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 apoptosis-inducing 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.     

Characterization of the mitochondrial association of hepatitis B virus X protein, HBx

Chronic infection with hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma (HCC). HBx, a protein encoded by HBV is believed to contribute to the development of HCC. HBx was recently shown to associate with mitochondria. In this study, we mapped region(s) of HBx necessary for mitochondrial targeting and showed that a putative transmembrane region (aa 54-70) is required for mitochondrial association. In addition, amino acids in the putative alpha helical regions (aa 75-88 and aa 109-131) seem to aid in the mitochondrial targeting of this protein. We further show that the majority of HBx localizes to the outer mitochondrial membrane based on its sensitivity to trypsin and resistance to alkaline treatment. These studies suggest that the association of HBx with the outer mitochondrial membrane is its intrinsic property. These characterizations define transmembrane and alpha-helical regions of this viral protein as domains of mitochondrial targeting. These studies are further useful in the investigations concerning the physiological significance of the HBx's association with mitochondria and its impact on liver disease pathogenesis.     

Hepatitis B virus regulatory HBx protein binds to adaptor protein IPS-1 and inhibits the activation of beta interferon

Hepatitis B virus (HBV) encodes the regulatory HBx protein, which is required for virus replication, although its specific role(s) in the replication cycle remains under investigation. An immunoprecipitation/mass spectrometry approach was used to identify four novel HBx binding proteins from the cytoplasmic fraction of HBx transgenic mouse livers. One of these HBx binding partners is beta interferon promoter stimulator 1 (IPS-1), an adaptor protein that plays a critical role in mediating retinoic acid-inducible gene I (RIG-I) signaling, which leads to the activation of beta interferon (IFN-β). The HBx-IPS-1 protein interaction was confirmed in plasmid-transfected HepG2 cells by reciprocal coimmunoprecipitation and Western blotting. We hypothesized that HBx might alter IPS-1 function since proteins of hepatitis C virus and hepatitis A virus similarly bind IPS-1 and target it for inactivation. The effect of HBx on IPS-1-mediated IFN-β signaling was tested in transfected 293T and HepG2 cells, and we show that HBx inhibits double-stranded DNA (dsDNA)-mediated IFN-β activation in a dose-dependent manner when expressed either alone or within the context of HBV replication. However, HBx does not inhibit poly(I:C)-activated IFN-β signaling. These results demonstrate that HBx interferes with the RIG-I pathway of innate immunity. Hepatitis B virus now joins hepatitis C virus and hepatitis A virus in targeting the same innate immune response pathway, presumably as a shared strategy to benefit replication of these viruses in the liver.     

Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria

Hepatitis B virus X protein (HBx) is essential for viral replication and plays an important role in viral pathogenesis. HBx transactivates many viral and cellular genes and participates in cellular signal transduction pathways, proliferation, and apoptosis. In the present study, we report that HBx induces apoptosis by enhancing the translocation of Bax to mitochondria, followed by inducing the loss of mitochondrial membrane potential and release of cytochrome C. In addition, Bcl-2, inhibitor of Bax, rescues the disruption of mitochondrial membrane potential and DNA fragmentation induced by serum starvation in HepG2-X cells expressing HBx. We also found that HBx binds directly to Bax and interferes with the interaction between Bax and 14-3-3epsilon to enhance the translocation of Bax to mitochondria. Taken together, our data suggest that HBx induces apoptosis by interacting with Bax and enhancing its translocation to mitochondria.     

Hepatitis B virus sensitizes hepatocytes to TRAIL-induced apoptosis through Bax

Hepatitis B virus (HBV) infection afflicts >300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.     

Mitochondrial membrane potential modulates regulation of mitochondrial Ca2+ in rat ventricular myocytes

