Inhibition of both protease and helicase activities of hepatitis C virus NS3 by an ethyl acetate extract of marine sponge Amphimedon sp

Abstract

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC₅₀ values of 1.5 and 24.9 µg/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC₅₀ values of 18.9 and 10.9 µg/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

Figure 1. Effect of the extract prepared from a marine sponge on viral replication in the replicon cell line derived from viral genotype 1b.

(A) Amphimedon sp. belongs to a marine sponge. The ethyl acetate fraction prepared from the marine organism was designated C-29EA in this study. (B) The Huh7 cell line, including the subgenomic replicon RNA of genotype 1b strain Con1, was incubated in medium containing various concentrations of C-29EA or DMSO (0). Luciferase and cytotoxicity assays were carried out as described in Materials and Methods. Error bars indicate standard deviation. The data represent three independent experiments.

Figure 2. Effect of C-29EA extract on viral replication in the replicon cell line derived from viral genotype 2a.

(A) The Huh7 cell line, including the subgenomic replicon RNA of genotype 2a strain JFH1, was incubated in medium containing various concentrations of C-29EA or DMSO (0). Luciferase and cytotoxicity assays were carried out as described in Materials and Methods. (B) The Huh7 OK1 cell line infected with HCVcc JFH1 was incubated with various concentrations of C-29EA or DMSO (0). The virus titers were determined by a focus-forming assay. The significance of differences in the means was determined by Student’s t-test. (C) Amounts of viral RNA and core protein were estimated by qRT-PCR and ELISA, respectively. Error bars indicate standard deviation. The data represent three independent experiments. Treatment with DMSO corresponds to ‘0′.

Figure 3. Effect of C-29EA on expression of viral proteins in replicon cell lines.

The Huh7 replicon cell lines derived from genotype 1b (A) and 2a (B) were incubated with C-29EA at 37°C for 72 h. The treated cells were harvested and then subjected to Western blotting. Treatment with DMSO corresponds to ‘0′. The bicistronic gene is transcribed under the control of the elongation factor 1α (EF1α) promoter. The upstream cistron encoding Renilla luciferase (RLuc) is translated by a cap-dependent mechanism. The downstream cistron encodes the fusion protein (Feo), which consists of the firefly luciferase (Fluc) and neomycin phosphotransferase (Neo^r), and is translated under the control of the EMCV IRES (C) or HCV IRES (D). The Huh7 cell line transfected with the plasmid (each above the panel in C and D) was established in the presence of G418. The cells were incubated for 72 h without (control) and with 15 µg/ml of C-29EA. Firefly or Renilla luciferase activity was measured by the method described in Materials and Methods and was normalized by the protein concentration. F/R: relative ratio of firefly luciferase activity to Renilla luciferase activity. F/R is presented as a percentage of the control condition. Error bars indicate standard deviation. The data represent three independent experiments.

Figure 4. Effect of C-29EA on interferon signaling pathway.

The Huh7 replicon cell line of genotype 1b was treated without (lane 3) or with 1, 10, 100, or 1000 U/mL interferon-alpha 2b (lanes 4–7), and 1.5 or 15 µg/ml C-29EA (lanes 1–2) for 48 h. Treatment with DMSO corresponds to ‘0′. The mRNAs of 2′, 5′-OAS, and GAPDH as an internal control were detected by RT-PCR. Error bars indicate standard deviation. The data represent three independent experiments.

Figure 5. Effect of C-29EA on unwinding activity of NS3 helicase.

(A) NS3 helicase activity was measured by PET assay. The reactions were carried out in the absence or presence of C-29EA. Helicase activity in the absence of C-29EA was defined as 100% helicase activity. Treatment with DMSO corresponds to ‘0′. The data are presented as the mean ± standard deviation for three replicates. (B) The unwinding activity of NS3 helicase was measured by an RNA unwinding assay using radioisotope-labeled RNA. The heat-denatured single-strand RNA (26-mer) and the partial duplex RNA substrate were applied to lanes 1 and 2, respectively. The duplex RNA was reacted with NS3 (300 nM) in the presence of C-29EA (lanes 4–9, 16–250 µg/ml). The resulting samples were subjected to native polyacrylamide gel electrophoresis. Treatment with DMSO corresponds to ‘0′.

Figure 6. Effect of C-29EA on ATPase and RNA-binding activities of NS3 helicase.

(A) The reaction mixtures were incubated with [γ-³²P] ATP as described in Materials and Methods. The reaction mixtures were subjected to thin-layer chromatography. The start positions and migrated positions of ATP and free phosphoric acid are indicated as ‘Origin’, ‘³²P-ATP’, and ‘³²P-Pi’, respectively, on the right side of the figure. The data represent three independent experiments. Treatment with DMSO corresponds to ‘0′. (B) Gel mobility shift assay for RNA-binding activity of NS3 helicase. The reaction was carried out with 0.5 nM labeled ssRNA at the indicated concentrations of C-29EA or DMSO. The reaction mixture was subjected to gel mobility shift assay. (C) The relative RNA-binding ability was calculated with band densities in each lane and presented as a percentage of RNA-NS3 in the total density. The data represent three independent experiments. Treatment with DMSO corresponds to ‘0′.

Figure 7. Effect of C-29EA on the activity of NS3 serine protease.

NS3/4A serine protease was mixed with various concentrations of C-29EA or DMSO (0) in the reaction mixture and then incubated at 37°C for 120 min. The initial velocity at each concentration of C-29EA was calculated during 120 min reaction. The initial velocity in the absence of C-29EA was defined as 100% of relative protease activity. The data are presented as the mean ± standard deviation for three replicates.

Figure 8. Effect of C-29EA on the antiviral activity of interferon-alpha.

(A) The Huh7 cell line, including the subgenomic replicon RNA of genotype 1b strain Con1, was incubated in medium containing various concentrations of C-29EA or DMSO (0) in the presence or the absence of interferon-alpha. Luciferase assay were carried out as described in Materials and Methods. Error bars indicate standard deviation. The data represent three independent experiments. (B) Isobole plots of 90% inhibition of HCV replication. The broken line indicates the additive effect in the isobologram.