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. 2009 Oct;100(10):1943-50.
doi: 10.1111/j.1349-7006.2009.01263.x. Epub 2009 Jun 26.

Identification of cellular and viral factors related to anti-hepatitis C virus activity of cyclophilin inhibitor

Kaku Goto  1 , Koichi Watashi, Daisuke Inoue, Makoto Hijikata, Kunitada Shimotohno
Affiliations

Identification of cellular and viral factors related to anti-hepatitis C virus activity of cyclophilin inhibitor

Kaku Goto et al. Cancer Sci. 2009 Oct.

Abstract

We have so far reported that an immunosuppressant cyclosporin A (CsA), a well-known cyclophilin (CyP) inhibitor (CPI), strongly suppressed hepatitis C virus (HCV) replication in cell culture, and that CyPB was a cellular cofactor for viral replication. To further investigate antiviral mechanisms of CPI, we here developed cells carrying CsA-resistant HCV replicons, by culturing the HCV subgenomic replicon cells for 4 weeks in the presence of CsA with G418. Transfection of total RNA from the isolated CsA-resistant cells to naïve Huh7 cells conferred CsA resistance, suggesting that the replicon RNA itself was responsible for the resistant phenotype. Of the identified amino acid mutations, D320E in NS5A conferred the CsA resistance. The replicon carrying the D320E mutation was sensitive to interferon-alpha, but was resistant to CsA and other CPIs including NIM811 and sanglifehrin A. Knockdown of individual CyP subtypes revealed CyP40, in addition to CyPA and CyPB, contributed to viral replication, and CsA-resistant replicons acquired independence from CyPA for efficient replication. These data provide important evidence on the mechanisms underlying the regulation of HCV replication by CyP and for designing novel and specific anti-HCV strategies with CPIs.

