Diverse effects of cyclosporine on hepatitis C virus strain replication

Abstract

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.

Figures

FIG. 1.

Schematic representation of the constructs of HCV subgenomic and genome-length replicon RNA. On the left, the constructs of each replicon RNA are shown. HCV strains, as well as genotypes from which the replicon RNA sequences are derived, are indicated in the second column. The names of replicon cell clones established with each replicon RNA are in the third column. The sensitivity to CsA of each replicon RNA revealed in this study is summarized in the fourth column. The replicon RNAs comprise the HCV 5′UTR, including HCV IRES, the neomycin phosphotransferase gene (Neor), EMCV IRES, or HCV IRES, the coding region for HCV proteins NS3 to NS5B (subgenomic) or core to NS5B (genome length or full genome), and HCV 3′UTR. MH-14 (NN/1b/SG), #50-1 (NN/1b/SG), MH14#W31 (NN/1b/SG), SN1 (Con1/1b/SG), sO (O/1b/SG), JFH1#4-1 (JFH1/2a/SG), and JFH1#2-3 (JFH1/2a/SG) cells carry subgenomic replicons, while NNC#2 (NN/1b/FL), SN1A#2 (Con1/1b/FL), and SNC#7 (Con1/1b/FL) cells have genome-length replicons. NNC#2 (NN/1b/FL) and SNC#7 (Con1/1b/FL) cells contain the replicon RNA without EMCV IRES.

FIG. 2.

CsA suppressed the replication of HCV genome, irrespective of the presence of the structural proteins. (A) Detection of HCV proteins from NNC#2 (NN/1b/FL) genome-length replicon. Core (a), E2 (b), NS3 (c), NS5A (d), NS5B (e), and tubulin (f) in Huh-7, NNC#2 (NN/1b/FL), and MH-14 (NN/1b/SG) cells analyzed by immunoblot analysis are shown. (B) HCV RNA in Huh-7, NNC#2 (NN/1b/FL), and MH-14 (NN/1b/SG) cells quantified by real-time RT-PCR analysis. The data represent the means of three independent experiments. (C) CsA decreased the production of HCV proteins in NNC#2 (NN/1b/FL), as well as in MH-14 (NN/1b/SG) cells. After treatment with 1-μg/ml CsA (+) for 5 days or without treatment (−), total-cell lysates of NNC#2 (NN/1b/FL) and MH-14 (NN/1b/SG) cells, together with Huh-7 cells as a negative control, were recovered to examine the production of HCV NS5A (top), NS5B (middle), and tubulin as an internal control (bottom) by immunoblot analysis. The same result was obtained at day 7 after treatment. (D) The sensitivity to CsA of HCV genome-length replicon was almost the same as that of the subgenomic replicon. HCV RNA was quantified by real-time RT-PCR analysis using total RNA from NNC#2 (NN/1b/FL), SN1A#2 (Con1/1b/FL), and SNC#7 (Con1/1b/FL) cells treated with various concentrations of CsA for 7 days. The relative amount of HCV RNA was plotted against the concentration of CsA (in micrograms per milliliter). (E) Effect of CsA on cell proliferation. NNC#2 (NN/1b/FL) cells were treated with various amount of CsA for 7 days. Cell numbers were counted, and cell numbers relative to those of cells without treatment were plotted against the concentration of CsA.

FIG. 3.

Replication of a genotype 2a strain, JFH1, was less sensitive to CsA. (A) Sensitivity to CsA of HCV genotype 1b and JFH1 replicons. SN1 (Con1/1b/SG), MH-14 (NN/1b/SG), sO (O/1b/SG), #50-1 (NN/1b/SG), JFH1#4-1 (JFH1/2a/SG), and JFH1#2-3 (JFH1/2a/SG) cells, carrying HCV subgenomic replicon, were treated with 1-μg/ml CsA for 7 days. HCV RNA titers were quantified by real-time RT-PCR analysis, and the relative amounts are shown. The bars represent the means of three independent experiments. White bars, no treatment; black bars, 1-μg/ml CsA. The numbers above the black bars indicate fold difference of the titer with 1-μg/ml CsA treatment compared to no treatment. (B) Levels of NS3 and tubulin as an internal control in MH14#W31 (NN/1b/SG) and JFH1#4-1 (JFH1/2a/SG) cells without (−) or with (+) 1-μg/ml CsA treatment for 5 days were detected by immunoblot analysis. (C) HCV RNA was quantified and plotted as described in the legend to Fig. 2D with genotype 1b replicon cells such as MH-14 (NN/1b/SG), #50-1 (NN/1b/SG), MH14#W31 (NN/1b/SG), SN1 (Con1/1b/SG), and sO (O/1b/SG) cells and JFH1-carrying replicon cells such as JFH1#4-1 (JFH1/2a/SG) and JFH1#2-3 (JFH1/2a/SG) cells. (D) Effect of CsA on cell proliferation. The growth of MH-14 (NN/1b/SG) and JFH1#4-1 (JFH1/2a/SG) cells were examined as described in the legend for Fig. 2E.

FIG. 4.

