Fig 1.
The structure of tannic acid and toxicity assays.
(A) Molecular structure of tannic acid. (B) Cell toxicity of tannic acid as measured by cell viability. The viability of Huh7.5 cells was determined using a CellTiter 96 Aqueous One solution cell proliferation assay as described in Materials and Methods. Each experiment was performed in triplicate and repeated at least three times. The CC50 based on these studies was 146.1 ± 6.2 μM. (C) The direct effect of tannic acid on Renilla luciferase (Rluc) assays: HCV JFH1-AM120-Rluc infected cell lysates containing Rluc protein was mixed with increasing concentrations of tannic acid for ten minutes at room temperature and luciferase activity was measured as described in Materials and Methods. Assays were done in triplicate and experiments done three times. Results are presented as the mean ± standard deviation (SD) (n = 9) of relative light units (RLU). No significant difference (Student’s t-test p>0.05) in Rluc assay results were observed with tannic acid concentrations ≤ 50 μM.
Fig 2.
Tannic acid inhibits HCV infection.
(A) The effect of tannic acid on an infectious HCV cell culture system. Huh7.5 cells in 96 well plates were infected with HCV JFH1-AM120-Rluc for two hours in the presence of the specified concentrations of tannic acid, cells were washed with PBS and incubated for 48 hours. Cells were lysed and luciferase activity was measured as described in Materials and Methods. The results for three experiments are shown as mean RLUs ± SD of triplicate cell culture assays in 96-well plates. The IC50 was 5.8 ± 0.38 μM. (B) The effect of tannic acid on the production of HCV NS3 protein. Huh7.5 cells were infected with JFH1-AM120-Rluc for two hours in the presence of the specified concentrations of tannic acid, cells were washed with PBS and incubated for 48 hours. Cells were lysed and Western blotting was done with anti-HCV NS3 antibodies as described in Materials and Methods. The image represents three different experiments. (C) The effect of tannic acid on the production of HCV RNA. Huh7.5 cells were infected with JFH1-AM120-Rluc for two hours in the presence of the specified concentrations of tannic acid, cells were washed with PBS and incubated for 48 hours. The total RNA was extracted and qPCR assays for HCV RNA were done as described in Materials and Methods. Error bars represent the mean ± SD of results from three experiments.
Fig 3.
Tannic acid inhibits an Early Step of HCV Infection.
Tannic acid 25μM was added to the culture medium of Huh7.5 cells for two hours before infection (pre-treatment), for two hours during infection (during-infection), and for two hours after infection with JFH1-AM120Rluc virus (post infection). Forty-eight hours post infection, cells were lysed and luciferase activity was measured. Infectivity is expressed as a percentage relative to luciferase activity measured in controls without tannic acid. Mock infection was the negative control. Mean values ± SD of three different experiments done in triplicate are presented. Results with a Student’s t-test P value < 0.01 are indicated by an asterisk.
Fig 4.
Tannic acid does not inhibit HCV replication.
(A) The effect of tannic acid on a HCV replicon, that displays intracellular replication events, was determined. HCV1b replicon RNA was electroporated into Huh7.5 cells followed by seeding of cells into six-well plates. After 48 hours of culture cells were trypsinized and seeded into twelve-well plates and tannic acid was added at concentrations of 0 μM, 3.1 μM, 6.3 μM, 12.5 μM and 25 μM and cells were cultured for 48 hours as described in Materials and Methods. Mock electroporation (no HCV1b replicon RNA) of cells provided controls. Cells were lysed and Western blots were done with anti-NS3 monoclonal antibodies as described in Materials and Methods. Experiments were performed three times and representative examples are shown. (B) The levels of NS3 were normalized to β-actin and the ratios of NS3/β-actin relative to 0 μM tannic acid (no inhibitor, 100% level of NS3) were plotted against inhibitor concentrations. The data are presented as mean ± SD (n = 3) of the NS3 levels normalized to β-actin for three independent experiments.
Fig 5.
Tannic acid blocks an early step of HCV entry.