Although recent studies focused on the contribution of mitochondrial Ca2+ to the mechanisms of ischemia-reperfusion injury, the regulation of mitochondrial Ca2+ under pathophysiological conditions remains largely unclear. By using saponin-permeabilized rat myocytes, we measured mitochondrial membrane potential (DeltaPsi(m)) and mitochondrial Ca2+ concentration ([Ca2+](m)) at the physiological range of cytosolic Ca2+ concentration ([Ca2+](c); 300 nM) and investigated the regulation of [Ca2+](m) during both normal and dissipated DeltaPsi(m). When DeltaPsi(m) was partially depolarized by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 0.01-0.1 microM), there were dose-dependent decreases in [Ca2+](m). When complete DeltaPsi(m) dissipation was achieved by FCCP (0.3-1 microM), [Ca2+](m) remained at one-half of the control level despite no Ca2+ influx via the Ca2+ uniporter. The DeltaPsi(m) dissipation by FCCP accelerated calcein leakage from mitochondria in a cyclosporin A (CsA)-sensitive manner, which indicates that DeltaPsi(m) dissipation opened the mitochondrial permeability transition pore (mPTP). After FCCP addition, inhibition of the mPTP by CsA caused further [Ca2+](m) reduction; however, inhibition of mitochondrial Na+/Ca2+ exchange (mitoNCX) by a Na+-free solution abolished this [Ca2+](m) reduction. Cytosolic Na(+) concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal DeltaPsi(m) and 7.6 mM at DeltaPsi(m) dissipation. We conclude that 1) the mitochondrial Ca2+ uniporter accumulates Ca2+ in a manner that is dependent on DeltaPsi(m) at the physiological range of [Ca2+](c); 2) DeltaPsi(m) dissipation opens the mPTP and results in Ca2+ influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes Ca2+ from the matrix even after DeltaPsi(m) dissipation.     

Rubella virus capsid associates with host cell protein p32 and localizes to mitochondria

Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.     

Hepatitis B virus X protein modulates upregulation of DHX9 to promote viral DNA replication

Hepatitis B virus (HBV) infection is a major cause of acute and chronic liver diseases. During the HBV life cycle, HBV hijacks various host factors to assist viral replication. In this research, we find that the HBV regulatory protein X (HBx) can induce the upregulation of DExH‐box RNA helicase 9 (DHX9) expression by repressing proteasome‐dependent degradation mediated by MDM2. Furthermore, we demonstrate that DHX9 contributes to viral DNA replication in dependence on its helicase activity and nuclear localization. In addition, the promotion of viral DNA replication by DHX9 is dependent on its interaction with Nup98. Our findings reveal that HBx‐mediated DHX9 upregulation is essential for HBV DNA replication.     

Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.     

Hepatitis B virus x protein induces perinuclear mitochondrial clustering in microtubule- and Dynein-dependent manners

The hepatitis B virus (HBV) X protein (HBx) is thought to play a key role in HBV replication and the development of liver cancer. It became apparent that HBx induces mitochondrial clustering at the nuclear periphery, but the molecular basis for mitochondrial clustering is not understood. Since mitochondria move along the cytoskeleton as a cargo of motor proteins, we hypothesized that mitochondrial clustering induced by HBx occurs by an altered intracellular motility. Here, we demonstrated that the treatment of HBx-expressing cells with a microtubule-disrupting drug (nocodazole) abrogated mitochondrial clustering, while the removal of nocodazole restored clustering within 30 to 60 min, indicating that mitochondrial transport is occurring in a microtubule-dependent manner. The addition of a cytochalasin D-disrupting actin filament, however, did not measurably affect mitochondrial clustering. Mitochondrial clustering was further studied by observations of HBV-related hepatoma cells and HBV-replicating cells. Importantly, the abrogation of the dynein activity in HBx-expressing cells by microinjection of a neutralizing anti-dynein intermediate-chain antibody, dynamitin overexpression, or the addition of a dynein ATPase inhibitor significantly suppressed the mitochondrial clustering. In addition, HBx induced the activation of the p38 mitogen-activated protein kinase (MAPK) and inhibition of the p38 kinase activity by SB203580-attenuated HBx-induced mitochondrial clustering. Taken together, HBx activation of the p38 MAPK contributed to the increase in the microtubule-dependent dynein activity. The data suggest that HBx plays a novel regulatory role in subcellular transport systems, perhaps facilitating the process of maturation and/or assembly of progeny particles during HBV replication. Furthermore, mitochondrion aggregation induced by HBx may represent a cellular process that underlies disease progression during chronic viral infection.     

Akt augments the oncogenic potential of the HBx protein of hepatitis B virus by phosphorylation

