Basic ResearchMitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein☆,☆☆,★
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.
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.
References (45)
Current therapy for chronic hepatitis C
Gastroenterology
(2000)- et al.
Hepatocellular mitochondrial alterations in patients with chronic hepatitis C: ultrastructural and biochemical findings
Am J Gastroenterol
(1999) - et al.
Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C
Am J Gastroenterol
(2000) - et al.
Superoxide dismutase in patients with chronic hepatitis C virus infection
Free Radic Biol Med
(1998) - et al.
A pilot study of the effects of d-alpha-tocopherol on hepatic stellate cell activation in chronic hepatitis C
Gastroenterology
(1997) - et al.
Apoptosis and hematopoiesis in murine fetal liver
Blood
(1993) - et al.
Tumor necrosis factor-alpha-inducible IkappaBalpha proteolysis mediated by cytosolic m-calpain. A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-kappab activation
J Biol Chem
(1999) - et al.
Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death
J Biol Chem
(1999) - et al.
Purification of mitochondria for apoptosis assays
Methods Enzymol
(2000) - et al.
Disruption of redox homeostasis in tumor necrosis factor-induced apoptosis in a murine hepatocyte cell line
Am J Pathol
(2000)
Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production
Biochem Biophys Res Commun
Ethanol stimulates the production of reactive oxygen species at mitochondrial complexes I and III
Free Radic Biol Med
Dolak JA, Waller RL. Mechanisms of carbon tetrachloride toxicity
Pharmacol Ther
Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein
Virology
The HCV core protein acts as a positive regulator of fas-mediated apoptosis in a human lymphoblastoid T cell line
Virology
Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins
J Biol Chem
Suppression of apoptotic cell death by hepatitis C virus core protein
Virology
Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein
Virology
Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (complex I)
J Biol Chem
Activation of intracellular signaling by hepatitis B and C viruses: C-viral core is the most potent signal inducer
Hepatology
Role of oxidative stress and antioxidant therapy in alcoholic and nonalcoholic liver diseases
Adv Pharmacol
In situ detection of lipid peroxidation in chronic hepatitis C: correlation with pathological features
J Clin Pathol
Cited by (0)
- ☆
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.