Figure 1.
Major role of CypA for HCV replication.
(A–C) Stable knock-down of CypA (left panel) or CypB (right panel) in Huh7.5 and Huh7-Lunet cells was achieved by retroviral transduction of shRNAs targeting the 3′ NTR of either mRNA. As a reference, cells were transduced with the retroviral vector encoding an unrelated shRNA sequence (sh-control). (A) Knock-down efficiency was determined by western blot with a CypA or a CypB specific antibody. Beta-actin was used as loading control. (B, C) Detection of CypA (B) and CypB (C) knock-down in Huh7-Lunet and Huh7.5 cells via indirect immunofluorescence analysis. Cells were seeded onto glass cover slips and fixed with methanol 72 h later. Immunostaining was performed with CypA or CypB specific antibodies.
Figure 2.
Impact of stable CypA or CypB knock-down on RNA replication of genotype 2a and 1b replicons.
(A) Huh7-Lunet cells (left panel) and Huh7.5 cells (right panel) either stably transduced with the unrelated shRNA control (sh-control) or a vector encoding the CypA- or CypB-specific shRNA or naive cells were transfected with a subgenomic luciferase reporter replicon derived from the HCV isolate JFH1 (sgNS3/JFH1-Luc, shown at the top). Cells were lysed at given time points after transfection, and luciferase activity (expressed in relative light units, RLU) in cell lysates was determined. Values are normalized to the 4 h value that reflects transfection efficiency and that was set to 100%. Means and standard deviations of a representative experiment are shown. Luc, firefly luciferase; sg, subgenomic replicon. (B) Impact of CypA or CypB knock-down on replication of Con1. Schematic diagrams of the two Con1-derived constructs are shown in the top. They are composed of the HCV 5′ NTR, the IRES of poliovirus for high-level expression of the firefly luciferase gene, the EMCV IRES, the HCV replicase coding region and the 3′ NTR. To enhance RNA replication, 3 cell culture adaptive mutations (labelled with asterisks) have been introduced in case of the wild type (ET) replicon [31]. The GND mutant contains an active site mutation destroying NS5B RdRp activity, thus serving as control to measure decay kinetics of luciferase activity expressed from transfected input RNA. Huh7-Lunet cells (left panel) and Huh7.5 cells (right panel) specified below the schematics of the replicons were transfected and replication was analyzed as described in the legend of panel A. One experiment of two independent repetitions, each performed in duplicate is shown.
Figure 3.
Increased inhibition of RNA replication of JFH1 full length genomes and NS2-containing replicons by CypA knock-down.
(A) Huh7-Lunet cells stably transduced with the control vector (sh-control) or the vector encoding the CypA- or CypB-specific shRNA were transfected with the Jc1-Luc genome (shown at the top). Replication of the genome was determined by luciferase assay with cell lysates prepared at given time points (left panel). Infectivity released from transfected cells was measured by inoculation of naive Huh7.5 cells with culture supernatants harvested at given time points after transfection and luciferase activity was determined in infected cells 72 h after inoculation (right panel). Means and standard deviations of two independent experiments are shown. The dark shaded region in the HCV genome map in the top indicates the J6-derived sequence; the white region corresponds to JFH1. (B) Huh7-Lunet cells were transfected with the Jc1 genome and accumulation of intracellular core protein amounts was determined by core-specific ELISA (upper panel). Kinetic of core release is shown in the middle panel. Core protein in culture supernatants of transfected cells was normalized to total core protein amounts (intra- plus extracellular core). Kinetic of release of infectious virus particles is shown in the bottom panel. Infectivity was determined by TCID50 assay. Background of the assay is indicated by the horizontal dashed line. (C) Huh7-Lunet cells were transfected with JFH1-derived subgenomic replicons encoding an NS3 to 5B or NS2 to 5B replicase of JFH1 (sgNS3/JFH1-Luc and sgNS2/JFH1-Luc, respectively) and replication was determined by luciferase assay as described in the legend to panel (A). Background of the assay was determined with the sgNS3-derived replicon with an active site mutation destroying NS5B RdRp activity (ΔGDD). A representative experiment of three independent repetitions, each performed in duplicate is shown.
Figure 4.
Rescue of HCV replication by overexpression of CypA wt but not CypA H126Q in stable knock-down cells.
(A) Huh7-Lunet cells with a stable knock-down of CypA (lower left) were stably transduced with a CypA wild type (wt) expression construct (CypA wt-rescue; upper right) or a CypA expression construct carrying the H126Q mutation (CypA H126Q-rescue; lower right) that are both resistant to the shRNA. For comparison, Huh7-Lunet cells stably transduced with the same expression vector containing the non-related control shRNA (sh-control) are shown (upper left). Cells were analyzed by immunofluorescence using a CypA-specific antibody. Note that CypA expression levels are higher in the ‘rescue cells’ as compared to the vector control cells (sh-control). (B) Lysates from cells specified in the top were analyzed by Western blot using a CypA-specific antibody. Lane 6 shows a Western blot of a lysate of Huh7-Lunet cells with a stable knock-down of CypA and transduced with an empty vector (cell line sh-CypA vector control-rescue). Beta-actin was used as a loading control. K-d, knock-down; rescue, stable CypA knock-down cells transduced with CypA wt or the H126Q mutant or empty expression vector, respectively. Loaded samples are specified above each lane. (C) Restoration of HCV replication by ectopic expression of CypA wt, but not CypA H126Q in stable CypA knock-down cells. Huh7-Lunet cells specified in the top were transfected with a bicistronic JFH1 reporter replicon (sgNS3/JFH1-Luc) and RNA replication was determined by luciferase assay 24, 48 and 72 h post transfection. Values are normalized for transfection efficiency by using the 4 h value. Means and standard deviations of a representative experiment of two independent repetitions are shown.