JFH1 replication was less sensitive to a CsA derivative, NIM811. (A) MH14#W31 (NN/1b/SG) and JFH1#4-1 (JFH1/2a/SG) cells were treated with 0.5-μg/ml NIM811 for 7 days. HCV RNA titers were quantified as described in the legend to Fig. 3A. White bars, no treatment; black bars, 0.5-μg/ml NIM811. (B and C) HCV RNA in replicon cells treated with various concentrations of NIM811 (B) or PSC833 (C) for 7 days was quantified and plotted against the concentration of NIM811 (B) or PSC833 (C) (in micrograms per milliliter) as described in the legend to Fig. 3C.

FIG. 5.

Interaction of HCV NS5B with CyPB in the JFH1 replicon. (A) Coimmunoprecipitation of endogenous CyPB with NS5B. Lysates from MH14#W31 (NN/1b/SG), JFH1#4-1 (JFH1/2a/SG), and Huh-7 cells as a negative control were used for immunoprecipitation with normal mouse immunoglobulin G (IgG) or anti-NS5B antibody (NS5B), followed by immunoblot analysis with either anti-CyPB (top) or anti-NS5B antibodies (bottom). IP, antibodies used for immunoprecipitation. (B) The interaction of CyPB with NS5B in JFH1 replicon was disrupted by CsA treatment. Coimmunoprecipitation between CyPB and NS5B was analyzed with MH14#W31 (NN/1b/SG) or JFH1#4-1 (JFH1/2a/SG) cells treated without CsA (lanes 1 and 5) or with CsA (0.3 μg/ml in lanes 2 and 6, 1 μg/ml in lanes 3 and 7, and 3 μg/ml in lanes 4 and 8).

FIG. 6.

CyPB in HCV replication of genotype 1b and JFH1. (A) Expression level of endogenous CyPB protein (top) and tubulin as an internal control (bottom) in MH14#W31 (NN/1b/SG), SN1 (Con1/1b/SG), sO (O/1b/SG), JFH1#4-1 (JFH1/2a/SG), and Huh-7 cells. (B) Knockdown of endogenous CyP proteins. MH14#W31 (NN/1b/SG) and JFH1#4-1 (JFH1/2a/SG) cells were transfected with siRNA specific for CyPA (si-CyPA), CyPB (si-CyP), a broad range of CyP subtypes [si-CyP(broad)], or a randomized siRNA (si-control). At 72 h posttransfection, CyPA (top), CyPB (middle) and tubulin as an internal control (bottom) were detected in total cell lysates of MH14#W31 (NN/1b/SG) (left) and JFH1#4-1 (JFH1/2a/SG) (right) cells by immunoblot analysis. (C) Depletion of CyPB did not affect HCV replication of JFH1 replicon. At 5 days posttransfection, HCV RNA titers in MH14#W31 (NN/1b/SG) (left) and JFH1#4-1 (JFH1/2a/SG) (right) cells were quantified by real-time RT-PCR analysis. no treatment, treatment with only the transfection reagent in the absence of siRNA. (D) Effect of siRNA on cell proliferation. Cell numbers of MH14W#31 (NN/1b/SG) and JFH1#4-1 (JFH1/2a/SG) cells treated with siRNA for 5 days were counted. Relative cell numbers were indicated.

FIG. 7.

RNA binding capacity of JFH1 NS5B was independent of CyPB. (A) An RNA-protein binding precipitation assay was performed using MH14#W31 (NN/1b/SG) cells (lanes 1 to 6) and JFH1#4-1 (JFH1/2a/SG) cells (lanes 7 to 12) as described in Materials and Methods. MH14#W31 (NN/1b/SG) and JFH1#4-1 (JFH1/2a/SG) cells preincubated without (lanes 1, 3, 5, 7, 9, and 11) or with (lanes 2, 4, 6, 8, 10, and 12) CsA were treated with digitonin, followed by digestion with proteinase K to isolate the replication complex. This fraction was then incubated with poly(U) RNA-Sepharose (lanes 5, 6, 11, and 12) or protein G-Sepharose as a negative control (lanes 3, 4, 9, and 10). Precipitates were detected by immunoblot analysis with anti-NS5B antibody. INP, one-sixth of the amount of cell lysate used in the precipitation assay; G and pU, samples with protein G-Sepharose and poly(U)-Sepharose, respectively. (B) An in vitro RNA binding assay was performed as described in Materials and Methods. In vitro-synthesized NS5B of MH-14 (lanes 1 to 6) or JFH1 (lanes 7 to 12) with the rabbit reticulocyte lysate in the presence of [35S]methionine was incubated with protein G-Sepharose (lanes 2 and 8) or poly(U)-Sepharose in the absence (lanes 3 and 9) or presence of various amounts of purified recombinant GST-CyPB (2 ng in panels 4 and 10, 10 ng in panels 5 and 11, and 50 ng in panels 6 and 12). The resultant precipitates were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by the detection of radiolabeled protein. (C) The density of the bands of NS5B in the RNA binding fraction was quantified and plotted against the amount of the recombinant GST-CyPB (in nanograms). Solid line, NS5B of MH-14; faint line, NS5B of JFH1.

FIG. 8.

Amino acid sequence alignment of NS5B encoded by HCV strains NN, Con1, O, and JFH1. The numbers above the sequence indicate the amino acid numbers. Conserved residues are shown by dashes. The region spanning 521 to 591 aa, which is involved in the interaction with CyPB, is boxed.