Hepatitis C Virus Core Protein Inhibits Mitochondrial Electron Transport and Increases Reactive Oxygen Species (ROS) Production
2005; Elsevier BV; Volume: 280; Issue: 45 Linguagem: Inglês
10.1074/jbc.m506412200
ISSN1083-351X
AutoresMasaaki Korenaga, Ting Wang, Yanchun Li, Lori Showalter, Teh‐Sheng Chan, Jiaren Sun, Steven A. Weinman,
Tópico(s)Liver Disease and Transplantation
ResumoHepatitis 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 Ca2+ 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 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 Ca2+ 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) 2The abbreviations used are: HCVhepatitis C virusROSreactive oxygen speciesERendoplasmic reticulumBSAbovine serum albuminGRglutathione reductaseSMPsubmitochondrial particlesDCFDAdihydrodichlorocarboxyfluorescein diacetateRTreverse transcriptionELISAenzyme-linked immunosorbent assay 2The abbreviations used are: HCVhepatitis C virusROSreactive oxygen speciesERendoplasmic reticulumBSAbovine serum albuminGRglutathione reductaseSMPsubmitochondrial particlesDCFDAdihydrodichlorocarboxyfluorescein diacetateRTreverse transcriptionELISAenzyme-linked immunosorbent assay infection produces acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (1Seeff L.B. Hepatology. 2002; 36: S35-S46Crossref PubMed Google Scholar). Severity and rate of progression of the disease are highly variable and may reflect both host and viral factors (2Cerny A. Chisari F.V. 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To clarify these questions, we investigated the interaction of HCV core protein with mitochondria in transgenic mice and by direct interaction of recombinant core protein with isolated mitochondria.HCV protein expression caused an increase in mitochondrial ROS production, an oxidation of the mitochondrial glutathione pool, inhibition of electron transport, and an increase in ROS production by mitochondrial electron transport complex I. Direct incubation of isolated mitochondria with HCV core protein resulted in an increase of Ca2+ influx and ROS production and reproduced glutathione oxidation and the reduction in complex I function. These results suggest that direct interaction of core protein with mitochondria is an important cause of the oxidative stress seen in chronic hepatitis C.MATERIALS AND METHODSGeneration of Transgenic Mice—The transgene, pAlbSVPA-HCV-S, containing the structural genes (core, E1, E2, and p7, nucleotides 342–2771) of hepatitis C virus genotype 1b, strain N, under the control of the murine albumin promoter/enhancer was described in detail by Lerat et al. (26Lerat H. Honda M. Beard M.R. Loesch K. Sun J. Yang Y. Okuda M. Gosert R. Xiao S.Y. Weinman S.A. Lemon S.M. Gastroenterology. 2002; 122: 352-365Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). This construct was injected into 1-cell F2 zygotes of C57BL/6J × C3H/HeJ mice. Fourteen transgene-positive pups were obtained, out of 121 live births, as screened by polymerase chain reaction, using two pairs of primers spanning the promoter to the core gene for one pair and the E2 gene for the other pair. Positive results were confirmed by Southern blot analysis. Each of these transgenic founder mice were backcrossed to C57BL/6J. One transgenic line, designated SL-139 was selected for subsequent experiments. Female HCV transgenic mice were used at 5–7 months of age and age matched non transgenic littermates were used as controls. All animal procedures were performed according to the National Institutes of Health Guidelines and were approved by the Institutional Animal Care and Use Committee.RNA Extraction and RT-PCR—Liver samples were collected from transgenic mice of the first backcross (N1) generation, following carbon dioxide euthanasia. RNA was extracted from the liver using TRIzol (Sigma). Contaminating DNA was removed by brief treatment with DNase, which was removed by phenol/chloroform extraction. Reverse transcription was performed using Omniscript (Qiagen, Valencia CA), followed by PCR amplification of the E2 region using RedTaq (Sigma) according to manufacturers' protocols. The PCR products were analyzed by electrophoresis on 2% agarose gels.Determination of Intrahepatic Core Protein Concentration—SL-139 mice were sacrificed by CO2 asphyxiation, and liver protein was extracted