Hepatitis B Virus X Protein Induces Cell Death by Causing Loss of Mitochondrial Membrane Potential

The hepatitis B virus X protein (HBx) has been implicated in the carcinogenicity of this virus as a causative factor by means of its transactivation function in development of hepatocellular carcinoma. However, we and others have recently reported that HBx is located in mitochondria and causes subsequent cell death (Takada, S., Shirakata, Y., Kaneniwa, N., and Koike, K. (1999) Oncogene 18, 6965–6973; Rahmani, Z., Huh, K. W., Lasher, R., and Siddiqui, A. (2000) J. Virol. 74, 2840–2846). In this study, we, therefore, examined the mechanism of HBx-related cell death. Using enhanced green fluorescent protein (EGFP) fusion constructs of HBx, the region required for its mitochondrial localization was mapped to amino acids (aa) 68–117, which is essential for cell death but inactive for transactivation function. In vitro binding analysis supported the notion that the recombinant HBx associates with isolated mitochondria through the region of aa 68–117 without causing redistribution of cytochrome c and apoptosisinducing factor (AIF). A cytochemical analysis revealed that mitochondrial membrane potential was decreased by HBx association with mitochondria, suggesting that HBx induces dysfunction of permeability transition pore (PTP) complex. Furthermore, PTP inhibitors, reactive oxygen species (ROS) scavengers and Bcl-xL, which are known to stabilize mitochondrial membrane potential, prevented HBx-induced cell death. Collectively, the present results suggest that location of HBx in mitochondria of hepatitis B virus-infected cells causes loss of mitochondrial membrane potential and subsequently induces mitochondria-dependent cell death. The hepatitis B virus X protein (HBx) has been implicated in the carcinogenicity of this virus as a causative factor by means of its transactivation function in development of hepatocellular carcinoma. However, we and others have recently reported that HBx is located in mitochondria and causes subsequent cell death (Takada, S., Shirakata, Y., Kaneniwa, N., and Koike, K. (1999) Oncogene 18, 6965–6973; Rahmani, Z., Huh, K. W., Lasher, R., and Siddiqui, A. (2000) J. Virol. 74, 2840–2846). In this study, we, therefore, examined the mechanism of HBx-related cell death. Using enhanced green fluorescent protein (EGFP) fusion constructs of HBx, the region required for its mitochondrial localization was mapped to amino acids (aa) 68–117, which is essential for cell death but inactive for transactivation function. In vitro binding analysis supported the notion that the recombinant HBx associates with isolated mitochondria through the region of aa 68–117 without causing redistribution of cytochrome c and apoptosisinducing factor (AIF). A cytochemical analysis revealed that mitochondrial membrane potential was decreased by HBx association with mitochondria, suggesting that HBx induces dysfunction of permeability transition pore (PTP) complex. Furthermore, PTP inhibitors, reactive oxygen species (ROS) scavengers and Bcl-xL, which are known to stabilize mitochondrial membrane potential, prevented HBx-induced cell death. Collectively, the present results suggest that location of HBx in mitochondria of hepatitis B virus-infected cells causes loss of mitochondrial membrane potential and subsequently induces mitochondria-dependent cell death. Epidemiological studies show that chronic hepatitis B virus (HBV) 1The abbreviations used are: HBV, hepatitis B virus; HBx, hepatitis B virus X protein; AIF, apoptosis-inducing factor; PTP, permeability transition pore; ROS, reactive oxygen species; VDAC, voltage-dependent anion channel; Tiron, 4,5-dihydroxy-1,3-benzenedisulfonic acid; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; EGFP, enhanced green fluorescent protein; EBFP, enhanced blue fluorescent protein; z-VAD-fmk, z-Val-Ala-Asp-fluoromethylketone; z-DEVD-fmk, z-Asp-Glu-Val-Asp-fluoromethylketon; aa, amino acid(s); RSV, Rous sarcoma virus; NAC, N-acetyl-l-cysteine.1The abbreviations used are: HBV, hepatitis B virus; HBx, hepatitis B virus X protein; AIF, apoptosis-inducing factor; PTP, permeability transition pore; ROS, reactive oxygen species; VDAC, voltage-dependent anion channel; Tiron, 4,5-dihydroxy-1,3-benzenedisulfonic acid; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; EGFP, enhanced green fluorescent protein; EBFP, enhanced blue fluorescent protein; z-VAD-fmk, z-Val-Ala-Asp-fluoromethylketone; z-DEVD-fmk, z-Asp-Glu-Val-Asp-fluoromethylketon; aa, amino acid(s); RSV, Rous sarcoma virus; NAC, N-acetyl-l-cysteine. infection is closely associated with development of hepatocellular carcinoma (3Blum H.E. Wieland S. Walter E. von Weizsacker F. Offensperger W-B. Moradpour D. Koshy R. Caselmann W.H. Hepatitis B Virus, Molecular Mechanisms in Disease and Novel Strategies for Therapy. Imperial College Press, London1998: 75-92Google Scholar), and the X gene has been implicated in the carcinogenicity of this virus because of its ability to transform rodent cells (4Shirakata Y. Kawada M Fujiki Y. Sano H. Oda M. Yaginuma K. Kobayashi M. Koike K. Jpn. J. Cancer Res. 1989; 80: 617-621Crossref PubMed Scopus (167) Google Scholar). Moreover, the development of hepatocellular carcinoma in at least one strain of transgenic mouse has been shown (5Kim C.M. Koike K. Saito I. Miyamura T. Jay G. Nature. 