Biliverdin inhibits hepatitis C virus nonstructural 3/4A protease activity: mechanism for the antiviral effects of heme oxygenase?

UNLABELLED: Induction of heme oxygenase-1 (HO-1) inhibits hepatitis C virus (HCV) replication. Of the products of the reaction catalyzed by HO-1, iron has been shown to inhibit HCV ribonucleic acid (RNA) polymerase, but little is known about the antiviral activity of biliverdin (BV). Herein, we report that BV inhibits viral replication and viral protein expression in a dose-dependent manner in replicons and cells harboring the infectious J6/JFH construct. Using the SensoLyte 620 HCV Protease Assay with a wide wavelength excitation/emission (591 nm/622 nm) fluorescence energy transfer peptide, we found that both recombinant and endogenous nonstructural 3/4A (NS3/4A) protease from replicon microsomes are potently inhibited by BV. Of the tetrapyrroles tested, BV was the strongest inhibitor of NS3/4A activity, with a median inhibitory concentration (IC(50)) of 9 µM, similar to that of the commercial inhibitor, AnaSpec (Fremont, CA) #25346 (IC(50) 5 µM). Lineweaver-Burk plots indicated mixed competitive and noncompetitive inhibition of the protease by BV. In contrast, the effects of bilirubin (BR) on HCV replication and NS3/4A were much less potent. Because BV is rapidly converted to BR by biliverdin reductase (BVR) intracellularly, the effect of BVR knockdown on BV antiviral activity was assessed. After greater than 80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of α-interferon in replicons. CONCLUSION: BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to the antiviral activity of HO-1. These findings suggest that BV or its derivatives may be useful in future drug therapies targeting the NS3/4A protease.

Hepatitis C virus core protein enhances Telomerase activity in Huh7 cells.

Hepatitis C is an oncogenic virus although the mechanisms responsible for this behavior are not clear. We studied the effects of hepatitis C virus (HCV) core protein expression on Telomerase, an enzyme closely associated with cellular immortalization and neoplasia. The aim of this study was to investigate the effects of HCV core protein on the regulation of Telomerase activity in human hepatoma cells. Regulation and expression of human Telomerase reverse transcriptase (TERT) was compared in Huh7 cells stably transfected with HCV core protein or cells expressing vector alone. Telomerase activity was measured using Quantitative Telomerase Detection (QTD) and telomere length was measured by fluorescence in situ hybridization (FISH). Transient transfection and luciferase assay were used to evaluate TERT promoter activity. Telomerase activity was increased twofold in Huh7 cells expressing HCV core protein compared to controls (P < 0.01). This was accompanied by a 1.4-fold increase of TERT mRNA and 1.9-fold increase in TERT protein (P < 0.01 in either case). Cellular fractionation and immunocytochemical studies showed increased localization of TERT in the nucleus of core-expressing cells as compared to controls. FISH assay confirmed that telomeres of HCV core-expressing Huh7 cells were relatively longer than those of control cells (0.22 + 0.05 vs. 0.12 + 0.03, P < 0.01). TERT promoter activity was enhanced about 30% in HCV core-expressing Huh7 cells compared to control cells (P < 0.02). HCV core protein is associated with increased Telomerase activity in hepatoma cells. These findings suggest that enhancement of Telomerase activity by HCV core protein may contribute to the oncogenicity of HCV.

Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury.

UNLABELLED: Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase-1 (HO-1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO-1 overexpression on HCV replication and hepatocellular injury. Full-length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3-3') were transfected with complete human HO-1 sequences or empty vector for control. Cell lines overexpressing HO-1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro-oxidant levels, and resistance to oxidative injury were assessed. HO-1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO-1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO-1 showed reduced prooxidant levels at baseline and increased resistance to oxidant-induced cytotoxicity. HO-1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO-1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO-1 had minimal effect on HCV replication. CONCLUSION: Overexpression or induction of HO-1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO-1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection.