Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

Abstract

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.

Figure 1

Intrabody-mediated inhibition of HCV RNA replicons. A. (left panel) SEAP activity secreted from HCV RNA replicon cells every 24 hours following transient transfection with seven NS3-inhibiting and one control non-inhibitory intrabodies. Results were corrected for DNA transfection efficiency as described in the text. Error bars represent the standard deviation of the data. (right panel) Real-time PCR analysis of replicon expression at 48 hours post transfection. The results are shown as the relative quantity of the HCV non-coding region normalized to the level of intrabody expression. Error bars represent the standard deviation of the data. B. Inhibition of HCV RNA replicon cells and control cells by two NS3-inhibiting and 3 control, non-inhibitory intrabodies. SEAP activities secreted from (left panel) HCV RNA replicon cells or (right panel) control Huh7/Et2A cells every 24 hours following transient transfection with intrabodies. Error bars represent the standard deviation of the data. C. Immunofluorescence detection of intrabody expression in replicon cells and related suppression of viral antigen expression. Replicon cells were transiently transfected with intrabodies 35, 171 or 53Y. Slides were fixed and stained with mouse mAb to core antigen (green) and rabbit polyclonal antibody to MBP antigen (red), followed by laser scanning confocal microscopy.

Figure 2

Intrabody-mediated inhibition of protease inhibitor-resistant HCV-N RNA replicons. Intrabodies 35 (filled squares) and 171 (open circles) were compared to the noninhibitory (control) intrabody 53Y (filled triangle) as the control for their effect on SEAP activity secreted from 4 different replicon cell lines (A, B, C, D) with NS3 inhibitorresistance; secreted SEAP activity was monitored every 24 hours following transient transfection and normalized as in Figure 1. Error bars represent the standard deviation of the data.

Figure 3

Cross genotypic intrabody-mediated inhibition of HCV RNA replicon amplification. Intrabodies 35 (filled squares) and 171 (open circles) were compared to the non-inhibitory (control) intrabody 53Y (filled triangle) as the control for their effect on SEAP activity secreted from HCV RNA replicon cells derived from genotypes (A) 1a (H77), (B) 1b (HCV-N), (C) 1b (Con1) and, (D) 2A (JFH1). SEAP activity in supernatant fluids was monitored every 24 hours following transient expression of the intrabodies and normalized as in Figure 1. Error bars represent the standard deviation of the data.

Figure 4

Intrabody-mediated inhibition of infectious virus production. A. (left panel) Inhibition of HJ3-5 virus production, as determined by FFU assay of virus released into media by HJ3-5 virus-infected cells following transfection with intrabodies. Percent of inhibition was determined compared to virus released by non transfected cells set as 100%. Error bars represent the standard deviation of the data (A, right panel). Real-time PCR analysis of HCV RNA expression level 48 hours post transfection with intrabodies. The results are shown as the relative quantity of the HCV non-coding region normalized to the level of intrabody expression. Error bars represent the standard deviation of the data. B. Double label immunofluorescence detection of intrabodies and viral core protein in virusinfected cells and transiently transfected with plasmids encoding intrabodies 35, 171 or 53Y. Slides stained with mouse mAb to core antigen and rabbit polyclonal antibody to MBP antigen and analyzed by laser scanning confocal microscopy, as in Figure 1C.

Figure 5

Inhibition of HCV replicon amplification in combination with Interferon α. SEAP activity secreted from HCV RNA replicon cells every 24 hours following transient transfection with intrabodies. Following transfection, 0 units/ml, 10 units/ml or 100 units/ml Interferon-α were added to the cells every 24 hours. Results were not corrected for DNA transfection efficiency in this experiment. Error bars represent the standard deviation of the data.

Figure 6

Effect of intrabodies on NS3 catalysis and cell-free HCV RNA synthesis. A and B. Analysis of NS3-inhibitory scFvs specific binding to MBP-scNS3 in an ELISA. A microtiter plate was coated with NS3 BK/HCV-N. Binding assays were performed with purified MBPscFv 171 (in A) and 35 (in B). MBP-scFvs were detected with anti-myc and HRP-conjugated anti-mouse antibodies. Error bars represent the standard deviation of the data. C. Inhibition of NS3 BK/HCV-N strains enzymatic activity in vitro by scFv inhibitors. Fluorescence in supernatant was used to calculate catalysis percentage. Error bars represent the standard deviation of the data. D. Inhibition of NS3 catalysis by intrabodies. Upper panel: 30ng of total protein from lysates of tetracycline-inducible NS3 expressing cells that were supplemented with 3 fold dilutions of tetracycline for 48 hours were analyzed by immunoblotting with mouse anti-EGFP (for the detection of EGFP-NS3) and mouse antiactin antibodies (loading control) followed by HRP-conjugated secondary antibodies and ECL development. Lower panel: Immunoblot analysis of naïve HEK293 cells or tetracyclineinducible NS3 HEK293 cells transfected with MBP-EGFP-full 1b NS5AB-CBD substrate or 27 cotransfected with the substrate and intrabody 35. Cells were induced with 0, 10 or 1000ng/ml tetracycline. Full-length substrate was detected with rabbit polyclonal anti-CBD and goat anti-rabbit antibodies. β actin was detected with mouse anti-β actin and goat anti mous antibodies. E. Effect of intrabodies on the HCV in vitro RNA synthesis assay. Increasing concentrations of a pyrimidine inhibitor of NS5B polymerase activity (lanes 1–3), SCH6 (lanes 4–6), control, non-inhibitory intrabody 53Y (lanes 10–14), or intrabody 171 (lanes 15–19) were incubated with an optimal amount of a P16 heavy membrane preparation made from lysates of subgenomic replicon cells. For RNA synthesis, membranes were incubated under standard transcription reaction condition. Following the reaction, viral [³²P]CTP-labeled RNA was extracted, precipitated and separated on a 1% agarose gel containing Glyoxal. Bands were detected by exposure of the dried gel to X-ray films. Control reactions (lanes 7–9) were carried out either in the absence or presence of 1% DMSO, as required to solubilize many small molecule compounds, or the elution buffer used in preparing the scFv proteins.