhibition of HCV entry by EGCG could also potentially result from an alteration of the virion. Unfortunately, the difficulties in identifying HCV particles by electron microscopy did not allow us to explore this hypothesis.

A major problem for liver transplantation caused by HCV is the reinfection of the graft, which is always observed with an accelerated progression of liver disease. 37 Thus, the ability of EGCG in inhibiting HCV cell-to-cell transmission is a major asset for an entry inhibitor. Furthermore, EGCG exhibits an antiviral activity against all HCV genotypes, tested in the HCVpp system, increasing its potential interest as a general anti-HCV agent. The combination of entry, replication, and polyprotein-processing inhibitors, in a context of a multidrug therapy, might be the way to reduce the risk of the emergence of resistant viruses. For all these reasons, after having tested its safety in HCV-infected patients, it would be interesting to further evaluate the antiviral activity of EGCG in combination with other DAA molecules.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proofs
  8. References
  9. Supporting Information

The authors thank J.K. Ball, R. Bartenschlager, F.L. Cosset, M. MacDonald, J. McKeating, and T. Wakita for providing essential reagents. The authors also thank P.E. Lobert for helpful discussion and M. Giard for technical assistance.

Note Added in Proofs

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proofs
  8. References
  9. Supporting Information

Since the first submission of this article, another report on the antiviral effect of EGCG on HCV entry has appeared (Ciesek S et al., Hepatology, in press).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proofs
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proofs
  8. References
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24803_sm_suppinfofig1.tif495KSupporting Information Figure 1
HEP_24803_sm_suppinfofig2.tif253KSupporting Information Figure 2
HEP_24803_sm_suppinfofig3.tif279KSupporting Information Figure 3
HEP_24803_sm_suppinfofig4.tif170KSupporting Information Figure 4

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