Volume 48, Issue 2, Pages 313-321 (October 2012) Hepatic Expression of HCV RNA-Dependent RNA Polymerase Triggers Innate Immune Signaling and Cytokine Production  Guann-Yi Yu, Guobin He, Chia-Yang Li, Martin Tang, Sergei Grivennikov, Wan-Ting Tsai, Ming-Sian Wu, Chih-Wen Hsu, Yu Tsai, Lily Hui-Ching Wang, Michael Karin  Molecular Cell  Volume 48, Issue 2, Pages 313-321 (October 2012) DOI: 10.1016/j.molcel.2012.07.032 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 HCV NS5B Expression Induces Liver Injury and Cytokine Production in Mice (A) Male mice (C57BL/6) were hydrodynamically injected with in vitro transcribed HCV subgenomic RNA, and liver tissue was collected 24 hr later for quantitative real-time PCR (RT-PCR) analysis (n = 3 ± SD). Replication-defective GND replicon RNA contains a single amino acid mutation (Aspartic acid changed to Asparagine) in the NS5B GDD motif. (B) Adenoviruses expressing GFP and HCV nonstructural proteins were injected into WT mice via the tail vein. Full-length NS proteins are marked with “ ∗ ” in the FLAG immunoblot. (C) WT mice were injected with adenoviruses expressing NS proteins. When indicated, the viruses were UV-irradiated for 1 hr on ice before injection (n = 3 ± SD). IL-6 and ALT were measured in serum samples at 16 hr and 24 hr post infection, respectively, and IFN-β RNA was measured in liver tissue collected at 24 hr post infection. (D) Mice were injected with adenoviruses; after 16 hr, livers were collected and subjected to RT-PCR analysis (n = 3 ± SD) and immunoblotting. Molecular Cell 2012 48, 313-321DOI: (10.1016/j.molcel.2012.07.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Type I IFN and IL-6 Are the Main Cytokines Induced by NS5B in Mouse and Human Hepatocytes (A) Primary mouse hepatocytes were infected with adenoviruses (moi = 10). NS5B-AAA is an NS5B substitution mutant in which the GDD motif was replaced with AAA, resulting in loss of RdRp activity. After 18 hr, IL-6 and type I IFN in culture medium were examined by ELISA or cell-based IFN activity assay (n = 3 ± SD). NS5B and GFP expression was detected by immunolotting. (B) Hepatocytes infected with the indicated adenoviruses were analyzed by RT-PCR for expression of type I IFNs and IFN-regulated genes (n = 3 ± SD). (C) Mice were infected with Ad-NS5B, Ad-Del, Ad-DM, and Ad-GFP (n = 3 ± SD). Blood samples and liver tissue collected at 16 hr and 24 hr post infection were subjected to cytokine analysis, ALT activity assay, and TUNEL staining. (D) Expression of NS5B and GFP in hepatocytes infected with the indicated adenoviruses was detected by immunoblotting. (E) NeHepLxHT human immortalized hepatocytes were infected with the indicated adenoviruses. After 18 hr, cytokines in culture supernatants were examined by ELISA (n = 3 ± SD), and protein expression was examined by immunoblotting. (F and G) NeHepLxHT (F) and Huh7 cells (G) were infected with HCV (Jc-1) and viral RNA, and cytokine mRNAs were measured by RT-PCR (n = 3 ± SD). Molecular Cell 2012 48, 313-321DOI: (10.1016/j.molcel.2012.07.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 RdRp Stimulates RNA Synthesis and TBK-1-Dependent IRF3 Activation and Cytokine Production (A) WT hepatocytes infected with the indicated recombinant adenoviruses were metabolically labeled with H3-uridine in the presence of actinomycin D, and 3H-uridine incorporation into cellular RNA was measured (n = 4 ± SD). (B and C) Hepatocytes from the indicated mouse strains (WT in B) were infected with the indicated adenoviruses, and IRF3 dimerization was examined 18 hr later. (D) Tbk1+/+/Tnf−/− and Tbk1−/−/Tnf−/− hepatocytes were infected with adenoviruses, and type I IFN and IL-6 secretion were analyzed (n = 3 ± SD). (E) Tbk1+/+/Tnf−/−, Tbk1+/−/Tnf−/− mice were infected with Ad-NS5B, and serum cytokines and ALT were measured (n = 3 ± SD). (F) dih/Stat3/1 cells harboring MAVS-specific (shMAVS) or control (shLuc) shRNAs were infected with the indicated adenoviruses. IRF3 dimerization and cytokine production were examined (n = 3 ± SD). Molecular Cell 2012 48, 313-321DOI: (10.1016/j.molcel.2012.07.032) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 RdRp Induces NF-κB-Dependent IL-6 Production and STAT3 Activation (A) Mice were infected with the indicated adenoviruses, and livers were collected 24 hr later. NF-κB activation was examined by EMSA. (B–D) The same livers as above were fixed, and paraffin-embedded sections were stained with phospho-p65 (B), IL-6 (C), and phospho-STAT3 antibodies (D). Arrows (→) mark hepatocytes with positive staining signal, and arrow heads (►) mark stained immune cells. Molecular Cell 2012 48, 313-321DOI: (10.1016/j.molcel.2012.07.032) Copyright © 2012 Elsevier Inc. Terms and Conditions