Volume 131, Issue 5, Pages 1584-1591 (November 2006) Silencing of USP18 Potentiates the Antiviral Activity of Interferon Against Hepatitis C Virus Infection  Glenn Randall, Limin Chen, Maryline Panis, Andrew K. Fischer, Brett D. Lindenbach, Jing Sun, Jenny Heathcote, Charles M. Rice, Aled M. Edwards, Ian D. McGilvray  Gastroenterology  Volume 131, Issue 5, Pages 1584-1591 (November 2006) DOI: 10.1053/j.gastro.2006.08.043 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 Inhibition of USP18 expression by USP18 siRNAs. (A) Huh-7.5 cells were transfected with siIRR (□) or USP18-specific (■, siUSP18) siRNAs, maintained for 30 hours, and then treated with the indicated IU of IFN/mL for 24 hours. RNA was purified and quantified by real-time PCR. USP18 RNAs were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA levels and the fold induction of USP18 RNA in response to IFN is represented. Data are the mean ± SD for duplicate samples and are representative of 3 independent experiments. (B) Huh-7.5 cells were treated with siIRR, or 4 individual USP18 siRNAs (siUSP18-a–d) for 45 hours. RNA was purified and USP18 and GAPDH RNAs were quantified. Data were normalized for GAPDH RNA levels and are represented as a ratio of USP18/GAPDH relative to siIRR-treated cells. Data are the mean ± SD of duplicate samples. Gastroenterology 2006 131, 1584-1591DOI: (10.1053/j.gastro.2006.08.043) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 USP18 silencing augments the antiviral effects of IFN against HCV infection. Huh-7.5 cells were transfected with siIRR (■) or siUSP18 (▴), maintained for 30 hours, and treated with the indicated concentrations of IU IFN/mL. After 15 hours, the cells were infected with HCV for 8 hours, then washed and maintained in media lacking IFN for 2 days, and HCV replication was measured. (A) Intracellular RNAs were purified and GAPDH and HCV RNAs were quantified by real-time PCR. (B) Infectious HCV in the supernatants of cells from A was quantified by limiting dilution assay and the 50% infectious dose/mL was calculated. The percentage of inhibition in response to IFN relative to untreated cells is shown. Data are the mean ± SD of (A) quadruplicate and (B) triplicate samples. The data are representative of 4 independent experiments. Gastroenterology 2006 131, 1584-1591DOI: (10.1053/j.gastro.2006.08.043) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 Multiple USP18 siRNAs enhance the antiviral effects of IFN against HCV. Cells were treated with siIRR (■), or 4 individual USP18 siRNAs (▴,▾,♦,● siUSP18-a–d, respectively) for 30 hours, then with indicated IU IFN/mL for 15 hours. Cells were rinsed, inoculated with HCV for 8 hours, rinsed again, and maintained for 2 days in media lacking IFN, and HCV replication was measured. (A) HCV RNA was quantified by real-time PCR and normalized to GAPDH levels. (B) Infectious HCV was quantified by limiting dilution assay and the 50% infectious dose/mL was calculated. The percentage of inhibition relative to untreated cells then was calculated for each. Data are the mean ± SD of (A) quadruplicate and (B) duplicate samples and are representative of 2 independent experiments. Gastroenterology 2006 131, 1584-1591DOI: (10.1053/j.gastro.2006.08.043) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 USP18 silencing augments the antiviral effects of IFN in cells previously infected with HCV. Huh-7.5 cells were infected with HCV at a multiplicity of infection of 2 for 6 hours. Infected cells then were transfected with siIRR (■) or siUSP18 (▴), maintained for 18 hours, and treated with the indicated concentrations of IU IFN/mL. After 24 hours, the cells were washed and maintained in media lacking IFN for 2 days, and HCV replication was measured. Intracellular RNAs were purified and GAPDH and HCV RNAs were quantified by real-time PCR. The percentage of inhibition in response to IFN relative to untreated cells is shown. Data are the mean ± SD of triplicate samples. The data are representative of 2 independent experiments. Gastroenterology 2006 131, 1584-1591DOI: (10.1053/j.gastro.2006.08.043) Copyright © 2006 AGA Institute Terms and Conditions

Figure 5 USP18 silencing enhances protein ISGylation and ISG15 induction by IFN. Huh-7.5 cells were treated as before with either siIRR or siUSP18, then with the indicated IU of IFN/mL. After 24 hours of IFN treatment, (A) protein lysates were collected, separated, and probed for ISG15 expression, or (B) RNA was purified and ISG15 and actin RNAs were quantified. RNA levels were normalized and presented as in Figure 1; the data are representative of 3 independent experiments. (B) □, siIRR; ■, siUSP18. Gastroenterology 2006 131, 1584-1591DOI: (10.1053/j.gastro.2006.08.043) Copyright © 2006 AGA Institute Terms and Conditions

Figure 6 General enhancement of ISG induction in USP18-silenced cells. Huh-7.5 cells were treated with either siIRR (□) or siUSP18 (■) for 30 hours, followed by the indicated IU of IFN/mL for 24 hours. RNA was purified and quantified by real-time PCR: (A) 2′-5′-oligoadenylate synthetase 3, (B) interferon-induced protein with tetratricopeptide repeats 1, (C) Viperin, (D) myxovirus resistance 1. RNA levels were normalized to β-actin RNA levels and expressed as in Figure 1; the data are representative of 2 independent experiments. Gastroenterology 2006 131, 1584-1591DOI: (10.1053/j.gastro.2006.08.043) Copyright © 2006 AGA Institute Terms and Conditions

Figure 7 USP18 silencing prolongs STAT1 activation after IFN treatment. Huh-7.5 cells were transfected with siIRR or siUSP18, maintained for 24 hours, and treated with 100 IU of IFN/mL. Protein lysates were harvested at the indicated time, separated electrophoretically, and probed for STAT1-phospho701. Blots then were stripped and probed for total STAT1 expression. Gastroenterology 2006 131, 1584-1591DOI: (10.1053/j.gastro.2006.08.043) Copyright © 2006 AGA Institute Terms and Conditions