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Figures

Figure 1
Figure 1
Schematic diagram outlining the production of individual cell clones carrying hepatitis C virus (HCV) subgenomic replicons. MH14#12 cells, carrying wild‐type HCV subgenomic repicon, were treated with 2 µg/mL cyclosporin A (CsA) in the presence of 1000 µg/mL G418 and CsR#11 cells were selected. Total RNA was extracted from CsR#11 cells and transduced into naïve Huh7 cells to select CsR#11‐3 cells, and sequencing of the replicon RNA in CsR#11 cells identified mutations, Q86R in NS3, I252T in NS3, and D320E in NS5A. Site‐directed mutagenesis followed by in vitro RNA synthesis generated HCV replicon RNA carrying Q86R, D320E, Q86R/D320E, and Q86R/I252T/D320E mutations. Transduction of the RNA into naïve Huh7 cells resulted in the production of Q86R, D320E, Q86R/D320E, and Q86R/I252T/D320E cells. The sensitivity of each replicon clone to CsA is presented as ‘CsA‐resistant’ or ‘CsA‐sensitive’.
Figure 2
Figure 2
Cyclophilin (CyP) inhibitor (CPI)‐resistant colony emergence. MH14#12 cells were treated either without (left panel) or with 2 µg/mL CPIs, cyclosporin A (CsA) (middle panel), and NIM811 (right panel), in the presence of 700 µg/mL G418. Following 4 weeks in culture, cells were fixed and stained with crystal violet.
Figure 3
Figure 3
Cyclosporin A (CsA) responses of the hepatitis C virus (HCV) replicon clones surviving the selection with CsA and G418. MH14#12 cells and three MH14#12‐derived cell clones that survived double selection with G418 and CsA, CsR#4, CsR#10, and CsR#11, were treated with 1 and 3 µg/mL CsA for 7 days, and the HCV‐RNA titers were measured by real‐time RT‐PCR. The data represent the percentage of HCV‐RNA level in cells either untreated or treated with CsA, and the dots represent the means of three independent experiments.
Figure 4
Figure 4
Hepatitis C virus (HCV) RNA alteration contributed to cyclosporin A (CsA)‐resistance. Total RNA extracted from CsA‐resistant CsR#11 cells or that from wild‐type MH14#12 cells as a control was transfected into Huh7 cells. Colonies established after 4‐week selection with G418 were isolated, propagated individually, and tested for CsA response. Three cell clones derived from MH14#12 cells, MH14#12‐1, MH14#12‐4, and MH14#12‐5 cells, and three cell clones from CsR#11 cells, CsR#11‐2, CsR#11‐3, and CsR#11‐5 cells, were treated with 1 and 3 µg/mL CsA for 7 days. HCV‐RNA titers were quantified by real‐time RT‐PCR analysis. The dots represent the means of three independent experiments.
Figure 5
Figure 5
The amino acid mutation D320E in NS5A conferred the cyclosporin A (CsA)‐resistance to hepatitis C virus (HCV) replicons. (a) Colony formation assay for replicons carrying mutations. Five‐microgram replicon RNA carrying the mutation(s), Q86R in NS3, I252T in NS3, D320E in NS5A, Q86R and D320E, or Q86R, I252T and D320E, or wild‐type RNA transcribed in vitro were transduced into Huh7 cells. After culture with G418 for 4 weeks, colonies were stained with crystal violet. (b) Cell clones with replicons carrying indicated mutations were treated with 1 and 3 µg/mL CsA for 7 days. HCV‐RNA titers were quantified by real‐time RT‐PCR analysis. The dots represent the means of three independent experiments. MH14#12‐4, wild‐type replicon; Q86R‐1 and Q86R‐4, replicon with Q86R mutation; D320E‐1 and D320E‐2, replicon with D320E mutation; Q86R/D320E‐2 and Q86R/D320E‐3, replicon with both Q86R and D320E mutations; Q86R/I252T/D320E‐1 and Q86R/I252T/D320E‐3, replicon with all three mutations, Q86R, I252T, and D320E.
Figure 6
Figure 6
Cyclosporin A (CsA)‐resistant replicons demonstrated cross resistance to additional cyclophilin (CyP) inhibitors, NIM811 and sanglifehrin A (SFA), but not interferon (IFN)‐α. MH14#12‐1, MH14#12‐4, D320E‐1 and D320E‐2 cells were treated with NIM811 at 0.1, 0.3, 0.5, 0.7 and 1 µg/mL (a), and MH14#12‐1, MH14#12‐4, Q86R/I252T/D320E‐1, and Q86R/I252T/D320E‐3 cells were treated with 1 µg/mL NIM811 (b), 1 µg/mL SFA (c), and 100 and 300 IU/mL IFN‐α (d) for 7 days. The amounts of hepatitis C virus (HCV) RNA were quantified by real‐time RT‐PCR analysis. The data represent the means of three independent experiments.
Figure 7
Figure 7
Cyclophilin (CyP) subtypes related to anti‐hepatitis C virus (HCV) effect of CyP inhibitor. MH14#12‐derived cells, MH14#12‐1 and MH14#12‐4 cells, and CsR#11‐2 and CsR#11‐3 cells, were transfected with siRNAs specific for CyPC (siCyPC), CyPE (siCyPE), CyPF (siCyPF), and CyPG (siCyPG) (a); or those specific for CyPA (siCyPA‐161, siCyPA‐285, and siCyPA‐459) and CyPB (siCyPB‐1 and siCyPB‐2) (c); or those specific for CyP40 (siCyP40‐3 and siCyP40‐4) (f); or randomized siRNA controls (siControl#1 and siControl#3). D320E cells were also transfected with the above siRNAs specific for either CyPA or CyPB (e). At 5 days post‐transfection, the levels of HCV‐RNA were quantified by real‐time RT‐PCR analysis. The mRNA levels of individual CyP subtypes, CyPC, CyPE, CyPF, and CyPG (CyPX corresponds to each CyP subtype indicated on the left side of the panels) (b), or CyPA and CyPB (d), or CyP40 (g) were measured using glyceraldehydes‐3‐phosphate dehydrogenase (GAPDH) as internal controls by RT‐PCR analysis at 5 days post‐transfection. The data represent the means of three independent experiments.
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