1991; 351: 317-320Crossref PubMed Scopus (1050) Google Scholar). Although there was an absence of tumors in other X transgenic mice so far analyzed, some of these mice exhibited an increased susceptibility to chemical carcinogens (6Slagle B.L. Lee T.H. Medina D. Finegold M.J. Butel J.S. Mol. 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EMBO J. 1990; 9: 497-504Crossref PubMed Scopus (130) Google Scholar, 12Arii M. Takada S. Koike K. Oncogene. 1992; 7: 397-403PubMed Google Scholar) and to activate several signal transduction pathways including mitogen-activated protein kinase, protein kinase C, and JAK/STAT pathways (13Kekule A.S. Lauer U. Weiss L. Luber B. Hofschneider P.H. Nature. 1993; 361: 742-745Crossref PubMed Scopus (335) Google Scholar, 14Luber B. Lauer U. Weiss L. Hohne M. Hofschneider P.H. Kekule A.S. Res. Virol. 1993; 144: 311-321Crossref PubMed Scopus (34) Google Scholar, 15Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10350-10354Crossref PubMed Scopus (402) Google Scholar, 16Lee Y.H. Yun Y. J. Biol. Chem. 1998; 273: 25510-25515Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 17Waris G. Huh K.W. Siddiqui A. Mol. Cell. Biol. 2001; 21: 7721-7730Crossref PubMed Scopus (284) Google Scholar). Although HBx transactivates a wide range of viral and cellular genes, including HBV itself, SV40, c-myc, and NF-κB, the mechanism of transactivation by HBx is still under debate (18Yen T.S. J. Biomed. Sci. 1996; 3: 20-30Crossref PubMed Scopus (145) Google Scholar). Because HBx is unable to directly bind any of the HBx-responsive elements of these genes, it has been proposed that HBx may stimulate transcription by interacting with several cellular sequence-specific transcription factors or with the basal transcriptional machinery (19Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar, 20Lin Y. Nomura T. Cheong J. Dorjsuren D. Iida K. Murakami S. J. Biol. Chem. 1997; 272: 7132-7139Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 21Shamay M. Borak O. Doitsh G. Ben-Dor I. Shaul Y. J. Biol. Chem. 2002; 277: 9982-9988Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). From these observations, it has been suggested that HBx may facilitate hepatocarcinogenesis through transactivating cellular oncogenes and signaling cascades that stimulate cell proliferation. In contrast to proliferative effects, several studies (1Takada S. Shirakata Y. Kaneniwa N. Koike K. Oncogene. 1999; 18: 6965-6973Crossref PubMed Scopus (161) Google Scholar, 22Su F. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8744-8749Crossref PubMed Scopus (287) Google Scholar, 23Bergametti F. Prigent S. Luber B. Benoit A. Tiollais P. Sarasin A. Transy C. Oncogene. 1999; 18: 2860-2871Crossref PubMed Scopus (73) Google Scholar) showed that HBx induces cell death or sensitizes cells to apoptotic stimuli such as tumor necrosis factor α. Our previous report revealed that expression of X gene brought some of apoptotic features to cells, such as TUNEL-positive nucleus, the decreased mitochondrial membrane potential, and the membrane blebbing. We also reported that the ectopically expressed HBx is located in mitochondria (1Takada S. Shirakata Y. Kaneniwa N. Koike K. Oncogene. 1999; 18: 6965-6973Crossref PubMed Scopus (161) Google Scholar). Recently, many reports have focused on mitochondria as an important player on apoptosis and programmed cell death, where many cell death signals reach to mitochondria. Mitochondria undergo major changes in membrane integrity before classical signs of cell death manifest. These changes concern both inner and outer mitochondrial membranes. Outer mitochondrial membrane permeabilization causes the redistribution of numerous apoptogenic factors such as cytochrome c, AIF, and Smac/DIABLO in the intermembrane space (24Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 25Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2770) Google Scholar). The redistribution of these apoptogenic factors is important step of cell death. For instance, cytochrome c release is essential for the generation of the apoptosome, the complex containing cytochrome c, Apaf-1, and caspase-9, and this apoptosome complex initiates the effector caspase activation (26Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6219) Google