Gastroenterology

Gastroenterology

Volume 122, Issue 2, February 2002, Pages 366-375
Gastroenterology

Basic Research
Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein,☆☆,

https://doi.org/10.1053/gast.2002.30983Get rights and content

Abstract

Background & Aims: The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood. Indirect evidence suggests that oxidative stress and mitochondrial injury play a role. The aim of this study was to determine if the HCV core protein itself alters mitochondrial function and contributes to oxidative stress. Methods: HCV core protein was expressed in 3 different cell lines, and reactive oxygen species (ROS) and lipid peroxidation products were measured. Results: Core expression uniformly increased ROS. In 2 inducible expression systems, core protein also increased lipid peroxidation products and induced antioxidant gene expression as well. A mitochondrial electron transport inhibitor prevented the core-induced increase in ROS. A fraction of the expressed core protein localized to the mitochondria and was associated with redistribution of cytochrome c from mitochondrial to cytosolic fractions. Sensitivity to oxidative stress was also seen in HCV transgenic mice in which increased intrahepatic lipid peroxidation products occurred in response to carbon tetrachloride. Conclusions: Oxidative injury occurs as a direct result of HCV core protein expression both in vitro and in vivo and may involve a direct effect of core protein on mitochondria. These results provide new insight into the pathogenesis of hepatitis C and provide an experimental rationale for investigation of antioxidant therapy.

GASTROENTEROLOGY 2002;122:366-375

Section snippets

Cell lines

Human hepatoma Huh-7 cells were maintained in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin G (100 U/mL), streptomycin (100 U/mL; Gibco-BRL, Rockville, MD) in a humidified 37°C/5% CO2 incubator. HeLa Tet-Off cells (Clontech, Palo Alto, CA) were cultured in the same medium except for the addition of 100 μg/mL G418. The complementary DNA (cDNA) fragment encoding full-length HCV core protein of an infectious cDNA clone (genotype 1b)8 was

Fluorescence microscopy

Cells were grown on plastic culture slides and incubated at 37°C for 30 minutes with MitoTracker Red (50 nmol/L; Molecular Probes, Inc.). Cells were fixed in 3.7% paraformaldehyde and permeabilized with 0.25% saponin in PBS. Fixed cells were subsequently incubated with mouse monoclonal anti-hepatitis C core antibody (Affinity Bioreagents, Golden, CO) at a dilution of 1:350 for 2 hours at room temperature and with Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Inc.) at a dilution of

Core protein expression in inducible cell lines

We developed clonal, stably transformed cell lines from human hepatoma (Huh-7/191-20) and human cervical carcinoma cells (HeLa/191-14) capable of conditionally expressing the full-length HCV core protein (amino acids 1–191) under control of the Tet-Off promoter. Figure 1A shows the tight control of core protein expression in both Huh-7/191-20 and HeLa/191-14 cells.

. HCV core protein expression and cellular DNA content. (A) Huh-7/191-20 and HeLa/191-14 cells were cultured in the presence (off) or

Discussion

This study used 2 different inducible cell culture systems to show that expression of the HCV core protein directly produces oxidative stress. This effect is blocked by an inhibitor of mitochondrial electron transport, and core protein itself localizes in mitochondria. Several possible mechanisms could explain a core-induced change in mitochondrial function. One explanation is that core protein alters signal transduction pathways that promote the mitochondrial permeability transition. Core

Acknowledgements

The authors thank S. Okuda for expert technical assistance, H. Fishman for assistance with confocal microscopy, S. Watowich for providing purified core protein, J. Sun for helpful discussions, and S. Ballinger and I. Boldough for their critical comments.

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    Address requests for reprints to: Steven A. Weinman, M.D., Department of Physiology and Biophysics, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0641. e-mail: [email protected]; fax: (409) 772-3381.

    ☆☆

    Supported in part by grants U19-AI40035 from the National Institute of Allergy and Infectious Diseases and AA12863 from the National Institute on Alcohol Abuse and Alcoholism.

    Drs. Okuda and Li authors contributed equally to this work.

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