Volume 133, Issue 4, Pages 1156-1165 (October 2007) Antiviral Activity and Hepatoprotection by Heme Oxygenase-1 in Hepatitis B Virus Infection  Ulrike Protzer, Stefan Seyfried, Maria Quasdorff, Gabriele Sass, Miriam Svorcova, Dennis Webb, Felix Bohne, Marianna Hösel, Peter Schirmacher, Gisa Tiegs  Gastroenterology  Volume 133, Issue 4, Pages 1156-1165 (October 2007) DOI: 10.1053/j.gastro.2007.07.021 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 HO-1 induction by CoPP in mouse livers. CH57Bl/6 mice were infected intravenously with 109 IU of AdHBV or control vector AdHBVk/o or injected with saline, and after 7 days injected intraperitoneally with 10 mg/kg CoPP. (A) Expression levels of HO-1 mRNA were quantified by real-time reverse-transcription PCR (n = 3; data are expressed as mean ± standard error of the mean). (B) HO-1, HBV core, and β-actin were analyzed by Western blot in pooled liver lysates. Gastroenterology 2007 133, 1156-1165DOI: (10.1053/j.gastro.2007.07.021) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 CoPP pretreatment inhibits HBV gene expression and the activity of alanine aminotransferase into sera of AdHBV-infected mice. Either 10 mg/kg CoPP or solvent was injected intraperitoneally 24 hours before infection with AdHBV in mice (n = 5; data are expressed as mean ± standard error of the mean). (A) At days 1, 5, and 26 postinfection, HO-1 mRNA expression was measured by real-time reverse-transcription PCR. Western blot analysis was performed of (B) pooled liver protein lysates, and (C) serum ALT activities were determined in mouse sera. (D) HBeAg serum levels were measured by an enzyme-linked immunosorbent–based assay. *P ≤ .05 CoPP-treated vs mock-treated AdHBV-infected mice. Gastroenterology 2007 133, 1156-1165DOI: (10.1053/j.gastro.2007.07.021) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 HO-1 induction by CoPP reduces AdHBV-induced hepatic necroinflammation and HBV core protein expression. Mice were treated with either 10 mg/kg CoPP or solvent intraperitoneally 24 hours before AdHBV infection. Histologic analysis (A–I, H&E staining) and HBV core antigen immunostaining (B–J) of liver sections of representative animals show persistent reduction of AdHBV-induced necroinflammatory lesions (E vs C and I vs G at day 5 and 26, respectively) and HBV core expression (F vs D and J vs H at day 5 and 26, respectively) in the CoPP-treated animals (A and B) solvent treated control without infection. Gastroenterology 2007 133, 1156-1165DOI: (10.1053/j.gastro.2007.07.021) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 HO-1 induction inhibits HBV replication in stably transfected hepatoma cells. HO-1 was induced by daily incubation with 10 μg/mL CoPP in parental HepG2 hepatoma cells and in HBV-replicating HepG2-H1.3 and HepG2.2.15 cells. Cells were lysed after 5 days of treatment. (A) Western blot analysis of HO-1 protein in CoPP-treated and untreated cells. (B) Real-time reverse-transcription PCR analysis of HO-1 mRNA. *P ≤ .05 CoPP-treated vs untreated cells (n = 3). Data are expressed as mean ± standard error of the mean. (C) Southern blot analysis of intracellular HBV DNA in HepG2.2.15 cells using a 32P-labeled HBV-DNA probe. Gastroenterology 2007 133, 1156-1165DOI: (10.1053/j.gastro.2007.07.021) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Analysis of the HBV replication cycle after induction and silencing of HO-1 in HepG2-H1.3 cells. HepG2-H1.3 hepatoma cells were transfected with HO-1–specific and nonsilencing (ns) scrambled siRNA, and treated with CoPP. (A) Western blot analysis of HO-1 and HBV core protein at day 5 posttransfection. (B) Northern blot analysis of intracellular HBV RNAs. (C) Nuclear HBV cccDNA and mitochondrial DNA analyzed by specific real-time PCRs. Agarose gel electrophoresis of PCR products is shown. (D) HepG2-H1.3 cells were grown to confluency and cultivated for 10 days to allow accumulation of HBV cccDNA before treatment with lamivudine (lam), CoPP, or both was started. HBV cccDNA was quantified relative to mitochondrial DNA in cellular lysates collected every second day. Regression curve analysis of mean values determined from 3 independent vials per time point and treatment is shown. •, LAM; , CoPP; ▾, LAM and CoPP; —, LAM; – – –, CoPP; - - -, LAM and CoPP. Gastroenterology 2007 133, 1156-1165DOI: (10.1053/j.gastro.2007.07.021) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Effect of HO-1 induction on stability of HBV core protein. (A) Autoradiography of HBV core protein in HepG2 cells, which were transfected with plasmid pCHcore (encoding the HBV core protein) and pulse-labeled for 24 hours, after a 6-hour or 24-hour chase reaction in the presence or absence of CoPP (upper panel). Immunoprecipitation was performed using rabbit antiserum against HBV core protein in cell lysates containing 200 μg of protein each. GAPDH expression was analyzed by Western blot in immunoprecipitation supernatants (lower panel). (B) HO-1 induction by CoPP as well as GAPDH expression as a loading control was analyzed by Western blot in cellular lysates. (C) Quantification of remaining HBV core protein after HO-1 induction for 6 hours from 3 independent experiments detected by autoradiography using the Gel Doc 2000 System. *P ≤ .05 for CoPP-treated vs untreated cells. Gastroenterology 2007 133, 1156-1165DOI: (10.1053/j.gastro.2007.07.021) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 HO-1 induction or over expression decreases HBV replication in HBV transgenic mice. HBV transgenic mice were injected with either 10 mg/kg CoPP 3 times intraperitoneally or once with 109 IU AdHO-1 or AdGFP intravenously. HO-1 and HBV core protein expression was determined after 5 days. (A) Western blot of protein liver lysates from 2 animals per group. (B) Southern blot analysis of hepatic HBV replication using a 32P-labeled HBV-DNA probe (40 μg pooled liver DNA/lane). (C) HBV viremia was measured by quantitative real-time PCR of HBV DNA in mouse sera (n = 4). Data are given as mean ± standard error of the mean. Gastroenterology 2007 133, 1156-1165DOI: (10.1053/j.gastro.2007.07.021) Copyright © 2007 AGA Institute Terms and Conditions