Scholar, 27Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1794) Google Scholar, 28Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar). On the other hand, the inner mitochondrial membrane permeabilization causes disruption of mitochondrial membrane potential (ΔΨm). The ΔΨm is indispensable for driving the ATP synthase, which phosphorylates ADP to ATP. Therefore the loss of mitochondrial membrane potential means the loss of ATP generation. In this paper, we show that the localization of HBx on mitochondria causes loss of mitochondrial membrane potential and subsequently causes cell death through a mitochondria-dependent pathway. Reagents and Plasmids—The X expression plasmid pCMVX, pCMVX-dAvaI/AvaII, and pCMVX-ΔRsaI were described previously (12Arii M. Takada S. Koike K. Oncogene. 1992; 7: 397-403PubMed Google Scholar). The Bcl-xL cDNA was cloned into pCMV vector. EGFP or EBFP fusion constructs were cloned into pEGFP or pEBFP vector (Clontech), respectively. Substitution of pEGFP-X(68–117LA) in HBx pEGFP-X(68–117) was carried out by PCR based site-directed mutagenesis and confirmed by DNA sequencing. A β-galactosidase expression plasmid, pCMVβ, was obtained from Clontech. Caspase inhibitors, z-VAD-fmk and z-DEVD-fmk were obtained from Enzyme Systems Products. Bongkrekic acid was obtained from Calbiochem, and cyclosporin A, 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), and N-acetyl-l-cysteine (NAC) were purchased from Sigma. Cell Culture and DNA Transfection—HuH7 cells and HepG2 cells were maintained in with and were carried out to the were for and to cytochemical or analysis Cell cells were with plasmid with the β-galactosidase expression plasmid, pCMVβ, at a of The of cell death was by the cells with apoptotic such as membrane of cells were and of cell death. was at least and obtained In of cells for β-galactosidase were with β-galactosidase cells were on the before at a of cells of and of or HBx constructs were pCMV was used as to the DNA to cells were and with was carried out with to the were as described previously (1Takada S. Shirakata Y. Kaneniwa N. Koike K. Oncogene. 1999; 18: 6965-6973Crossref PubMed Scopus (161) Google Scholar). The c has been used at of protein were with the enhanced to the were with and and are obtained from Molecular were with or for at and a was a with which was by Mitochondria and a mouse was for of mitochondria is by S. Y. Y. A. H. Y. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, M. S. T. T. R.J. H. Y. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar) with the was with and with a in of mitochondria cells and were by at for at The were at for at to the mitochondria. The mitochondria was in of mitochondria A of mouse mitochondria was with the recombinant protein in of the for at the end of the was and the mitochondria was in of and of was to the The were and to were as fusion were in and on by The protein was by PubMed Scopus Google Scholar). of the previous report (1Takada S. Shirakata Y. Kaneniwa N. Koike K. Oncogene. 1999; 18: 6965-6973Crossref PubMed Scopus (161) Google Scholar) that X gene expression brought some apoptotic changes to cells such as TUNEL-positive nucleus, the decreased mitochondrial membrane potential, and the membrane blebbing. of the membrane was examined the cells and the because this is to cells which are with DNA and is to many cells for the of We carried out the analysis of HBx-induced cell death. cells were with an X gene expression plasmid, pCMVX, with β-galactosidase expression plasmid, The β-galactosidase was to cells with shown in the cells showed and that are from that of of is known to of apoptosis J. A. 1994; PubMed Scopus Google Scholar). in the of β-galactosidase cells with membrane a in The for cell death analysis were carried out at of HBx for Cell we previously some of HBx, such as aa and as are for transactivation function (12Arii M. Takada S. Koike K. Oncogene. 1992; 7: 397-403PubMed Google Scholar), and that one of these transactivation we, therefore, examined the transactivation function of HBx is required for HBx-induced cell death. The and the were carried out one is a pCMVX-dAvaI/AvaII, which has an from aa to and the other is pCMVX-ΔRsaI that has from aa to with previous report (12Arii M. Takada S. Koike K. Oncogene. 1992; 7: 397-403PubMed Google Scholar), these were unable to activate the these still cell death with that of HBx Furthermore, we the of HBx expression plasmid to cell death or transactivation and The of to transactivation cell death is in this to cell death HBx expression plasmid is required as with transactivation that the transactivation ability of HBx is directly with its cell death function. A of HBx for Cell and reported previously (1Takada S. Shirakata Y. Kaneniwa N. Koike K. Oncogene. 