The isomerase active site of cyclophilin A is critical for hepatitis C virus replication

Abstract

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.

FIGURE 1.

Respective contribution of Cyp members to HCV replication. A, Huh7 cells containing the subgenomic HCV Con1 replicon were transfected with an irrelevant control siRNA or siRNAs that target CypA (siRNA CypA), CypB (siRNA CypB), CypC (siRNA CypC), or CypD (siRNA CypD). The cells were washed 24 h post-transfection. Seven days post-transfection, the cells were collected an dialyzed. To ensure that the siRNA treatments did not nonspecifically influence growth and viability of transfected hepatocytes, the cells were counted and analyzed for trypan blue uptake at the time of collection. The cell lysates were standardized for protein content and analyzed for Cyp content by Western blot using antibodies directed against CypA, CypB, CypC, or CypD. B, same as A except that cell lysates were analyzed for HCV protein expression using antibodies directed against NS5A and NS5B. C, naïve Huh7 cells were electroporated with 10 μg of in vitro transcribed genomic Con1 RNA. Twenty-four hours post-HCV RNA electroporation, the cells were transfected with siRNA Cyp and then retransfected 24 h later. At the indicated time points, intracellular HCV RNA was analyzed via reverse-transcription quantitative polymerase chain reaction and presented as genome equivalents (GE)/μg of total RNA.

FIGURE 2.

HCV, like HIV-1, requires host CypA to fully replicate in human cells. A, naïve Huh7 cells were transduced with an vesicular stomatitis virus G-pseudotyped HIV-1-based vector containing an irrelevant control shRNA or shRNAs that target CypA, CypB, or CypC. Transduced cells were selected for 7 weeks under puromycin (1 μg/ml). Stable cell lines were analyzed for CypA, CypB, and CypC content by Western blotting. B, stable Cyp-KD cell lines were electroporated with 10 μg of in vitro transcribed genomic Con1 RNA. At the indicated time points, intracellular HCV RNA was analyzed via reverse-transcription quantitative polymerase chain reaction and presented as genome equivalents (GE)/μg of total RNA. C, top panels, Cyp-KD cells lines were exposed to cell-free vesicular stomatitis virus G-HIV-1-GFP (left panel) or HSV-1-GFP (right panel) at a multiplicity of infection of 0.1. Forty-eight hours post-infection, the percentage of GFP-positive cells was quantified by FACS. Bottom left panel, cells were exposed to cell-free Dengue-2 (multiplicity of infection of 0.1). Three days post-infection, Dengue-2 infection was quantified by measuring the amounts of intracellular Dengue-2 capsid using an IFSA. Bottom right panel, Cyp-KD cells lines were electroporated with 10 μg of in vitro transcribed genomic Con1 RNA. Seven days post-transfection, HCV infection was quantified by measuring the amounts of intracellular HCV NS5B. The data are expressed as percentages of infected cells (GFP-, capsid-, or NS5B-positive cells) by fixing arbitrary infection of parental cells at 100. The results are representative of three independent experiments.

FIGURE 3.

HCV requires the isomerase activity of CypA to replicate in hepatocytes. A, parental or CypA knockdown Huh7 cell lines were electroporated with 10 μg of in vitro transcribed genomic Con1 RNA. Twenty-four hours post-HCV RNA electroporation, the cells were transfected with shRNA-resistant wild-type or H126Q CypA-HA in the presence or absence of the Cyp inhibitors CsA (2.5 μm) or Debio 025 (2 μm). At the indicated time points, intracellular HCV RNA was analyzed via reverse-transcription quantitative polymerase chain reaction and presented as genome equivalents (GE)/μg of total RNA. B, the inability of the H126Q CypA mutant to bind to HIV-1 Gag in hepatocytes was examined by measuring amounts of CypA incorporated into released particles. Huh7 cells were co-transfected with HIV-1 together with wild-type (WT) or H126 CypA-HA in the presence or absence of Cyp inhibitors CsA (10 μm) or Debio 025 (2 μm). Forty-eight post-transfection, both transfected cells and released virions were analyzed for CypA content by Western blotting using anti-HA antibodies. The cell lysates were standardized for protein content, whereas virions were standardized for HIV-1 capsid content by p24 enzyme-linked immunosorbent assay.

FIGURE 4.

The HCV NS5B polymerase associates with CypA via its enzymatic pocket. Parental Huh7 cells (3 million) were co-transfected with NS5B-Myc (5 μg of DNA) and wild-type (WT) CypA-HA or H126Q CypA-HA (5 μg of DNA) in the presence or absence of the Cyp inhibitor Debio 025 (2 μm). Three days post-transfection, the cells were collected and lysed. The cell lysates were precleared with agarose beads. CypA-NS5B association was assessed by co-immunoprecipitation using the Pierce HA tag Co-IP kit. Bound material was eluted and analyzed by Western blotting using anti-HA and anti-Myc antibodies. The results are representative of three independent transfections.