The cyclophilin inhibitor SCY-635 disrupts hepatitis C virus NS5A-cyclophilin A complexes

Abstract

The nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication both in vitro and in vivo and represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arose in vitro under SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA.

Fig 1

(A) Ten micrograms of in vitro-transcribed subgenomic Con1 or genomic JFH-1 or RNA was electroporated into Huh7 cells in the presence of increasing concentrations of SCY-635 (0.3125, 0.625, 1.25, and 2.5 μM). At the indicated time points, intracellular HCV RNA was analyzed via reverse transcription-quantitative PCR; the results are presented as genome equivalents (GE) per microgram of total RNA. (B) After day 7, JFH-1-electroporated cells were fixed and stained with anti-NS5A human IgG (E1) (1:1,000) for 2 h at room temperature. After 3 washes, cells were incubated for 1 h at room temperature with secondary Alexa Fluor 555-conjugated goat anti-human IgG (1:1,000). Cells were washed 3 times; cells and plates were stored in the dark at 4°C until pictures were taken. The number of JFH-1-positive foci was counted by fluorescence microscopy. Panel C is the same as panel B, except that typical fluorescence microscopy immunostainings of JFH-1-positive foci are presented. These results are representative of 2 independent experiments. Error bars represent standard errors of triplicate determinations.

Fig 2

Huh7-Con1 cells were passaged eight consecutive times without G418 in the presence or absence of two concentrations of CsA, SCY-635, or telaprevir (0.5 and 1 μM). At each passage, five clones were collected, and RNA was extracted and analyzed by RT-qPCR for replicon content. After eight passages, drugs were removed and cells were analyzed for viral RNA content by qPCR for two additional passages. Data are representative of two independent experiments. Error bars represent standard errors of duplicate determinations.

Fig 3

(A) GST-CypA or GST-CypB (1 μg) was mixed with Con1 NS5A-His (200 ng) for 3 h at 4°C in the presence or absence of increasing concentrations of SCY-635. Glutathione beads were added to the GST-Cyp–NS5A mixture for 30 min at 4°C and washed. Bound material was eluted and analyzed by Western blotting using anti-His and anti-GST antibodies. (B) Plates were coated with GST-CypA or GST-CypB (10 μg/ml) for 16 h at 4°C. Recombinant Con1 NS5A-His (1 ng/ml) was added to wells for 16 h at 4°C. Captured NS5A-His was detected using mouse anti-His antibodies (1 μg/ml) followed by anti-mouse HRP-conjugated antibodies (1:1,000 dilution). Adsorbed levels of GST-CypA or GST-CypB were monitored using mouse anti-GST IgG (1 μg/ml) and rabbit anti-CypA or mouse anti-CypB antibodies (1 μg/ml) followed by anti-mouse and anti-rabbit HRP-conjugated antibodies (1:1,000 dilution). These results are representative of two independent experiments. Error bars represent standard errors of triplicate determinations.

Fig 4

(A) Same as Fig. 2A, except that GST-CypA (1 μg) was mixed with recombinant NS5A-His (200 ng) derived from genotypes 1a, 1b, 2a, and 2b with increasing concentrations of SCY-635 (from 0.3125 to 0.625 μM) for 3 h at 4°C. Glutathione beads were added to the GST-CypA–NS5A mixture for 30 min at 4°C and washed. Bound material was eluted and analyzed by Western blotting using anti-His and anti-GST antibodies. (B) Same as Fig. 2B. These results are representative of two independent experiments. (C) To calculate SCY-635 and CsA IC₅₀s, NS5A-His (1 ng/ml) was added to wells in the presence of increasing concentrations of SCY-635 or CsA (0.078, 0.156, 0.3125, 0.625, 1.25, and 2.5 μM) for 16 h at 4°C. Captured NS5A-His was subsequently detected using mouse anti-His and rabbit anti-mouse HRP-conjugated antibodies. The amounts of SCY-635 necessary to inhibit 50% of NS5A binding in the absence of drug were calculated (IC₅₀). Results (duplicate determinations) are representative of two independent experiments. Error bars represent standard errors of triplicate determinations.

Fig 5

Wild-type and D320E and Y321N mutant Con1 RNAs were electroporated into Huh7.5.1 cells, and the HCV RNA replication was monitored over time by measuring luciferase activity in cell lysates. SCY-635 was added to cells at the time of the cell seeding, immediately after the electroporation. Results are representative of two independent experiments. Error bars represent standard errors of triplicate determinations.

Fig 6

(A) Same as Fig. 4A, except that GST-CypA (1 μg) was mixed with recombinant wild-type or D320E or Y321N mutant NS5A-His (200 ng) with increasing concentrations of SCY-635 (from 0.3125 to 0.625 μM) for 3 h at 4°C. Glutathione beads were added to the GST-CypA–NS5A mixture for 30 min at 4°C and washed. Bound material was eluted and analyzed by Western blotting using anti-His and anti-GST antibodies. (B) Same as Fig. 4B. These results are representative of two independent experiments. Error bars represent standard errors of triplicate determinations.

Fig 7

Wild-type and D320E and Y321N mutant Con1 RNAs were electroporated into parental, CypB-KD, or CypA-KD Huh7.5.1 cells treated with or without SCY-635 (1 μM), and HCV RNA replication was monitored over time by measuring luciferase activity in cell lysates. Results are representative of two independent experiments. Error bars represent standard errors of triplicate determinations.