Development of a drug assay system with hepatitis C virus genome derived from a patient with acute hepatitis C
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- Mori, K., Ueda, Y., Ariumi, Y. et al. Virus Genes (2012) 44: 374. doi:10.1007/s11262-012-0712-2
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We developed a new cell culture drug assay system (AH1R), in which genome-length hepatitis C virus (HCV) RNA (AH1 strain of genotype 1b derived from a patient with acute hepatitis C) efficiently replicates. By comparing the AH1R system with the OR6 assay system that we developed previously (O strain of genotype 1b derived from an HCV-positive blood donor), we demonstrated that the anti-HCV profiles of reagents including interferon-γ and cyclosporine A significantly differed between these assay systems. Furthermore, we found unexpectedly that rolipram, an anti-inflammatory drug, showed anti-HCV activity in the AH1R assay but not in the OR6 assay, suggesting that the anti-HCV activity of rolipram differs depending on the HCV strain. Taken together, these results suggest that the AH1R assay system is useful for the objective evaluation of anti-HCV reagents and for the discovery of different classes of anti-HCV reagents.
KeywordsHCVAcute hepatitis CAnti-HCV drug assay systemAnti-HCV activity of rolipram
Hepatitis C virus (HCV) infection frequently causes chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive single-stranded 9.6 kb RNA genome, which encodes a large polyprotein precursor of approximately 3,000 amino acid (aa) residues [1, 2]. This polyprotein is cleaved by a combination of the host and viral proteases into at least 10 proteins in the following order: Core, envelope 1 (E1), E2, p7, non-structural 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B .
Human hepatoma HuH-7 cell culture-based HCV replicon systems derived from a number of HCV strains have been widely used for various studies on HCV RNA replication [3, 4] since the first replicon system (based on the Con1 strain of genotype 1b) was developed in 1999 . Genome-length HCV RNA replication systems (see Fig. 2 for details) derived from a limited number of HCV strains (H77, N, Con1, O, and JFH-1) are also sometimes used for such studies, as they are more useful than the replicon systems lacking the structural region of HCV, although the production of infectious HCV from the genome-length HCV RNA has not been demonstrated to date [3, 4]. Furthermore, these RNA replication systems have been improved enough to be suitable for the screening of anti-HCV reagents by the introduction of reporter genes such as luciferase [3, 4, 6]. We also developed an HuH-7-derived cell culture assay system (OR6) in which genome-length HCV RNA (O strain of genotype 1b derived from an HCV-positive blood donor) encoding renilla luciferase (RL) efficiently replicates . Such reporter assay systems could save time and facilitate the mass screening of anti-HCV reagents, since the values of luciferase correlated well with the level of HCV RNA after treatment with anti-HCV reagents. Furthermore, OR6 assay system became more useful as a drug assay system than the HCV subgenomic replicon-based reporter assay systems developed to date [3, 4], because the older systems lack the Core-NS2 regions containing structural proteins likely to be involved in the events that take place in the HCV-infected human liver. Indeed, by the screening of preexisting drugs using the OR6 assay system, we have identified mizoribine , statins , hydroxyurea , and teprenone  as new anti-HCV drug candidates, indicating that the OR6 assay system is useful for the discovery of anti-HCV reagents.
On the other hand, we previously established for the first time an HuH-7-derived cell line (AH1) that harbors genome-length HCV RNA (AH1 strain of genotype 1b) derived from a patient with acute hepatitis C . In that study, we noticed different anti-HCV profiles of interferon (IFN)-γ or cyclosporine A (CsA) between AH1 and O cells supporting genome-length HCV RNA (O strain) replication . From these results, we supposed that the diverse effects of IFN-γ or CsA were attributable to the difference in HCV strains .
To test this assumption in detail, we first developed an AH1 strain-derived assay system (AH1R) corresponding to the OR6 assay system, and then performed a comparative analysis using AH1R and OR6 assay systems. In this article, we report that the difference in HCV strains causes the diverse effects of anti-HCV reagents, and we found unexpectedly by AH1R assay that rolipram, an anti-inflammatory drug, is an anti-HCV drug candidate.
Materials and methods
IFN-α, IFN-γ, and CsA were purchased from Sigma-Aldrich (St. Louis, MO). Rolipram was purchased from Wako Pure Chemical Industries (Osaka, Japan).
The plasmid pAH1RN/C-5B/PL,LS,TA,(VA)3 was constructed from pAH1 N/C-5B/PL,LS,TA,(VA)3 encoding genome-length HCV RNA clone 2 (See Fig. 2) obtained from AH1 cells , by introducing a fragment of the RL gene from pORN/C-5B into the AscI site before the neomycin phosphotransferase (NeoR) gene as previously described .
The plasmid pAH1RN/C-5B/PL,LS,TA,(VA)3 DNA was linearized by XbaI, and used for RNA synthesis with T7 MEGAscript (Ambion, Austin TX) as previously described .
AH1R and OR6 cells supporting genome-length HCV RNAs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.3 mg/mL of G418 (Geneticin; Invitrogen, Carlsbad, CA). AH1c-cured cells, which were created by eliminating HCV RNA from AH1 cells  by IFN-γ treatment, were also cultured in DMEM supplemented with 10% FBS.