in cold RPMI 1640. Quantity of HCV core protein was determined with an HCV core ELISA kit (trak-C™, Ortho Clinical Diagnostics, Raritan, NJ). Briefly, samples were mixed with a pretreatment buffer containing detergents and incubated at 56 °C for 30 min. A set of six standards, supplied by the manufacturer, which contained 100, 50, 15, 5, 1.5, and 0 pg/ml of HCV core antigen, was prepared. Standards, pretreated samples, and controls were transferred to a microwell plate coated with capturing monoclonal antibodies against HCV core protein, and incubated at 25 °C for 60 min. After washing, monoclonal antibody F(ab′)2 fragments conjugated to horseradish peroxidase were added and incubated at 25 °C for 30 min. After a final wash step, wells were incubated in the dark with substrate for 30 min. To stop color development, sulfuric acid was added, and absorbance was measured with a microwell plate reader at a wavelength of 490 nm with a reference wavelength of 620 nm. The concentration of HCV core antigen in each sample was determined from the standard curve.Isolation of Mitochondria—Liver mitochondria were isolated by a modification of the method of Johnson and Hardy (27Martin E.J. Racz W.J. Forkert P.G. J. Pharmacol. Exp. Ther. 2003; 304: 121-129Crossref PubMed Scopus (17) Google Scholar, 28Rolo A.P. Oliveira P.J. Seica R. Santos M.S. Moreno A.J. Palmeira C.M. Toxicol. Appl. Pharmacol. 2002; 182: 20-26Crossref PubMed Scopus (11) Google Scholar, 29Johonson D. Lardy H. Methods Enzymol. 1967; 10: 94-96Crossref Scopus (1189) Google Scholar). In brief, liver (400 mg) was minced on ice and transferred (10% w/v) to isolation buffer (250 mm sucrose, 10 mm HEPES, 0.5 mm EGTA, 0.1% BSA, pH 7.4). The sample was gently homogenized by 3–4 strokes with a Dounce homogenizer and loose fitting pestle. The homogenate was centrifuged at 500 × g for 5 min at 4 °C. The supernatant fraction was retained, whereas the pellet was washed with isolation buffer and centrifuged again. The combined supernatant fractions were centrifuged at 7800 × g for 10 min at 4 °C to obtain a crude mitochondria pellet. The mitochondria pellet was resuspended in isolation buffer (without EGTA and BSA) and centrifuged again at 7800 × g for 10 min. An aliquot was removed for determination of protein concentration by the Bio-Rad assay kit, using bovine serum albumin as the standard.Determination of Glutathione Content—Liver tissue samples (50–75 mg) and mitochondrial samples (2 mg) were sonicated using a Branson Sonifer 450 (VWR Scientific Products, West Chester PA) for 15 s at power setting 3 in ice-cold 5% trichloroacetic acid and centrifuged at 3000 × g at 4 °C for 10 min. The concentration of reduced GSH was measured by the thioester method using the GSH-400 kit (Oxis International Inc., Portland, OR). Total glutathione content of samples was measured by the glutathione reductase-DTNB recycling assay (30Anderson M.E. Dolphin D. Poulson R. Avramovic O. Glutathione: Chemical, Biochemical and Medical Aspects. Part A. 1989: 339-365Google Scholar) using a commercial kit (GSH-412, Oxis International).To measure the effect of recombinant core protein (amino acids 1–179, kindly provided by S. Watowich) on mitochondrial glutathione, freshly isolated mitochondria were suspended in phosphate-buffered saline and incubated at 25 °C for 5 min with or without core protein. Proteins were precipitated and thiols stabilized by subsequent addition of sulfosalicylic acid to a final concentration of 5%. To confirm that decreases in reduced GSH measured by the thioester method were indeed a result of oxidation, parallel mitochondrial samples were either further oxidized by exposure to 0.2 mm tBOOH for 5 min, or reduced by freeze-thaw followed by incubation with glutathione reductase (4.1 units/ml) and NADPH (1 mm) for 5 min at 25 °C. Following reduction, samples were precipitated with sulfosalicylic acid and processed as described. Control experiments showed that this tBOOH treatment fully oxidized the glutathione pool under these conditions and it was used to determine the background value for the assay.NADPH and Glutathione Reductase Measurement—NADPH was measured in isolated mouse liver mitochondria by the method described by Zhang et al. (31Zhang Z. Yu J. Stanton R.C. Anal. Biochem. 