Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro

Abstract

Cyclosporine (CsA) and its derivatives potently suppress hepatitis C virus (HCV) replication. Recently, CsA-resistant HCV replicons have been identified in vitro. We examined the dependence of the wild-type and CsA-resistant replicons on various cyclophilins for replication. A strong correlation between CsA resistance and reduced dependency on cyclophilin A (CyPA) for replication was identified. Silencing of CyPB or CyPC expression had no significant effect on replication, whereas various forms of small interfering RNA (siRNA) directed at CyPA inhibited HCV replication of wild-type but not CsA-resistant replicons. The efficiency of a particular siRNA in suppressing CyPA expression was correlated with its potency in inhibiting HCV replication, and expression of an siRNA-resistant CyPA cDNA rescued replication. In addition, an anti-CyPA antibody blocked replication of the wild-type but not the resistant replicon in an in vitro replication assay. Depletion of CyPA alone in the CsA-resistant replicon cells eliminated CsA resistance, indicating that CyPA is the chief mediator of the observed CsA resistance. The dependency on CyPA for replication was observed for both genotype (GT) 1a and 1b replicons as well as a GT 2a infectious virus. An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to CsA treatment in wild-type but not in resistant replicons was detected. These findings reveal the molecular mechanism of CsA resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection.

FIG. 1.

An siRNA directed at CyPA inhibits replication of wild-type but not CsA-resistant replicons. (A) RS2 replicon is less sensitive than GS5 replicon to CsA. The two replicon cell lines were treated with CsA at the indicated concentrations for 4 days before being analyzed for NS5A expression by Western blotting. (B) Various shRNAs directed at three CyPs or firefly luciferase were introduced into the replicon cells and expressed for 7 days. The cells were then lysed and probed for the indicated proteins. (C) Suppression of CyPC RNA expression by sh-C454. Total RNA was then extracted 7 days posttransduction and subjected to real-time RT-PCR analysis to determine the level of CyPC mRNA. (D) After introduction of the shRNAs as in panel B, the cells were subjected to double selection with puromycin and G418. At the end of the selection, the cell colonies were stained with violet blue. (E) In vitro replication reaction of GS5 or RS2 lysate was allowed to progress in the presence of either IgG negative control or an anti-CyPA antibody for 4 h. The ³²P-labeled products of replication were detected as a single band of expected size on an RNA gel.

FIG. 2.

HCV replicon expression is correlated with CyPA expression level. (A) Schematic representation of the target sites of anti-CyPA shRNAs used in this study. The target sites of previously reported siRNAs are also shown. (B) GS5 cells were transduced with the indicated shRNAs for 7 days. Total protein lysates were then subjected to SDS-PAGE and immunoblotting. (C) Effects of synthesized siRNA duplexes directed at either the 161 or the 285 site on HCV replication. The detection of NS5A, actin, and CyPA was performed 7 days after transfection of the siRNA duplexes into GS5 cells. (D) An siRNA-resistant CyPA cDNA rescued NS5A expression in the presence of sh-A161. A mammalian expression plasmid bearing a CyPA cDNA (Myc-A#) that contained silent mutations in the recognition site of sh-A161 was introduced into GS5 cells, which had been transduced with sh-A161, to examine the effect of restoring CyPA expression on HCV replication. (E) Expression of CyPA# cDNA in CyPA-knock-down cells partially rescues HCV RNA replication. In vitro-transcribed Rep1b RNA was coelectroporated into Huh-7.5/sh-A161 cells with the Myc-A# cDNA expression plasmid. RNA was extracted at 7 h and 4 days postelectroporation for analysis of HCV IRES and GAPDH RNA level. After normalization to GAPDH levels, the ratio of the day 4 HCV RNA versus the 7-h HCV RNA was calculated and plotted. A parallel electroporation of Rep1b RNA into Huh-7.5/sh-Luc cells was used as a positive control, which is set at 100%.

FIG. 3.

