1. Volume 135, Issue 5, Pages 1710-1718.e2 (November 2008)
Antiviral Suppression vs Restoration of RIG-I Signaling by Hepatitis C Protease and Polymerase Inhibitors 
Yuqiong Liang, Hisashi Ishida, Oliver Lenz, Tse–I Lin, Origène Nyanguile, Kenny Simmen, Richard B. Pyles, Nigel Bourne, MinKyung Yi, Kui Li, Stanley M. Lemon 
Gastroenterology 
Volume 135, Issue 5, Pages e2 (November 2008)
DOI: /j.gastro
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2. Figure 1
TMC restoration of SenV activation of the IFN-β promoter in UNS3-4A cells. (A) Schematic showing experimental design. (B) Immunoblots of NS3 and MAVS in lysates of cells grown in 0 (lanes 1 and 2), (lanes 3–8), or 2.0 μg/mL tetracycline (lane 9). Cells were treated with TMC at the concentrations indicated for 46 hours prior to lysis. An asterisk indicates the MAVS product of NS3/4A cleavage; “o” indicates uncleaved NS3-4A. GAPDH was included as a loading control. (C) SenV-induced activation of the IFN-β promoter. Cells were grown in various concentrations of tetracycline and TMC prior to challenge with SenV, as shown in panels A and B. Results shown are mean fold induction of the IFN-β promoter compared with baseline activity in similarly treated, mock-infected cells, ± SD, and are representative of several independent experiments.
Gastroenterology  , e2DOI: ( /j.gastro )
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3. Figure 2
The MAVS fragment produced by NS3/4A cleavage is not a dominant negative inhibitor of RIG-I signaling. (A) Immunoblot of Flag-tagged MAVS and full-length wild-type (wt) MAVS in transfected HEK293 cells. (B) IFN-β promoter activity in cells ectopically expressing wt MAVS or MAVS 1–508, or mixtures thereof, as indicated: “0” indicates cells were transfected with 100% wt MAVS expression vector, whereas “100” indicates cells were transfected with 100% MAVS 1–508 expression vector. “EV,” empty vector. (C) SenV-induced IFN-β promoter activity in cells expressing wt MAVS or MAVS 1–508, or mixtures thereof, as in panel B. Results shown in panels B and C are mean luciferase activity, normalized to β-galactosidase activity expressed by a cotransfected control vector, ± SD.
Gastroenterology  , e2DOI: ( /j.gastro )
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4. Figure 3
TMC restoration of SenV activation of the IFN-β promoter in 2-3 replicon cells. (A) Schematic showing experimental design; 2-3 replicon and cured 2-3c cells were studied simultaneously. (B) Fold induction of IFN-β promoter activity following SenV infection of 2-3 replicon cells (darkly shaded columns) and 2-3c cells (lightly shaded columns) that do not contain replicating HCV RNA. Results shown are mean fold induction ± SD and are representative of several independent experiments.
Gastroenterology  , e2DOI: ( /j.gastro )
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5. Figure 4
Comparative analysis of the rescue of SenV-induced activation of the IFN-β promoter in 2-3 replicon cells treated with the NS3/4A inhibitor TMC vs a nonnucleoside NS5B inhibitor, Tib-3. (A) Schematic showing experimental design. (B) Fold induction of IFN-β promoter activity following SenV infection of 2-3 replicon cells (darkly shaded columns) or cured 2-3c cells (lightly shaded columns) treated with TMC (C) Fold induction of IFN-β promoter activity in similarly infected cells treated with Tib-3. Results shown in panels B and C are mean fold induction ± SD and are representative of independent experiments. (D) Immunoblots showing NS3 and MAVS expression in 2-3 cells treated with TMC or Tib-3. The positions of the intact wt MAVS and the MAVS 1–508 cleavage product are shown at the right. The absence of detectable NS3 in lane 4 is likely due to a transfer artifact; GAPDH was included as a loading control.
Gastroenterology  , e2DOI: ( /j.gastro )
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6. Figure 5
Rescue of SenV-induced activation of the IFN-β promoter by TMC in FT3-7 cells infected with the genotype 2a JFH-1 virus. (A) Schematic showing experimental design; the protease inhibitor was added at the time of reporter plasmid transfection, 47 hours prior to lysis of the cells and 72 hours after JFH-1 infection at a multiplicity of infection of 1.0. (B) IFN-β promoter activity following SenV infection of infected (left-hand columns) vs mock-infected cells (right-hand 2 columns). Results shown are mean fold induction ± SD. (C) Immunoblots showing MAVS and NS3 expression in infected vs mock-infected cells, following treatment with TMC The positions of the intact MAVS and the MAVS 1–508 cleavage product are shown at the right. GAPDH was included as a loading control.
Gastroenterology  , e2DOI: ( /j.gastro )
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7. Supplementary Figure 1
Inhibition of scNS3 protease-mediated cleavage of full-length MAVS and NS5A-5B by TMC (A) [35S]-labeled MAVS and NS5A-5B were synthesized in a cell-free translation system, then subjected to proteolysis with increasing concentrations of MBP-scNS3 (single-chain genotype 1a NS3 protease from H77 virus fused to maltose binding protein). Parallel reactions were carried out with catalytically inactive MBP-scNS3-S139A which contains a Ser-139 to A1a substitution in NS3 (see Supplementary Materials and Methods included in the On-line Supporting Information). Reaction products were separated by SDS-PAGE and visualized by autoradiography. Solid symbols represent full-length, uncleaved molecules, while open symbols indicate products of NS3/4A cleavage. (B) Quantitative analysis of the SDS-PAGE results shown in panel A. Results are shown as percent cleavage of the total MAVS or NS5A-5B product. Data fitted to a 3 parameter Hill equation yielded IC50 (50% inhibition of cleavage) values of 150 nM and 140 nM for MAVS and NS5A/5B, respectively. The IC50 values obtained in the cell-free cleavage assays are higher than the antiviral EC50 values determined in the cell-based replicon and infectivity assays (Table 1), presumably reflecting the higher concentration of protease in the cell-free assay. Importantly, there was no significant difference in inhibition of the protease cleavage of the viral versus the cellular substrate by TMC
Gastroenterology  , e2DOI: ( /j.gastro )
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