Figure 5.
CypA is the target of the cyclosporine analogue DEBIO-025.
(A) Dose-response curve for cyclosporine A (CsA) and DEBIO-025. Huh7-Lunet cells transfected with JFH1 reporter replicon (sgNS3/JFH1-Luc, shown in the top of Fig. 2A) were incubated 4 h post electroporation with escalating concentrations of CsA or DEBIO-025 and 72 h later cells were lysed and luciferase activity was determined. (B) Naive Huh7-Lunet cells or cells transduced with a retroviral vector encoding a CypA- or CypB-specific shRNA or transduced with the retroviral vector control (sh-control) were transfected with a HCV luciferase reporter replicon (sgNS3/JFH1-Luc). Four hours post transfection various concentrations of CsA or DEBIO-025 were added, cells were incubated for 72 h and lysed. Luciferase activities were determined and normalized to the values obtained with mock-treated cells, which was set to 100%. The background of the assay is indicated with the grey horizontal line. (C) Huh7-Lunet cells with a stable knock-down of CypA and transduced with the empty vector (control-rescue) or the shRNA-resistant CypA wt or CypA H126Q encoding vector were transfected with sgNS3/JFH1-Luc. Thereafter cells were treated with various concentrations of DEBIO-025 and 72 h later, cells were lysed and luciferase activity was determined. Naive Huh7-Lunet cells were analyzed in parallel. (A–C) Means and standard deviations of a representative experiment (2 independent repetitions) are shown.
Figure 6.
Identification and characterization of mutations conferring resistance against the antiviral activity of DEBIO-025.
Table 1.
Mutations identified in JFH1/RFP replicons after selection for DEBIO-025 resistance.
Figure 7.
A mutation close to the NS5A/B cleavage site confers resistance to DEBIO-025 and renders HCV replication less dependent on CypA.
(A) Replication of sgNS3/JFH1-Luc wt and the V2440L replicon in Huh7-Lunet cells with a stable knock-down of CypA or transduced with the retroviral vector control (sh-control). Cells were lysed at given time points after transfection with each of the replicon RNAs and luciferase activity in cell lysates was determined. Values were normalized to the 4 h value to exclude differences due to transfection efficiency. Note the higher replication of the V2440L mutant as compared with the wild type in CypA knock-down cells. (B) Experimental setup as in A, with the difference that the V2440A- and not the V2440L-mutant was used. Means and standard deviations of two independent experiments are shown in each panel.
Figure 8.
Alteration of the kinetics of polyprotein processing at the NS5A-B cleavage site by mutations conferring DEBIO-025 resistance.
(A, B) Huh7-Lunet/T7 cells were transfected with subgenomic NS3 to NS5B JFH1 expression constructs derived from the wild type or mutants specified at the top of each panel or with the empty vector (pTM1-2; lane 1). After 24 h cells were pulse-labeled for 90 min with [35S] methionine/cysteine. Cells were lysed immediately (0) or incubated with non-radioactive medium for 1 or 2 additional hours. Immunoprecipitation was performed using either an NS5A- (A) or NS5B-specific antibody (B). Molecular weights are given on the left, arrows to the right point to the respective HCV proteins detected. (C) Amounts of unprocessed NS5AB and NS4B5AB precursors were quantified by phosphoimaging. Values obtained with the phosphoimage displayed in the upper panel are shown in panel C. Similar ratios were obtained with the phosphoimage of the NS5B-specific immunoprecipitation (data not shown).
Figure 9.
Properties of NS5A-B cleavage site mutants.
(A) Alignment of amino acid consensus (con) sequences of various HCV genotypes or individual HCV isolates (H77, Con1, JFH1) at the P-side of the NS5A-B cleavage site. JFH1 (upper lane) was used as master sequence. Numbers at the top refer to the P-side positions. Amino acid sequences of cleavage site mutants are given in the lower part of this panel (CONSTRUCTS). (B) Time course of replication of subgenomic sgNS3/JFH1-Luc replicons specified in the top. Only the early time points up to 24 h post infection are shown. (C) Dose-response assay of cleavage site mutants. Subgenomic replicons were transfected into Huh7-Lunet cells and treated 4 h later with escalating concentrations of DEBIO-025. After 72 h cells were lysed and luciferase activity was determined. Values are normalized to transfected control cells that were mock treated. The means and standard deviations of two independent experiments are shown in panel B and C. (D) Replication kinetic of cleavage site mutants in Huh7-Lunet cells with a stable knock-down of CypA. Cells transfected with constructs specified in the top were lysed at given time points and luciferase activity was determined. Values were normalized for transfection efficiency as determined with the 4 h value. (E) Impact of NS5A-B cleavage site mutants on release of infectivity. Genomes depicted at the bottom of the panel were transfected into Huh7-Lunet cells and release of infectious HCV particles into the supernatant was determined by TCID50 assay at time points specified in the top. (D–E) The background of the assay is indicated with the horizontal dashed line. Means and standard deviations of a representative experiment are shown.
Table 2.
Summary of phenotypes of NS5A mutants.