2000; 285: 163-167Crossref PubMed Scopus (98) Google Scholar). Mitochondrial pellets were suspended in 0.1 m Tris, 10 mm EDTA, 1% Triton X-100, pH 7.6, and then centrifuged at 20,000 × g for 10 min to remove membrane debris and obtain clear supernatant. Absorbance at 340 nm was determined in untreated supernatants (A1), and after specific oxidation of NADPH to NADP+ with glutathione reductase and GSSG (A2). A1 – A2 represented the amount of NADPH in the sample (31Zhang Z. Yu J. Stanton R.C. Anal. Biochem. 2000; 285: 163-167Crossref PubMed Scopus (98) Google Scholar). Glutathione reductase (GR) activity was measured as the rate of decrease in absorbance at 340 nm caused by the oxidation of NADPH (GR assay kit, Sigma). A reaction with assay buffer instead of mitochondrial sample was run as a blank.Measurement of Oxygen Consumption—Oxygen consumption of isolated mitochondria was measured at 25 °C using a model 782 oxygen meter system and model 1302 Microcathode oxygen electrode (Strathkelvin, Glasgow, UK). Mitochondrial pellet (1–1.5 mg/ml) was added to the 1-ml sample chamber filled with respiration buffer (130 mm sucrose, 50 mm KCl, 5 mm MgCl2, 5 mm KH2PO4, 0.05 mm EDTA, and 5 mm HEPES, pH 7.4) and allowed to equilibrate with magnetic stirring. Complex I-supported state 4 respiration was initiated by addition of 5 mm glutamate and 5 mm malate to the sample chamber. Subsequent addition of 100 nmol of ADP initiated complex I-supported state 3 respiration. After returning to state 4 respiration, maximum oxygen consumption (uncoupled respiration) was measured by adding 5 μm carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Similarly, complex II-supported state 3 and 4 respiration was measured using 5 mm succinate.Effects of tBOOH and HCV Core Protein on Mitochondrial Respiration—Isolated hepatic mitochondria were incubated with 100 μm tBOOH and/or 1, 10, or 100 ng of recombinant HCV core protein per mg of mitochondrial protein at 25 °C for 5 min. Aliquots of the mitochondrial suspension were added to the sample chamber for analysis of rates of oxygen consumption. P:O ratio and FCCP-induced consumption rates were calculated as described by Estabrook (32Estabrook R.W. Methods Enzymol. 1967; 10: 41-47Crossref Scopus (1888) Google Scholar).Measurement of Complex I and III Activity—Enzyme activity assays were performed at 25 °C by previously established methods (33Jarreta D. Orus J. Barrientos A. Miro O. Roig E. Heras M. Moraes C.T. Cardellach F. Casademont J. Cardiovasc. Res. 2000; 45: 860-865Crossref PubMed Scopus (159) Google Scholar, 34Trounce I.A. Kim Y.L. Jun A.S. Wallace D.C. Methods Enzymol. 1996; 264: 484-509Crossref PubMed Google Scholar). Submitochondrial particles (SMPs) were prepared from mitochondria by incubation for 3 min at 37 °C followed by sonication in a microcentrifuge tube immersed in ice water. Submitochondrial particles were pelleted at 15,000 × g for 10 min, and 50 μg were used for each assay. In some instances SMPs were reduced by incubation with dithiothreitol (100 μm) for 10 min at 0 °C. Complex I activity (NADH-decylubiquinone oxidoreductase) was measured as the initial (5 min) rate of decrease of A340 using the acceptor 2,3-dimethoxy-5-methyl-6-n-decyl-1,4-benzoquinone (DB 80 μm) and 200 μm NADH as the donor in 10 mm Tris (pH 8.0) buffer containing 1 mg/ml BSA, 0.24 mm KCN, and 0.4 μm antimycin A. Complex III activity (ubiquinol cytochrome c reductase) was measured at 550 nm using 40 μm oxidized cytochrome c as the acceptor and 80 μm decylubiquinol as the donor in 10 mm KH2PO4 (pH 7.8), 1 mg/ml BSA, 2 mm EDTA, in the presence of 0.24 mm KCN, 4 μm rotenone, 0.2 mm ATP for 2 min. The addition of 1 μm antimycin A allowed us to distinguish between the reduction of cytochrome c catalyzed by complex III and the nonenzymatic reduction of cytochrome c by the reduced quinine. Extinction coefficients were 6200 liters/mol·cm for NADH and 2.11 × 104 liters/mol·cm for oxidized cytochrome c.Measurement of ROS Production in Mitochondria—Mitochondrial ROS production was determined with the oxidation sensitive fluorogenic precursor dihydrodichlorocarboxyfluorescein diacetate (DCFDA, Molecular Probes) (35Young T.A. Cunningham C.C. Bailey S.M. Arch. Biochem. Biophys. 