Hepatitis C Virus Core Protein Inhibits Mitochondrial Electron Transport and Increases Reactive Oxygen Species (ROS) Production

Abstract

Hepatitis C infection causes a state of chronic oxidative stress, which may contribute to fibrosis and carcinogenesis in the liver. Previous studies have shown that expression of the HCV core protein in hepatoma cells depolarized mitochondria and increased reactive oxygen species (ROS) production, but the mechanisms of these effects are unknown. In this study we examined the properties of liver mitochondria from transgenic mice expressing HCV core protein, and from normal liver mitochondria incubated with recombinant core protein. Liver mitochondria from transgenic mice expressing the HCV proteins core, E1 and E2 demonstrated oxidation of the glutathione pool and a decrease in NADPH content. In addition, there was reduced activity of electron transport complex I, and increased ROS production from complex I substrates. There were no abnormalities observed in complex II or complex III function. Incubation of control mitochondria in vitro with recombinant core protein also caused glutathione oxidation, selective complex I inhibition, and increased ROS production. Proteinase K digestion of either transgenic mitochondria or control mitochondria incubated with core protein showed that core protein associates strongly with mitochondria, remains associated with the outer membrane, and is not taken up across the outer membrane. Core protein also increased Ca 2؉ uptake into isolated mitochondria. These results suggest that interaction of core protein with mitochondria and subsequent oxidation of the glutathione pool and complex I inhibition may be an important cause of the oxidative stress seen in chronic hepatitis C. Hepatitis C virus (HCV)2 infection produces acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (1). Severity and rate of progression of the disease are highly variable and may reflect both host and viral factors (2) but the mechanisms of pathogenesis are incompletely understood. Because current antiviral treatment can only eliminate the virus in about 50% of patients (3-5), therapies to reduce disease progression in chronically infected individuals would be of great benefit. Thus, understanding the mechanisms of HCV pathogenesis is an important goal of HCV research. Numerous studies have shown that oxidative stress is present in chronic hepatitis C to a greater degree than in other inflammatory liver diseases (6, 7) and a prospective study showed improvement in liver injury in chronic hepatitis C with antioxidant treatment (8). Previous studies from our laboratory and others (9 -12) have shown that HCV core protein induces the production of reactive oxygen species (ROS) but the mechanism of the core-associated increase in ROS production is not understood.HCV core protein localizes to ER (13, 14), fat droplets (15, 16), and nucleus (17) as well as mitochondria (9,18,19). It has been shown to produce multiple cellular effects including changes in gene transcription, signal transduction, immune presentation, cell cycle regulation, and apoptosis (20 -25). Despite this evidence, it is not known ...

Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3

“…This would not be unexpected because the core protein itself has been implicated as a major cause of HCV-induced oxidative stress. 26,49,50 In conclusion, this study has identified FOXO3 as a novel protective factor for alcoholic liver injury. Loss of FOXO3 expression in mice results in a phenotype in which alcohol consumption causes severe liver injury that is histologically similar to human alcoholic hepatitis.…”

“…HCV transgenic mice (SL-139 line) were generated on a C57BL/6J background, as previously described. 26 These mice are a hemizygous model and express HCV structural proteins (core, E1, E2, and p7) in hepatocytes under control of the murine albumin enhancer/promoter. Sod2 þ/À mice on a C57BL/6J background have been previously described.…”

“…We used mice that have liver-specific expression of the HCV protein core, E1, E2, and p7 and display increased mitochondrial oxidative stress. 26 To maximize the chance to observe an oxidative liver injury, we also crossed the HCV mice with Sod2 heterozygous knockout mice (Sod2 þ/À ). The latter mice have a mild, subclinical oxidative stress phenotype and have previously shown utility in toxicological studies.…”

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein

“…Mitochondria were purified using Nycodenz (Nycomed Pharma, Zürich, Switzerland) according to the protocols reported by Okado-Matsumoto et al 17 For transient transfection experiments, HepG2 cells were transfected with a core-expression plasmid using TransIT-LT1 (Mirus Bio, Madison, WI). Huh7 cells harboring HCV genotype 1b full-genomic (RCYM1) 18 or subgenomic replicon (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), and livers of 3-month-old core-gene transgenic mice 2 were also used for the analysis.…”

“…In livers of transgenic mice harboring the core gene, increased ROS production has been observed. [7][8][9] A recent study found, by the proteomic profiling of biopsy specimens, that an impairment in key mitochondrial processes, including fatty acid oxidation and oxidative phosphorylation, and in the response to oxidative stress occurs in HCV-infected human liver with advanced fibrosis. 10 Therefore, it is probable that the HCV core protein affects mitochondrial functions because such pathogenesis is observed in both HCV core-transgenic mice and HCV-infected patients.…”