Ikuo Shoji

Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors.

A baculovirus (Autographa californica nucleopolyhedrovirus) vector containing a strong promoter, the CAG promoter, was developed to introduce foreign genes into mammalian cells. Recombinant baculoviruses carrying a reporter gene under the control of the CAG promoter were inoculated into various mammalian cell lines. High-level expression was observed not only in hepatocytes but also in other non-hepatic cell lines tested. Expression of the reporter gene was detected even 14 days after infection. The infectious titre of the recovered baculoviruses decreased significantly after infection, indicating that the baculoviruses did not replicate in mammalian cells. We then compared the efficiencies of gene expression by the baculovirus vector with that of a replication-defective adenovirus vector by using the same expression unit. The same level of expression was observed in HepG2, HeLa and COS7 cells by both vectors. Efficient expression and proper processing were observed in mammalian cells infected with baculoviruses carrying genes coding for structural regions of hepatitis C virus. These results suggest that the baculovirus vector is a good tool for gene delivery into various mammalian cells in order to study the function of foreign genes.

Hepatitis C Virus Infection Induces Apoptosis through a Bax-Triggered, Mitochondrion-Mediated, Caspase 3-Dependent Pathway

ABSTRACT We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).

Molecular biology of hepatitis C virus

Infection with hepatitis C virus (HCV), which is distributed worldwide, often becomes persistent, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. For many years, the characterization of the HCV genome and its products has been done by heterologous expression systems because of the lack of a productive cell culture system. The development of the HCV replicon system is a highlight of HCV research and has allowed examination of the viral RNA replication in cell culture. Recently, a robust system for production of recombinant infectious HCV has been established, and classical virological techniques are now able to be applied to HCV. This development of reverse genetics-based experimental tools in HCV research can bring a greater understanding of the viral life cycle and pathogenesis of HCV-induced diseases. This review summarizes the current knowledge of cell culture systems for HCV research and recent advances in the investigation of the molecular virology of HCV.

E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

ABSTRACT Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

Hepatitis C Virus Infection Promotes Hepatic Gluconeogenesis through an NS5A-Mediated, FoxO1-Dependent Pathway

ABSTRACT Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Here, using Huh-7.5 cells either harboring HCV-1b RNA replicons or infected with HCV-2a, we showed that HCV transcriptionally upregulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. In this way, HCV enhanced the cellular production of glucose 6-phosphate (G6P) and glucose. PEPCK and G6Pase gene expressions are controlled by the transcription factor forkhead box O1 (FoxO1). We observed that although neither the mRNA levels nor the protein levels of FoxO1 expression were affected by HCV, the level of phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells compared to the control cells, resulting in an increased nuclear accumulation of FoxO1, which is essential for sustaining its transcriptional activity. It was unlikely that the decreased level of FoxO1 phosphorylation was mediated through Akt inactivation, as we observed an increased phosphorylation of Akt at Ser473 in HCV-infected cells compared to control cells. By using specific inhibitors of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS), we demonstrated that HCV infection induced JNK activation via increased mitochondrial ROS production, resulting in decreased FoxO1 phosphorylation, FoxO1 nuclear accumulation, and, eventually, increased glucose production. We also found that HCV NS5A mediated increased ROS production and JNK activation, which is directly linked with the FoxO1-dependent increased gluconeogenesis. Taken together, these observations suggest that HCV promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathway.

HCV replication suppresses cellular glucose uptake through down-regulation of cell surface expression of glucose transporters☆