2002; 405: 65-72Crossref PubMed Scopus (109) Google Scholar). Briefly, each well of a 96-well microtiter plate was filled with respiration buffer containing 1 μm DCFDA and 0.5 mg/ml of mitochondrial particles (final volume, 0.2 ml). The reaction was started by addition of 5 mm glutamate or 5 mm succinate and then incubated at 30 °C in a shaker for 30–60 min. Fluorescence was measured with a CytoFluorII fluorescence plate reader (PerSeptive Biosystems, Inc., Framingham, MA) at excitation of 485 nm and emission of 530 nm. Some experiments included inhibitors, 5 μm FCCP, 1 μm rotenone, or 10 μm BAPTA-AM (Molecular Probes). In some experiments mitochondria from control mice were incubated for 5 min with HCV core protein. For Ca2+-induced ROS production, the mitochondrial suspension was first exposed to 125 μm Ca2+ for 30 min on ice.Measurement of Mitochondrial Ca2+—For Ca2+ determination, mitochondria (0.5 mg/ml) were incubated for 1 h at 4 °C with the mitochondrial Ca2+ indicator Rhod-2 AM (4 μm, Molecular Probes), washed twice in 0.25 m sucrose, 2 mm K-Hepes buffer, and diluted to a final concentration of 0.33 mg of protein/ml in 100 mm KCl, 20 mm Tris, 20 mm Hepes, 10 mm NaCl, 5 mm sodium succinate, 1 mm KH2PO4, 20 μm potassium EGTA, 2 μm rotenone, and 1 μg/ml oligomycin, pH 7.2. The mitochondria were exposed to further treatment with or without exogenous HCV core protein (10 ng/mg protein) on ice for 5 min, and FCCP (5 μm) on ice for 30 min as indicated. Mitochondrial suspensions were subsequently exposed to 125 μm Ca2+ containing respiration buffer for 30 min on ice. The red fluorescence of Rhod-2 was measured in 96-well plates in a CytoFluorII fluorescence plate reader at excitation 530 nm and emission 590 nm.Western Blotting—Samples were lysed in 625 mm Tris, pH 7.4, 2% SDS, 1 mm EDTA, and 1% protease inhibitor mixture (Sigma). Mitochondria and liver lysates were centrifuged at 7900 × g for 10 min, and the supernatants (30 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad), blocked overnight at 4 °C with 5% nonfat dried milk, 0.1% Tween 20 in phosphate-buffered saline and subsequently incubated for 2 h at room temperature with mouse monoclonal antibody to human hepatitis C virus core protein (1:450, Anogen, Mississauga, ON), rabbit polyclonal anti-Tom20 antibody (1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse monoclonal anti-cytochrome c antibody (1:2000, R&D System, Inc., Minneapolis, MN), mouse monoclonal IgG anti-complex III core 2 subunit antibody (1:5000, Affinity Molecular Probes, Inc.) or mouse monoclonal antimitochondrial heat shock protein 70 antibody (1:2000, affinity BioReagents, Golden, CO). The membranes were washed, incubated with appropriate secondary antibodies, and detected with the ECLplus chemiluminescence system (Amersham Biosciences).Assessment of Core Protein Localization by Proteolysis—Isolated mitochondria from transgenic liver were incubated in respiration buffer with proteinase K (50 μg/ml) for 30 min at 4 °C. After incubation, protease activity was inhibited by addition of phenylmethylsulfonyl fluoride to a final concentration of 2 μm, followed by incubating on ice for an additional 10 min. Then 10 μg of the mitochondrial suspension was subjected to Western blotting without centrifugation. 1% Triton X-100 was used in some experiments to disrupt the mitochondrial membranes.Statistics—Results are expressed as mean ± S.E. Student's t test was used for statistical analyses. p < 0.05 was considered significant.RESULTSCharacteristics of HCV Transgenic Mice—We developed a new transgenic mouse model to study the effects of viral proteins on native liver mitochondria. Lerat et al. (26Lerat H. Honda M. Beard M.R. Loesch K. Sun J. Yang Y. Okuda M. Gosert R. Xiao S.Y. Weinman S.A. Lemon S.M. Gastroenterology. 2002; 122: 352-365Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) reported the occurrence of steatosis and hepatocellular carcinogenesis in a transgenic line S-N/863 encoding the structural genes of HCV. Transgene RNA expression in the liver of this lineage is detectable by RT-PCR, and Northern blot analysis, but protein expression was not detectable by immunoblotting and thus is lower than that typically seen in patients with chronic hepatitis C (36Lau J.Y. Krawczynski K. Negro F. Gonzalez-Peralta R.P. J. Hepatol. 