1999; 18: 6965-6973Crossref PubMed Scopus (161) Google Scholar) the mitochondrial localization of HBx of mitochondria, and as described that the transactivation function of HBx is to cell death we to out the of HBx localization on mitochondria is for cell death. We an expression plasmid, that an fusion into cells and with a mitochondria. shown in protein over the and its location with the mitochondria. to the localization of protein to and at the of the revealed that mitochondria also to at the of the mitochondrial is with to cells protein show with the membrane as shown in HBx protein exhibited a to that of HBx, we to a region of HBx protein required for localization on mitochondria plasmid constructs containing HBx or a of HBx fusion HBx constructs and of shown in the for transactivation were as The region required for cell death is shown as a blue amino acid of C, the region of HBx required for mitochondrial of is The plasmid was into cells, and cells were with and under a fluorescent The are shown in the cell death of these EGFP fusion HBx constructs were as shown in The were into cells with The which to on mitochondria, cell death shown in a from the showed decreased to mitochondria. from the of HBx showed association with mitochondria of the its ability to to mitochondria. containing the region of aa 68–117 to EGFP mitochondria suggesting that aa 68–117 are for the localization mitochondria. We to a required region for localization on mitochondria, but of the aa 68–117 region out to in the mitochondria We of of or amino acid in this region any on HBx We also to in this or of a on its localization However, and were with this in loss of mitochondrial localization suggesting that in this region are important for localization of HBx on mitochondria. These HBx plasmid constructs were to cells with β-galactosidase expression plasmid, and cell was shown in that expressed in mitochondria, and were to cell death. On the other hand, and which with mitochondria, were unable to cell death. The cell death of these is to the of protein Because analysis showed that the which are unable to cell death, to in cells that mitochondrial localization of HBx as well as its are important for cell of HBx with Mitochondria to c and we recombinant HBx and its and ability to associate with isolated mouse mitochondria in the was and the mitochondrial was by with HBx-related that the aa 68–117 region and were with the protein this was a notion that the aa 68–117 region is important for association with mitochondria and is also with the cytochemical obtained with HBx constructs On the other hand, the was for redistribution of cytochrome c and by Although cytochrome c and are from isolated mitochondria with as described previously A. G. Cell 1999; 6: PubMed Scopus Google Scholar), cytochrome c and release from mitochondria are by with recombinant HBx or any HBx that HBx is unable to directly cytochrome c and release from isolated mitochondria. it has been well cytochrome c, Apaf-1, and ATP or a apoptosome (26Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6219) Google Scholar), which and induces apoptotic cell death. The of cytochrome c release that HBx-induced cell death is pathway. cells were with and and were with caspase inhibitors, z-DEVD-fmk or z-VAD-fmk A z-DEVD-fmk, cell death, a caspase z-VAD-fmk, HBx-induced cell death in that HBx induces cell death through a cytochrome pathway. of HBx with Mitochondria of cytochrome c and release from mitochondria suggested that are in cell death. we reported previously (1Takada S. Shirakata Y. Kaneniwa N. Koike K. Oncogene. 1999; 18: 6965-6973Crossref PubMed Scopus (161) Google Scholar) that decreased mitochondrial membrane potential was expression of HBx, we localization of HBx on mitochondria is directly to decreased mitochondrial membrane A cytochemical analysis was carried out to mitochondrial membrane potential by a fluorescent which is a and to changes of mitochondria. at at in and in the of a so that are associated with a in to M. C. A. M. PubMed Scopus Google Scholar, S. A. C. A. 1997; PubMed Scopus Google Scholar). mitochondrial membrane potential by HBx constructs were to of the green with shown in expression of and and were on loss of mitochondrial membrane potential, expression of any of mitochondrial membrane that association of HBx with mitochondria decreased mitochondrial membrane of PTP and from HBx-induced Cell decreased mitochondrial membrane potential is a to HBx-induced cell death, of ΔΨm is to HBx-induced cell death. this PTP inhibitors, acid and cyclosporin A, which stabilize mitochondrial membrane potential, were to analysis of HBx-induced cell death. DNA these were to the and cell death was shown in A and both PTP prevented cells from HBx-induced cell death in a suggesting that decreased mitochondrial membrane potential is a major