CyPA is the key mediator of CsA resistance in RS2 cells. (A) RS2 cells and two derivative cell lines stably expressing sh-Luc and sh-A161 were treated with increasing amounts of CsA for 4 days before being fixed for FACS analysis. The percentage of GFP-positive cells at the end of the treatments is normalized to the value of the untreated sample. (B) Western blot results confirmed the substantial knock-down of CyPA in the RS2/sh-A161 cells and their heightened CsA sensitivity. (C) CsA treatment of RS2 cells transiently transduced with sh-A161. The RS2 cells were transduced with either sh-Luc or sh-A161 for 4 days; half of the cells were then treated with 0.5 μg/ml of CsA for 3 more days before being subjected to FACS analysis of NS5A-GFP expression.

FIG. 4.

CyPA is essential for replication of both GT 1a and GT 1b replicons. (A) Silencing of CyP targets in stable Huh-7.5 cells transduced with shRNAs directed at CyPA, CyPB, and CyPC. (B) sh-A161 blocks the ability of GT 1a and 1b replicons to replicate and form colonies in stable Huh-7.5 cells. One microgram of replicon RNA was used in each electroporation, and the selection was allowed to proceed for 4 weeks before the plates were stained. (C) Inhibition of Con1 and H77 replication by transiently expressed sh-A161. The Con1 and H77 replicon cells were transduced with either the sh-Luc or the sh-A161 vector for 7 days. The cells were lysed, and total proteins were subjected to immunoblotting for the detection of viral proteins NS5A and the two CyPs shown.

FIG. 5.

Suppression of CyPA expression prevents infection by HCVcc/JFH-1. (A to C) The indicated stable cell lines were infected with JFH-1 for 9 to 12 days (9 days for panels A and B and up to 12 days for panel C), at which time (A) total RNA was isolated and subjected to RT-PCR detection of either HCV RNA or a cellular gene, RNA helicase A (RHA). (B) The cells were fixed with paraformaldehyde and stained with an anticore antibody. DAPI, 4′,6′-diamidino-2-phenylindole. (C) Total proteins were extracted and subjected to immunoblotting with an anti-NS3 antibody. (D) Infection of Huh-7.5/sh-A161 cells by vesicular stomatitis virus (VSV). Huh-7.5 cells with or without sh-A161 were subjected to infection by serial dilutions of VSV, ranging from 50 to 5 × 10⁴ PFU. A plaque assay was then performed to quantify infection results. (E) Inhibition of an existing infection by sh-A161. Huh-7.5 cells were first infected with HCVcc/JFH-1 for 10 days and then transduced with either sh-Luc or sh-A161 vector. One week after transduction, total protein was extracted for immunoblotting for detection of the indicated proteins.

FIG. 6.

CyPA interacts with NS5B and HCV RNA. (A) HCV RNA from replicon cells could be coprecipitated with CyPA. GS5 replicon cells were used, and HCV IRES was detected by RT-PCR. (B) Interaction between CyPA and HCV replicon RNA is sensitive to CsA treatment in GS5 but not in RS2 cells. The co-IP experiments were performed with anti-CyPA in the presence or absence of 4 μg/ml CsA. (C) CyPA interacts with NS5B in vitro. A GS5 replicon lysate was incubated with equal amounts of GST or GST-CyPA in vitro. The binding proteins were then purified with glutathione beads and subjected to Western blot assays to detect NS5B. FT, flowthrough; FW, final wash; B, bound. (D) CsA abolishes CyPA's interaction with GS5 NS5B but not with RS2 NS5B. The GST pull-down assay in panel C was performed in the presence of increasing amounts of CsA. Lysates from GS5 or RS2 cells were used. (E) NS5B interacts with GST-CyPA in the absence of other HCV proteins or RNA. 293-T cells were cotransfected with plasmids expressing Con1 NS5B and Flag-tagged CyPA for 48 h. The cells were lysed and subjected to IP by anti-Flag monoclonal antibody-conjugated beads.

FIG. 7.

Relative expression levels of CyPA, CyPB, and CyPC in HCV replicon cells. (A) CyPA is expressed at a much higher level than is CyPB or CyPC. Total RNA was extracted from GS5 and RS2 cells and subjected to reverse transcription with oligo(dT) primers. The cDNA was then divided into four equal parts for the detection of CyPA, CypB, CyPC, and GAPDH mRNA with real-time PCR.