(A) Schematic drawing of the experiment design. Inhibition of HCV JFH1-AM120-Rluc entry into Huh7.5 cells by tannic acid (25 μM) and heparin controls (200 μg/ml) was compared using four different experimental conditions (indicated by I through IV). Virus binding to cells was performed for 1 h at 4°C in the absence (II to IV) or presence (I) of compounds. Subsequently, cells were washed with PBS and shifted to 37°C to allow HCV entry to proceed. Inhibitors were either added directly, at 1,2 or 3 hours as indicated (II, III and IV, respectively). Dotted lines indicate the time that an inhibitor was present; black arrows indicate the addition and removal of the HCV inoculum. (B) Effect of different times of tannic acid or heparin treatment on HCV infection. The experimental design describe in Panel A was followed, luciferase assays were done at 48 hours post infection and measures of infection are expressed relative to HCV infection in the absence of inhibitors. The data are presented as mean ± SD of three experiments done in triplicate. Results with a Student’s t-test P value < 0.01 are indicated by an asterisk. (C) Effect of tannic acid or heparin treatment on HCV binding to cells as measured by HCV RNA. Huh7.5 cells were inoculated with HCV JFH1-AM120-Rluc at a MOI of 10 for one hour at 4°C in presence of 50 μM of tannic acid, 500 μg/ml of heparin or no inhibitor as a control. Cells were washed twice with ice-cold PBS and total RNA was extracted. HCV bound to cells was measured by qPCR analysis of HCV RNA. Relative binding is expressed as the percentage of the control for (100%). Results are shown as the mean ± SD of three different experiments done in triplicate. Results with a P value < 0.01 are indicated by an asterisk. (D) Effect of preincubating HCV with tannic acid on their infectivity. JFH1-AM120-Rluc pre-treated with 50 μM tannic acid for 15 minutes and then diluted to decrease the concentration of tannic acid to 5 μM prior to the inoculation of Huh-7.5 cells. Cells were inoculated for two hours with HCV JFH1-AM120-Rluc was preincubated with tannic acid (as described) or without tannic acid then incubated with (5 μM) or without tannic acid as controls. The virus titers were kept constant in the three different conditions. At 48 hours post infection, cells were lysed and luciferase activity was measured. Infectivity is expressed as the percentage of the control (no tannic acid) which was assigned a 100% value. Mean values ± SD of three independent experiments done in triplicate are presented. Results with a P value < 0.05 are indicated by an asterisk.
Fig 6.
Effect of tannic acid on cell-to-cell spread of HCV.
(A) The effect of tannic acid on cell-to-cell spread of HCV as measured by viral protein staining. Huh7.5 cells were infected with HCV using an MOI of 0.005 at 37°C. The inoculum was removed two hours post infection and replaced with a 1% agarose medium overlay containing 25 μM tannic acid. Controls had no tannic acid. Cells were incubated at 37°C for 72 hours as described in Materials and Methods. Infected cells were labeled by indirect immuno-fluorescence using an anti-HCV NS5A monoclonal antibody (red), nuclei were stained with DAPI (blue) and photographs were taken with a fluorescence microscope as described in Materials and Methods. (B) The mean and standard deviation of infected cells/focus was determined by visual counting of 30 foci present on randomly selected fields of cover slips for each condition. P-values were calculated using the Student’s ttest (asterisk indicates P = 0.000019). Experiments were performed three times and representative examples are shown in A and B.
Fig 7.
Comparison of the effect of gallic and tannic acid on HCV infection.
(A) Effect during inoculation in an HCV cell culture system. Huh7.5 cells were infected with JFH1-AM120-Rluc for two hours in the presence of the specified concentrations of gallic or tannic acid, cells were washed and further incubated for 48 hours as described in Materials and Methods. Cells were lysed and luciferase activity was used to measure HCV infection as in Fig 2. Results are presented as the mean ± SD of relative light units (RLU) from three experiments. (B) Effect after inoculation in an HCV cell culture system. Huh7.5 cells were infected with JFH1-AM120-Rluc for two hours, cells were washed, gallic or tannic acid was added in the specified concentrations and cells were incubated for 48 hours. Cells were lysed and luciferase activity was measured as described previously. Results are presented as the mean ± SD of relative light units (RLU) from three experiments. (C) Effect in an HCV replicon system. HCV1b replicon RNA was electroporated into Huh7.5 cells, seeded into six-well plates and cultured for 48 hours. Cells were trypsinized, seeded in twelve-well plates, cultured overnight and gallic acid at concentrations of 0 μM, 3.1 μM, 6.3 μM, 12.5 μM and 25 μM was added at following day. The cells were cultured with gallic acid for 48 hours prior to analysis. Mock electroporation (no HCV1b replicon RNA) of cells served as controls. Cells were lysed and Western blots done with an anti-NS3 monoclonal antibody as described in Materials and Methods. Experiments were performed three times and representative examples are shown. (D) The levels of HCV NS3 protein were normalized to β-actin and the ratios of NS3/β-actin relative to a 0 μM tannic acid control (designated 100%) were plotted against inhibitor concentrations. The data are presented as mean ± SD (n = 3) of the NS3 levels normalized to β-actin for three independent experiments.