of HBx-induced cell death. It has been reported that decreased mitochondrial membrane potential results in a of including generation of and decreased of ATP. the generation of has been shown to cell death we of NAC, and on HBx-induced cell death. shown in and these and Tiron, also prevented cells from HBx-induced cell death in a suggest that HBx association with mitochondria the PTP complex and causes in cell death. it is known that and Bcl-xL stabilize mitochondrial membrane potential and cells from HBx induces loss of mitochondrial membrane potential, expression of and Bcl-xL cells from HBx-induced cell death. A Bcl-xL expression plasmid was with HBx expression plasmid, and the of cell death was that expression of Bcl-xL protein cells from under the of HBx expression in that loss of mitochondrial membrane potential is to HBx-induced cell death. that the mitochondrial membrane potential under the cells are with and with acid Although cells with HBx expression plasmid showed decreased mitochondrial membrane potential with acid cells from of mitochondrial membrane shown some cells with acid show mitochondrial membrane potential mitochondria are These cells with ΔΨm were without acid The expression of Bcl-xL also exhibited a on of mitochondrial membrane potential also suggested that of mitochondrial membrane potential, which the PTP is the of HBx-induced cell death. the present results that HBx-induced cell death is to localization of HBx on mitochondria. We mapped that aa 68–117 are for HBx localization on mitochondria and cell death Our show that cell death by HBx is of its transactivation Although several have out that the of HBx with its transactivation F. Prigent S. Luber B. Benoit A. Tiollais P. Sarasin A. Transy C. Oncogene. 1999; 18: 2860-2871Crossref PubMed Scopus (73) Google Scholar, H. C. K. P. D. C. Oncogene. 1999; 18: PubMed Scopus Google Scholar, R. W.H. S. Oncogene. 2000; PubMed Scopus Google Scholar), that the transactivation ability of HBx is required for its cell death suggesting that the mechanism of cell death is to transactivation of the cell genes, such as we reported that expression of HBx release of cytochrome c from mitochondria (1Takada S. Shirakata Y. Kaneniwa N. Koike K. Oncogene. 1999; 18: 6965-6973Crossref PubMed Scopus (161) Google Scholar). with such as cytochrome c release by HBx is a Although HBx on mitochondria its cytochrome c release from mitochondria was Although cytochrome c release was from a of mitochondria, the of mitochondria still cytochrome Our analysis in this revealed that HBx association with isolated mitochondria redistribution of cytochrome c and These results suggested that HBx-induced cell death is on mitochondria, but of of other to In mitochondrial membrane permeabilization is a and a step of several apoptotic pathways G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2770) Google Scholar). the or of mitochondrial membrane permeabilization the outer and inner of mitochondria. Outer mitochondrial membrane permeabilization and that in the intermembrane such as cytochrome c and The redistribution of cytochrome c through the outer mitochondrial membrane is the for mitochondria-dependent caspase The inner mitochondrial membrane permeabilization to but the mitochondrial Our show that HBx release cytochrome c and from the mitochondrial intermembrane space in the association of HBx with mitochondria decreased mitochondrial membrane potential in These suggest that HBx the inner mitochondrial membrane permeabilization but the outer membrane Furthermore, we showed that PTP inhibitors, scavengers and Bcl-xL, which stabilize mitochondrial membrane potential, cells from HBx-induced cell death, the that decreased mitochondrial membrane potential, the inner membrane is the major of HBx-induced cell death. Although the of outer and inner mitochondrial membrane as well as to cell death, are still a of the HBx-induced cell death that the inner membrane permeabilization of mitochondria is implicated in cell death. We examined the that HBx and Bcl-xL with the mitochondrial protein isolated mouse mitochondria, we any these in association with mitochondria that Bcl-xL is to indirectly HBx-induced cell death, such as of mitochondrial membrane It has been shown that several viral and cell death at the mitochondrial by to mitochondrial membranes. Huh K.W. R. Siddiqui A. J. Virol. 2000; PubMed Scopus Google Scholar) reported that HBx with one voltage-dependent anion by a The is one of the PTP complex in the outer mitochondrial Our results suggested that HBx inner membrane but it is to that the of HBx with may inner membrane cell death or apoptosis a major in development and as well as in many of apoptosis have been implicated in many such as and M. P. N. B. C. M. A. 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