Volume 131, Issue 6, Pages 1887-1898 (December 2006) Interferons α and λ Inhibit Hepatitis C Virus Replication With Distinct Signal Transduction and Gene Regulation Kinetics  Tobias Marcello, Arash Grakoui, Giovanna Barba–Spaeth, Erica S. Machlin, Sergei V. Kotenko, Margaret R. Macdonald, Charles M. Rice  Gastroenterology  Volume 131, Issue 6, Pages 1887-1898 (December 2006) DOI: 10.1053/j.gastro.2006.09.052 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 Schematic of the HCV full-length replicon, HCVcc genome and IFN pathways. (A) HCV-Con1 (top) is a full-length genotype 1b bicistronic replicon containing the HCV 5′nontranslated region plus nucleotides 342 to 389 of the core coding region, a neomycin resistance gene, the EMCV-IRES, the entire HCV polyprotein coding sequence, and the HCV 3′nontranslated region. The FL-J6/JFH-5’C19Rluc2AUbi HCVcc genotype 2a genome (J6/JFH1 HCVcc, bottom) contains J6-derived structural genes and JFH1-derived nonstructural and nontranslated regions as indicated. The amino terminus of the polyprotein contains a ubiquitin- and self-cleavable (2AUbi) Renilla luciferase cassette (Rluc). (B) Binding of IFN-α, IFN-γ, and IFN-λ to their distinct heterodimeric receptors leads to activation and translocation of STAT molecules that induce the expression of interferon-inducible genes (ISG) through the interferon-stimulated response element (ISRE) or γ-activated sequence (GAS). Gastroenterology 2006 131, 1887-1898DOI: (10.1053/j.gastro.2006.09.052) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 Comparison of the antiviral effects on the replicon-harboring FL-neo cell line. (A) Incubation of FL-neo cells with IFN-λ or IFN-α for 48 hours resulted in dose-dependent antiviral effects. TaqMan RT-PCR was performed on the extracted RNA. HCV RNA levels are shown as relative percentages of the untreated control. For each sample, TaqMan analysis was performed in triplicate. The mean value obtained from 2 independent experiments is plotted; error bars indicate the SEM. (B) Western blot analysis of HCV NS5A protein expression was performed on protein extracts from cells that were treated for 48 hours with varying IFN doses. β-actin is shown as a loading control. The experiment shown is representative of 4 independent experiments. (C) FL-neo cells were incubated for varying lengths of time in the presence or absence of IFN-λ or IFN-α at 10 or 5 ng/mL, respectively. All cultures had reached comparable levels of confluency after the given duration of treatment, and media replete with each cytokine was replaced every 48 hours. Total RNA extracts were prepared and HCV RNA levels were determined by TaqMan RT-PCR analysis. The data shown are from 1 experiment, with 6 samples for no IFN and 1 sample for each time point for each cytokine. The results are shown as relative percentages of the untreated control for each time point. For each sample, TaqMan analysis was performed in triplicate; error bars indicate the SD. Gastroenterology 2006 131, 1887-1898DOI: (10.1053/j.gastro.2006.09.052) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 Treatment of Huh-7.5 cells with IFN-α or IFN-λ inhibits replication of genotype 2a HCV cell culture virus. Huh-7.5 cells were treated for 12 hours with the indicated concentrations of cytokine and then were infected with FL-J6/JFH-5’C19Rluc2AUbi HCVcc virus expressing Renilla luciferase. After 48 hours, luciferase activity was determined and is presented as the percentage of the activity present in untreated infected cells. Symbols show the mean value of triplicate wells; error bars show the SD. Gastroenterology 2006 131, 1887-1898DOI: (10.1053/j.gastro.2006.09.052) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 Low doses of IFN-λ or IFN-α enhance the antiviral efficacy of IFN treatment. Huh-7.5 cells were treated for 12 hours with the indicated concentrations of cytokines and then were infected with FL-J6/JFH-5’C19Rluc2AUbi HCVcc virus expressing Renilla luciferase. After 48 hours, luciferase activity was determined and is presented as the percentage of the activity present in untreated infected cells. Solid lines represent the single cytokine data shown in Figure 3, whereas dashed lines show luciferase activity after treatment with additional low-dose (0.1 ng/mL) cytokine. Symbols show the mean value of triplicate wells; error bars show the SD. Gastroenterology 2006 131, 1887-1898DOI: (10.1053/j.gastro.2006.09.052) Copyright © 2006 AGA Institute Terms and Conditions