RNA transfection and selection of G418-resistant cells
Genome-length HCV (AH1RN/C-5B/PL,LS,TA,(VA)3) RNA synthesized in vitro was transfected into AH1c cells by electroporation, and the cells were selected in the presence of G418 (0.3 mg/mL) for 3 weeks as described previously .
RL assay for anti-HCV reagents
To monitor the effects of anti-HCV reagents, RL assay was performed as described previously . Briefly, the cells were plated onto 24-well plates (2 × 104 cells per well) in triplicate and cultured with the medium in the absence of G418 for 24 h. The cells were then treated with each reagent at several concentrations for 72 h. After treatment, the cells were subjected to a luciferase assay using the RL assay system (Promega, Madison, WI). From the assay results, the 50% effective concentration (EC50) of each reagent was determined.
Quantification of HCV RNA
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis for HCV RNA was performed using a real-time LightCycler PCR (Roche Applied Science, Indianapolis, IN, USA) as described previously . The experiments were done in triplicate.
IFN-α treatment to evaluate the assay systems
To monitor the anti-HCV effect of IFN-α on AH1R cells, 2 × 104 cells and 5 × 105 cells were plated onto 24-well plates (for luciferase assay) and 10 cm plates (for quantitative RT-PCR assay) in triplicate, respectively, and cultured for 24 h. The cells were then treated with IFN-α at final concentrations of 0, 1, 10, and 100 IU/mL for 24 h, and subjected to luciferase and quantitative RT-PCR assays as described above.
Western blot analysis
The preparation of cell lysates, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunoblotting analysis with a PVDF membrane were performed as described previously . The antibodies used in this study were those against HCV Core (CP11 monoclonal antibody; Institute of Immunology, Tokyo), NS5B, and E2 (generous gifts from Dr. M. Kohara, Tokyo Metropolitan Institute of Medical Science, Japan). Anti-β-actin antibody (AC-15; Sigma, St. Louis, MO, USA) was used as a control for the amount of protein loaded per lane. Immunocomplexes were detected with the Renaissance enhanced chemiluminescence assay (Perkin-Elmer Life Sciences, Boston, MA).
WST-1 cell proliferation assay
The cells were plated onto 96-well plates (1 × 103 cells per well) in triplicate and then treated with rolipram at several concentrations for 72 h. After treatment, the cells were subjected to the WST-1 cell proliferation assay (Takara Bio, Otsu, Japan) according to the manufacturer’s protocol. From the assay results, the 50% cytotoxic concentration (CC50) of rolipram was estimated. The selective index (SI) value of rolipram was also estimated by dividing the CC50 value by the EC50 value.
RT-PCR and sequencing
To amplify the genome-length HCV RNA, RT-PCR was performed separately in two fragments as described previously [7, 15]. Briefly, one fragment covered from 5′-untranslated region to NS3, with a final product of approximately 6.2 kb, and the other fragment covered from NS2 to NS5B, with a final product of approximately 6.1 kb. These fragments overlapped at the NS2 and NS3 regions and were used for sequence analysis of the HCV open reading frame (ORF) after cloning into pBR322MC. PrimScript (Takara Bio) and KOD-plus DNA polymerase (Toyobo, Osaka, Japan) were used for RT and PCR, respectively. The nucleotide sequences of each of the three independent clones obtained were determined using the Big Dye terminator cycle sequencing kit on an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA).
Differences between AH1R and OR6 cell lines were tested using Student’s t test. P values <0.05 were considered statistically significant.
Development of a luciferase reporter assay system that facilitates the quantitative monitoring of genome-length HCV-AH1 RNA replication
We first demonstrated that AH1R cells expressed sufficient levels of HCV proteins (Core, E2, and NS5B) by Western blot analysis for the evaluation of anti-HCV reagents, and the expression levels were almost equivalent to those in OR6 cells (Fig. 1b). In this analysis, we confirmed that the size of the E2 protein in AH1R cells was 7 kDa larger than that in OR6 cells (Fig. 1b), as observed previously . This result indicates that AH1R cells express AH1 strain-derived E2 protein possessing two extra N-glycosylation sites . We next demonstrated good correlations between the levels of RL activity and HCV RNA in AH1R cells (Fig. 1c), as we previously demonstrated in OR6 cells treated with IFN-α for 24 h . These correlations indicate that AH1R cells were as useful as OR6 cells as a luciferase assay system.
Aa substitutions detected in genome-length HCV RNA in AH1R cells
Comparison between the AH1R and OR6 assay systems regarding the sensitivities to IFN-α, IFN-γ, and CsA
Using quantitative RT-PCR analysis, we previously examined the anti-HCV activities of IFN-α, IFN-γ, and CsA in AH1 and O cells, and noticed different anti-HCV profiles of IFN-γ and CsA between AH1 and O cells . In that study, AH1 cells seemed to be more sensitive than the O cells to CsA (significant difference was observed when 0.063, 0.12, or 0.25 μg/mL of CsA was used). Conversely, AH1 cells seemed to be less sensitive than the O cells to IFN-γ (significant difference was observed when 1 or 10 IU/mL of IFN-γ was used). However, we were not able to determine precisely the EC50 values of these reagents, because of the unevenness of the data obtained by RT-PCR.