BACKGROUND/AIMS Persistent infection with hepatitis C virus (HCV) causes extrahepatic diseases, including diabetes. We investigated the possible effect(s) of HCV replication on cellular glucose uptake and expression of the facilitative glucose transporter (GLUT) 2 and 1. METHODS We used Huh-7.5 cells harboring either an HCV subgenomic RNA replicon (SGR) or an HCV full-genomic RNA replicon (FGR), HCV-infected cells, and the respective cells treated with interferon (IFN). We also used liver tissue samples obtained from patients with or without HCV infection. RESULTS Glucose uptake and surface expression of GLUT2 and GLUT1 were suppressed in SGR, FGR and HCV-infected cells compared to the control cells. Expression levels of GLUT2 mRNA, but not GLUT1 mRNA, were lower in SGR, FGR and HCV-infected cells than in the control. Luciferase reporter assay demonstrated decreased GLUT2 promoter activities in SGR, FGR and HCV-infected cells. IFN treatment restored glucose uptake, GLUT2 surface expression, GLUT2 mRNA expression and GLUT2 promoter activities. Also, GLUT2 expression was reduced in hepatocytes of liver tissues obtained from HCV-infected patients. CONCLUSIONS HCV replication down-regulates cell surface expression of GLUT2 partly at the transcriptional level, and possibly at the intracellular trafficking level as suggested for GLUT1, thereby lowering glucose uptake by hepatocytes.

17β-estradiol inhibits the production of infectious particles of hepatitis C virus.

Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh‐7.5 cells, the possible inhibitory effect(s) of female hormones such as 17β‐estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E2, but not P4, significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E2–mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ERα‐selective agonist 4, 4′, 4″‐ (4‐propyl‐[1H]‐pyrazole‐1, 3, 5‐triyl)trisphenol (PPT), but not with the ERβ‐selective agonist 2, 3‐bis (4‐hydroxyphenyl)‐propionitrile (DPN) or the G protein‐coupled receptor 30 (GPR30)‐selective agonist 1‐(4‐[6‐bromobenzo 1, 3 dioxol‐5‐yl]‐3a, 4, 5, 9b‐tetrahydro‐3H‐cyclopenta [c] quinolin‐8‐yl)‐ethanone (G‐1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E2, inhibits the production of HCV infectious particles in an ERα–dependent manner.

Anti-hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species

Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti‐HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti‐HCV activity with 50%‐inhibitory concentrations (IC50) of 20.0 and 8.0 μg/mL, respectively. Through bioactivity‐guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti‐HCV compounds, their IC50 being 8.8, 7.2, 4.6 and 16.4 μg/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti‐HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti‐HCV activity, their IC50 being 2.5 and 6.2 μg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti‐HCV activity, with an IC50 of 3.7 μg/mL. Time‐of‐addition analysis revealed that all Glycyrrhiza‐derived anti‐HCV compounds tested in this study act at the post‐entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV.

Proteasomal Turnover of Hepatitis C Virus Core Protein Is Regulated by Two Distinct Mechanisms: a Ubiquitin-Dependent Mechanism and a Ubiquitin-Independent but PA28γ-Dependent Mechanism

ABSTRACT We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28γ, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways.

Involvement of PA28γ in the propagation of hepatitis C virus

We have reported previously that the proteasome activator PA28γ participates not only in degradation of hepatitis C virus (HCV) core protein in the nucleus but also in the pathogenesis in transgenic mice expressing HCV core protein. However, the biological significance of PA28γ in the propagation of HCV has not been clarified. PA28γ is an activator of proteasome responsible for ubiquitin‐independent degradation of substrates in the nucleus. In the present study, knockdown of PA28γ in cells preinfection or postinfection with the JFH‐1 strain of HCV impaired viral particle production but exhibited no effect on viral RNA replication. The particle production of HCV in PA28γ knockdown cells was restored by the expression of an small interfering RNA (siRNA)‐resistant PA28γ. Although viral proteins were detected in the cytoplasm of cells infected with HCV, suppression of PA28γ expression induced accumulation of HCV core protein in the nucleus. HCV core protein was also degraded in the cytoplasm after ubiquitination by an E3 ubiquitin ligase, E6AP. Knockdown of PA28γ enhanced ubiquitination of core protein and impaired virus production, whereas that of E6AP reduced ubiquitination of core protein and enhanced virus production. Furthermore, virus production in the PA28γ knockdown cells was restored through knockdown of E6AP or expression of the siRNA‐resistant wild‐type but not mutant PA28γ incapable of activating proteasome activity. Conclusion: Our results suggest that PA28γ participates not only in the pathogenesis but also in the propagation of HCV by regulating the degradation of the core protein in both a ubiquitin‐dependent and ubiquitin‐independent manner. (HEPATOLOGY 2010;)