Figure 5 Expression of and signaling through IFN receptors IFN-αR2, IL-10R2, or IFN-γR1 in FL-neo cells. (A) IFN-αR2, IFN-γR1, and IL-10R2 expression levels on the cell surface of FL-neo cells were evaluated by flow cytometry. The receptor expression was detected by incubating the cells with primary antibodies directed against the indicated receptor chains (solid lines). Nonspecific sera served as a negative control (dashed lines). The experiment shown is representative of 3 independent experiments. (B) FL-neo cells were incubated for 1 hour in the presence or absence of inhibitory antibodies to IFN-αR2, IFN-γR1, IL-10R2 or a combination of antibodies to both IFN-αR2 and IFN-γR1 as indicated (α-, γ-, λ-, or α/γ block) before the addition of IFN-α or IFN-γ (5 ng/mL) or IFN-λ (10 ng/mL) for 30 minutes. Western blot analysis was performed on protein extracts using an antibody specific for the phosphorylated form of STAT1 to detect the downstream signal mediated by receptor activation. Subsequent blotting with an antibody specific for total STAT1 is shown to ensure equal loading. The experiment shown is representative of 3 independent experiments. (C) FL-neo cells were incubated in the presence or absence of inhibitory antibodies to IFN-αR2, IFN-γR1, IL-10R2 or a combination of antibodies to both IFN-αR2 and IFN-γR1 for 1 hour before the addition of IFN-α or IFN-γ (5 ng/mL) or IFN-λ (10 ng/mL) for 48 hours. HCV RNA levels were determined by TaqMan RT-PCR analysis and are shown as relative percentage compared with the untreated control. Mean values are plotted; error bars for the no (zero) IFN data represent SEM of 3 samples measured in duplicate TaqMan assays, whereas error bars for the IFN-treated samples represent the range of duplicate TaqMan assays on single samples. Gastroenterology 2006 131, 1887-1898DOI: (10.1053/j.gastro.2006.09.052) Copyright © 2006 AGA Institute Terms and Conditions

Figure 6 Kinetics of STAT phosphorylation mediated by IFN-λ and IFN-α. Western blot analysis was performed on protein extracts of FL-neo cells that were treated for varying lengths of time with IFN-λ or IFN-α at 10 or 5 ng/mL, respectively. The blots were sequentially incubated with antibodies to the phosphorylated form of STAT1 and STAT2. Subsequent probing with β-actin is shown as a loading control. The experiment shown is representative of 2 independent experiments. Gastroenterology 2006 131, 1887-1898DOI: (10.1053/j.gastro.2006.09.052) Copyright © 2006 AGA Institute Terms and Conditions

Figure 7 Microarray analysis of gene induction by IFN-α and IFN-λ. (A) Visualization of the intensity matrix plot (“heat map”) of 66 genes in response to IFN-α (5 ng/mL) or IFN-λ (10 ng/mL) at 3, 12, or 24 hours. Only genes that were induced (red) or reduced (green) more than 2-fold compared with a baseline (mean of untreated controls at the particular time points) in any of the 6 samples are shown. Genes are sorted in a decreasing order on the basis of their fold regulation in response to IFN-λ at 24 hours (see text for detail). (B) Table showing the 58 IFN-induced genes identified by microarray analysis in response to IFN-α (5 ng/mL) or IFN-λ (10 ng/mL) at 3, 12, or 24 hours. Only genes that were induced at least 2-fold in any of the 6 samples are shown. Genes are organized in clusters of maximum induction in response to IFN-α at the given time point Color codes (shown below) have been used to visualize the induction peak. (C) Histogram plot comparing global transcript regulation profiles in IFN-α (light grey)- or IFN-λ (dark grey)-treated cells at 3, 12, and 24 hours. Histogram classes represent intervals between whole numbers of fold change in transcript abundance compared with the baseline. The number of transcripts that fall within these intervals is shown. Gastroenterology 2006 131, 1887-1898DOI: (10.1053/j.gastro.2006.09.052) Copyright © 2006 AGA Institute Terms and Conditions