Anti-HCV activity of rolipram was clearly observed in the AH1R assay, but not in the OR6 assay
In the present study, we developed for the first time a drug assay system (AH1R), derived from the HCV-AH1 strain (from a patient with acute hepatitis C), in which HCV-AH1 RNA is efficiently replicated. Using this system, we found that rolipram, an anti-inflammatory drug, had potential anti-HCV activity. This potential had not been detected by preexisting assay systems such as OR6, in which HCV-O RNA was derived from an HCV-positive blood donor. Since an HCV replicon harboring the sAH1 cell line, the parent of the AH1R cell line, was obtained from OR6-cured cells , the divergence in rolipram’s effects between AH1R and OR6 cells is probably attributable to the difference in HCV strains rather than to the difference in cell clones. Indeed, rolipram’s anti-HCV activity was not observed in another ORL8 assay system (O strain), which was recently developed using a new hepatoma Li23 cell line (data not shown) . Therefore, we propose that multiple assay systems derived from different HCV strains are required for the discovery of anti-HCV reagents such as rolipram or for the objective evaluation of anti-HCV activity.
Comparative evaluation analysis of anti-HCV activities of IFN-α, IFN-γ, and CsA using AH1-strain-derived AH1R and O-strain-derived OR6 assay systems demonstrated that each of these anti-HCV reagents showed significantly diverse antiviral effects between the two systems. Regarding IFN-γ and CsA, the present results obtained using a luciferase reporter assay fully supported our previous findings  using quantitative RT-PCR analysis. However, in the present analysis, we noticed that IFN-α also showed significantly diverse effects (especially at less than 1 IU/mL) between the AH1R and OR6 assays. The differences in IFN-α sensitivity may be attributable to the difference in aa sequences in the IFN sensitivity-determining region (ISDR; aa 2209-2248 in the HCV-1b genotype), in which aa substitutions correlate well with IFN sensitivity in patients with chronic hepatitis C , because the AH1 strain possesses three aa substitutions (T2217A, H2218R, and A2224 V) in ISDR, whereas the O strain possesses no aa substitutions. However, no report has demonstrated the correlation between IFN sensitivity and the substitution numbers in ISDR using the cell culture-based HCV RNA replication system.
Alternatively, Akuta et al.  reported that aa substitutions at position 70 and/or position 91 in the HCV Core region of patients infected with the HCV-1b genotype are pretreatment predictors of null virological response (NVR) to pegylated IFN/ribavirin combination therapy. In particular, substitutions of arginine (R) by glutamine (Q) at position 70, and/or leucine (L) by methionine (M) at position 91, were common in NVR. The patients with position-70 substitutions often showed little or no decrease in HCV RNA levels during the early phase of IFN-α treatment . Regarding this point, it is interesting that position 70 in the AH1 strain is R (wild type) and that in the O strain is Q (mutant type), whereas position 91 is L (wild type) in both strains. Therefore, wild-type R in position 70 of the AH1 strain may contribute to the high sensitivity to IFN-α in the AH1R assay. Regarding positions 70 and 91 of the HCV Core, it is noteworthy that, among all of the HCV strains used thus far to develop HCV replicon systems, only the AH1 strain possesses double wild-type aa (data not shown). Therefore, the AH1R assay system may be useful for further study of sensitivity to IFN/ribavirin treatment.
The anti-HCV activity of rolipram, which is currently used as an anti-inflammatory drug, is interesting, although its anti-HCV mechanism is unclear. As a selective PDE4 inhibitor , rolipram may attenuate fibroblast activities that can lead to fibrosis and may be particularly effective in the presence of transforming growth factor (TGF)-β1-induced fibroblast stimulation . On the other hand, HCV enhances hepatic fibrosis progression through the generation of reactive oxygen species and the induction of TGF-β1 . Taken together, the previous and present results suggest that rolipram may inhibit both HCV RNA replication and HCV-enhanced hepatic fibrosis. However, it is unclear that rolipram shows anti-HCV activity against the majority of HCV strains, because rolipram has been effective for AH1 strain, but not for O strain. Although rolipram’s anti-HCV activity would be HCV-strain-specific, it is not clear which HCV strain is the major type regarding the sensitivity to rolipram. Since developed assay systems using genome-length HCV RNA-replicating cells are limited to several HCV strains including O and AH1 strains to date, further analysis using the assay systems of other HCV strains will be needed to clarify this point.
In this study, we demonstrated that the AH1R assay system, which was for the first time developed using an HCV strain derived from a patient with acute hepatitis C, showed different sensitivities against anti-HCV reagents in comparison with assay systems in current use, such as OR6 assay. Therefore, AH1R assay system would be useful for various HCV studies including the evaluation of anti-HCV reagents and the identification of antiviral targets.
This study was supported by grants-in-aid for research on hepatitis from the Ministry of Health, Labor, and Welfare of Japan. K. M. was supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science.