1996; 24: 43-51Crossref PubMed Google Scholar). Using the same construct (Fig. 1A), we derived five transgenic lines that expressed HCV RNA, as demonstrated by RT-PCR, three of which expressed the HCV core protein detectable by immunoblotting. Northern blot analysis shows that these mouse lines produced two HCV-specific RNA transcripts, 3.4 and 4.2 kb (Fig. 1B). The spleen was the only other organ that was positive by RT-PCR, at a relatively reduced level (data not shown). One of the three lines, designated as SL-139 was backcrossed to C57BL/6J, and the offspring were used in all experiments reported here. Core protein concentration determined by ELISA in liver extracts was 0.59 ± 0.23 pg core/μg liver protein (mean ± S.D.).Presence of Core Protein in Transgenic Mitochondria—To test for expression of HCV core protein in the mitochondria, Western blot analysis was performed on crude mitochondria from transgenic and control livers (Fig. 2A). As previously reported, HCV core protein was present in the mitochondrial pellet (9Okuda M. Li K. Beard M.R. Showalter L.A. Scholle F. Lemon S.M. Weinman S.A. Gastroenterology. 2002; 122: 366-375Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar, 18Schwer B. Ren S. Pietschmann T. Kartenbeck J. Kaehlcke K. Bartenschlager R. Yen T.S. Ott M. J. Virol. 2004; 78: 7958-7968Crossref PubMed Scopus (129) Google Scholar) and two forms of HCV core protein (p23 and p21) (13Santolini E. Migliaccio G. La Monica N. J. Virol. 1994; 68: 3631-3641Crossref PubMed Google Scholar, 14Moradpour D. Englert C. Wakita T. Wands J.R. Virology. 1996; 222: 51-63Crossref PubMed Scopus (190) Google Scholar, 17Yasui K. Wakita T. Tsukiyama-Kohara K. Funahashi S.I. Ichikawa M. Kajita T. Moradpour D. Wands J.R. Kohara M. J. Virol. 1998; 72: 6048-6055Crossref PubMed Google Scholar, 37Hussy P. Langen H. Mous J. Jacobsen H. Virology. 1996; 224: 93-104Crossref PubMed Scopus (127) Google Scholar, 38Liu Q. Tackney C. Bhat R.A. Prince A.M. Zhang P. J. Virol. 1997; 71: 657-662Crossref PubMed Google Scholar) were detected. Core protein relative abundance was greater in mitochondria than in whole liver homogenate (Fig. 2A) and comparison to recombinant core (amino acids 1–179) standards suggested that p21 is similar in size to core 1–179. Content of core protein was on the order of 2–5 ng core/mg mitochondrial protein (Fig. 2B).FIGURE 2HCV core protein expression in transgenic mouse liver and mitochondria. Immunoblot demonstration of HCV core protein expression in liver mitochondria. CON, control mice; TgM, transgenic mice; L, liver lysate; M, mitochondrial lysate. A, each lane was loaded with 100 μg of either total lysate or mitochondrial protein. Two forms of core protein were detected in transgenic but not control samples. HCV core protein in mitochondria was ∼4-fold more abundant in mitochondria than in liver lysate. B, purified HCV core protein (1–179) was added to control mitochondrial lysate (10 ng or 5 ng/mg mitochondrial protein) and compared with two separate mitochondrial lysates.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Oxidant Status of Transgenic Mitochondria—To determine if SL-139 mice have altered oxidative status of their mitochondria we measured total hepatic and mitochondrial content of reduced (GSH) and total (GSH + GSSG) glutathione. There was no significant difference in whole liver total glutathione between control and transgenic mice (36.6 ± 1.9 versus 32.7 ± 3.4 nmol/mg protein), ∼97% of the whole liver glutathione pool was in the reduced form, and there was no effect of HCV transgene expression on the proportion of reduced versus total glutathione (Fig. 3, A and B). Liver mitochondrial total glutathione was 8.2 ± 1.2 nmol/mg protein in control animals and was not different in transgenic animals (Fig. 3C). However, mitochondrial reduced GSH content was significantly decreased in transgenics (Fig. 3D, p < 0.01) demonstrating a baseline oxidation of the mitochondrial glutathione pool in these animals.FIGURE 3Glutathione content in HCV transgenic mouse liver. Glutathione content was measured in freshly isolated whole liver homogenate (A and B) or mitochondrial fractions (C and D) as described under “Materials and Methods.” Total glutathione (GSH + GSSG) was expressed as GSH equivalents (A and C). Reduced glutathione (GSH only) is shown in B and D. Values in transgenic liver samples were compare
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