Volume 130, Issue 3, Pages 883-892 (March 2006) Hepatitis C Virus Core Protein Is a Potent Inhibitor of RNA Silencing-Based Antiviral Response  Yue Wang, Naoya Kato, Amarsanaa Jazag, Narayan Dharel, Motoyuki Otsuka, Hiroyoshi Taniguchi, Takao Kawabe, Masao Omata  Gastroenterology  Volume 130, Issue 3, Pages 883-892 (March 2006) DOI: 10.1053/j.gastro.2005.12.028 Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 1 The RNAi targets both the IRES and the replicative intermediate of HCV. (A) In vitro-transcribed HCV IRES and total RNA of Huh7 were incubated with Dicer RNase enzyme (lanes 2 and 4). Lanes 1 (IRES) and 3 (total RNA) are the controls without digestion. (B) Top: Diagram of pRSV-HCV1b. RSV, Rous sarcoma virus long terminal repeat promoter; Rluc, Renilla luciferase gene; Fluc, firefly luciferase gene; IRES, internal ribosomal entry site. Bottom: Organization of the HCV subgenomic replicon pRep-Feo.24,25 UTR, untranslated region; ΔC, truncated HCV core region; Fluc, firefly luciferase gene; NPT, neomycin phosphotransferase gene; EMCV, encephalomyocarditis virus. (C) Total RNA extracted from Huh7 cells transfected with pRSV-HCV1b or pRep-Feo was used to conduct HCV-related siRNA detection assay. Dicer expression was confirmed on Western blots using the anti-human Dicer serum. Replicon RNA was detected by an RNase protection assay using the NS5B probe. Gastroenterology 2006 130, 883-892DOI: (10.1053/j.gastro.2005.12.028) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 2 HCV core protein suppresses the RNAi system. (A) Huh7, HepG2, or HeLa cells were transfected with plasmids expressing firefly luciferase, luciferase silencing shRNA, and each of the HCV proteins. The relative luciferase activities were determined by setting the activity of control cell lysate, without HCV protein expression, as 1. The results are expressed as the mean ± SD of 6 experiments (*P = .001; **P = .002; ***P = .003). (B) Top: Huh7, HepG2, or HeLa cells were transfected with plasmids expressing firefly luciferase and luciferase-silencing shRNA, and 0 μg, 0.05 μg, 0.1 μg, or 0.2 μg of the plasmid expressing the HA-tagged core protein (0.1 μg:*P = .043; **P = .039; ***P = .048. 0.2 μg: *P = .005; **P = .002; ***P = .004). Bottom: The expression of HA-tagged core protein and GAPDH in Huh7, HepG2, and HeLa cells. (C) The expression of p53 and HA-tagged core protein in normal Huh7 cells or in Huh7 cells in which p53 expression was stably knocked down, after transfection with increasing amounts of the plasmid expressing HA-tagged core protein. Gastroenterology 2006 130, 883-892DOI: (10.1053/j.gastro.2005.12.028) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 3 HCV core protein suppresses the RNAi system via a physical interaction with Dicer. (A) Top: Huh7 cells were transfected with plasmids expressing firefly luciferase, luciferase–silencing shRNA, and N-terminal–deleted and/or C-terminal–truncated core protein mutants expressed as HA-tagged fusion proteins (*P = .005; **P = .043). Bottom: The subcellular localizations of N-terminal–deleted and/or C-terminal–truncated core proteins by indirect immunofluorescence assay. (B) Huh7, HepG2, or HeLa cells were transfected with a plasmid expressing firefly luciferase, 50 nmol/L of siRNA duplex, and plasmid expressing N-terminal–deleted and/or C-terminal–truncated core protein mutants expressed as HA-tagged fusion proteins. (C) Huh7 cells were transfected with plasmids expressing T7-tagged Dicer and each of the HA-tagged N-terminal–deleted and/or C-terminal–truncated core protein mutants. Immunoprecipitation was performed using anti-HA antibody; the bound proteins were processed for immunoblot analysis and probed with anti-T7 antibody. (D) The subcellular localizations of Dicer and HCV core protein were assessed by immunofluorescent staining. Gastroenterology 2006 130, 883-892DOI: (10.1053/j.gastro.2005.12.028) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 4 Dicer and/or core protein expression affects the replication of the HCV subgenomic replicon. (A) Huh7 cells were cotransfected with in vitro-transcribed HCV subgenomic RNA and with the empty control plasmid (mock; solid circles); the Dicer-expressing plasmid (open circles); the core-expressing plasmid (solid squares); the Dicer-silencing plasmid (solid triangle), or both the Dicer- and core-expressing plasmids (open squares). The luciferase activities were expressed by setting the activity of the cell lysate at 24 hours after transfection with HCV subgenomic replicon and the empty control plasmid as 1. The results presented are the mean ± SD of 3 experiments. (B) Huh7 cells were cotransfected with in vitro-transcribed HCV subgenomic RNA and with the empty control plasmid, the Dicer-expressing plasmid, the core-expressing plasmid, the Dicer-silencing plasmid, or both the Dicer- and core-expressing plasmids. G418-resistant Huh7 cells harboring the HCV subgenomic replicon were selected and stained at 3 weeks after transfection. Gastroenterology 2006 130, 883-892DOI: (10.1053/j.gastro.2005.12.028) Copyright © 2006 American Gastroenterological Association Terms and Conditions