Hepatitis B virus X protein (HBx) is a putative viral oncoprotein that plays an important role in various cellular processes, including modulation of the phosphatidylinositol 3-kinase/Akt signalling pathway. However, the molecular mechanism of Akt activation remains elusive. Here we show that HBx interacts with Akt1 kinase and is phosphorylated at serine 31 as indicated by mutational analysis of the Akt recognition motif (creating the HBxS31A mutant) or immunoblotting of HBx immunoprecipitates using Akt motif-specific antibody. The Akt-dependent phosphorylation of HBx was abrogated in the presence of the phosphatidylinositol 3-kinase inhibitor LY294002 or Akt1 gene silencing by specific siRNA. Co-immunoprecipitation studies provided evidence for HBx-Akt interaction in a cellular environment. This interaction was also confirmed in hepatoma HepG2.2.15 cells in which HBx was expressed at physiological levels from the integrated hepatitis B viral genome. The HBx-Akt interaction was essential for Akt signalling, and involved displacement of the Akt-bound negative regulator 'C-terminal modulator protein' by HBx. HBx-activated Akt phosphorylated its downstream target glycogen synthase kinase 3β, leading to stabilization of β-catenin, while p65 phosphorylation resulted in enhanced promoter recruitment and expression of target genes encoding cyclin D1 and Bcl-XL. Further, the oncogenic potential of HBx was significantly augmented in the presence of Akt in a soft agar colony formation assay. Together, these results suggest that oncogenic co-operation between HBx and Akt may be important for cell proliferation, abrogation of apoptosis and tumorigenic transformation of cells.     

Transgenic UCP1 in white adipocytes modulates mitochondrial membrane potential

To test if mitochondrial uncoupling in white adipocytes is responsible for obesity resistance of the aP2-Ucp transgenic mice expressing ectopic uncoupling protein 1 (UCPI) in white fat, mitochondrial membrane potential (delta psi(m)) was estimated by flow cytometry in adipocytes isolated from gonadal fat. Ectopic UCP1 (approximately 0.8 mol UCP1/mol respiratory chain) decreased the delta psi(m) and rendered the potential sensitive to GDP and fatty acids. These ligands of UCP1 had no effect on delta psi(m) in white adipocytes from non-transgenic mice, suggesting that the function of endogenous UCP2 in adipocytes was not affected. The results support the hypothesis that mitochondrial uncoupling in white fat may prevent development of obesity.     

Dopamine modifies oxygen consumption and mitochondrial membrane potential in striatal mitochondria

Dopamine is a neurotransmitter that has been related to mitochondrial dysfunction. In this study, striatal intact mitochondria and submitochondrial membranes were incubated with different dopamine concentrations, and changes on mitochondrial function, hydrogen peroxide, and nitric oxide production were evaluated. A 35% decrease in state 3 oxygen uptake (active respiration state) was found after 1 mM dopamine incubation. In addition, mitochondrial respiratory control significantly decreased, indicating mitochondrial dysfunction. High dopamine concentrations induced mitochondrial depolarization. Also, evaluation of hydrogen peroxide production by intact striatal mitochondria showed a significant increase after 0.5 and 1 mM dopamine incubation. Incubation with 0.5 and 1 mM dopamine increased nitric oxide production in submitochondrial membranes by 28 and 49%, respectively, as compared with control values. This study provides evidence that high dopamine concentrations induce striatal mitochondrial dysfunction through a decrease in mitochondrial respiratory control and loss of membrane potential, probably mediated by free radical production.     

Hepatitis B virus X protein modulates peroxisome proliferator-activated receptor gamma through protein-protein interaction

Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARgamma activation has not been characterized in hepatitis B virus (HBV)-associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARgamma ligand in HBx-associated HCC cells. We found that HBx bound to DNA binding domain of PPARgamma and HBx/PPARgamma interaction blocked nuclear localization and binding to recognition site of PPARgamma. HBx significantly suppressed a PPARgamma-mediated transactivation. These results suggest that HBx modulates PPARgamma function through protein-protein interaction.     

The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.     

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.     

Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential

Understanding the mechanism(s) of action of the hepatitis B virus (HBV)-encoded protein HBx is fundamental to elucidating the underlying mechanisms of chronic liver disease and hepatocellular carcinoma caused by HBV infection. In our continued attempts to identify cellular targets of HBx, we have previously reported the identification of a novel cellular protein with the aid of a yeast two-hybrid assay. This cellular gene was identified as a third member of the family of human genes that encode the voltage-dependent anion channel (HVDAC3). In the present study, physical interaction between HBx and HVDAC3 was established by standard in vitro and in vivo methods. Confocal laser microscopy of transfected cells with respective expression vectors colocalized HVDAC3 and HBx to mitochondria. This novel, heretofore unreported subcellular distribution of HBx in mitochondria implies a functional role of HBx in functions associated with mitochondria. Using a stable cationic fluorophore dye, CMXRos, we show that HBx expression in cultured human hepatoma cells leads to alteration of mitochondrial transmembrane potential. Such functional roles of HBx in affecting mitochondrial physiology have implications for HBV-induced liver injury and the development of hepatocellular carcinoma.