vMIA,a viral inhibitor of apoptosis targeting mitochondria

Human cytomegalovirus encodes a powerful cell death suppressor vMIA (viral mitochondria-localized inhibitor of apoptosis), also known as pUL37x1. vMIA, a product of the immediate early gene UL37 exon 1, is predominantly localized in mitochondria, where it appears to form a complex with adenine nucleotide translocator, believed to be a component of the mitochondrial transition pore complex. vMIA suppresses apoptosis by blocking permeabilization of the mitochondrial outer membrane. Expression of vMIA protects cells against apoptosis triggered by diverse stimuli, including ligation of death receptors, exposure to certain cytotoxic drugs, and infection with an adenovirus mutant deficient in E1B19K. Deletion mutagenesis of vMIA revealed two domains that are necessary and, together, sufficient for its anti-apoptotic activity. The first domain contains a mitochondrial targeting signal. The function of the second domain is still unknown. vMIA does not share any significant amino acid sequence homology with Bcl-2, and, unlike Bcl-2 or Bcl-x(L), it does not bind BAX or VDAC. These structural and functional differences between vMIA and Bcl-2 suggest that vMIA represents a separate class of cell death suppressors. Experiments with vMIA-deficient CMV (human cytomegalovirus) mutants provide strong evidence that the anti-apoptotic function of vMIA is required to prevent CMV-induced apoptosis, and is necessary for viral replication. In addition to vMIA, UL37 encodes two longer splice-variant proteins, gpUL37 and GP37(M). Biological functions of these proteins have not yet been identified, and may be unrelated to their anti-apoptotic activity. The identification of vMIA and the finding that its anti-apoptotic function is required for CMV replication provides a rationale for the development of anti-CMV pharmaceuticals that would inactivate vMIA and thus restore apoptosis in cells infected with CMV.     

Insights into the mechanisms of CMV-mediated interference with cellular apoptosis

Apoptosis has the potential to function as a defence mechanism during viral infection. Identification of CMV mutants that cause the apoptotic death of infected cells confirmed that viral infection activates apoptotic pathways and that this process is counteracted by CMV to ensure efficient viral replication. The recent identification of CMV-encoded proteins that suppress cell death has greatly enhanced our understanding of the mechanisms used by this family of viruses to prevent apoptosis. CMV do not encode homologues of known death-suppressing proteins, suggesting that the CMV family has evolved novel, more sophisticated strategies for the inhibition of apoptosis. The identification and characterization of the human CMV (HCMV)-encoded antiapoptotic proteins UL36 (viral inhibitor of caspase-8 activation [vICA]) and UL37 (viral mitochondria-localized inhibitor of apoptosis [vMIA]) have confirmed that CMV target unique apoptotic control points. For example, vMIA inhibits apoptosis by binding Bax and sequestering it at the mitochondrial membrane as an inactive oligomer. This knowledge not only provides a more complete understanding of the CMV replication process but also allows the identification of previously unrecognized apoptotic checkpoints. Because HCMV is an important cause of birth defects and an increasingly important opportunistic pathogen, a firm grasp of the mechanisms by which it affects cellular apoptosis may provide avenues for the design of improved therapeutic strategies. Here, we review the recent progress made in understanding the role of CMV-encoded proteins in the inhibition of apoptosis.     

An anti-apoptotic viral protein that recruits Bax to mitochondria

The viral mitochondria-localized inhibitor of apoptosis (vMIA), encoded by the UL37 gene of human cytomegalovirus, inhibits apoptosis-associated mitochondrial membrane permeabilization by a mechanism different from that of Bcl-2. Here we show that vMIA induces several changes in Bax that resemble those found in apoptotic cells yet take place in unstimulated, non-apoptotic vMIA-expressing cells. These changes include the constitutive localization of Bax at mitochondria, where it associates tightly with the mitochondrial membrane, forming high molecular weight aggregates that contain vMIA. vMIA recruits Bax to mitochondria but delays relocation of caspase-8-activated truncated Bid-green fluorescent protein (GFP) (t-Bid-GFP) to mitochondria. The ability of vMIA and its deletion mutants to associate with Bax and to induce relocation of Bax to mitochondria correlates with their anti-apoptotic activity and with their ability to suppress mitochondrial membrane permeabilization. Taken together, our data indicate that vMIA blocks apoptosis via its interaction with Bax. vMIA neutralizes Bax by recruiting it to mitochondria and "freezing" its pro-apoptotic activity. These data unravel a novel strategy of subverting an intrinsic pathway of apoptotic signaling.     

HtrA2/Omi terminates cytomegalovirus infection and is controlled by the viral mitochondrial inhibitor of apoptosis (vMIA)

Viruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle.     

The C-terminal moiety of HIV-1 Vpr induces cell death via a caspase-independent mitochondrial pathway

Previous biochemical studies suggested that HIV-1-encoded Vpr may kill cells through an effect on the adenine nucleotide translocase (ANT), thereby causing mitochondrial membrane permeabilization (MMP). Here, we show that Vpr fails to activate caspases in conditions in which it induces cell killing. The knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated killing. In contrast, the cytotoxic effects of Vpr are reduced by transfection-enforced overexpression of two MMP-inhibitors, namely the endogenous protein Bcl-2 or the cytomegalovirus-encoded ANT-targeted protein vMIA. Vpr, which can elicit MMP through a direct effect on mitochondria, and HIV-1-Env, which causes MMP through an indirect pathway, exhibit additive (but not synergic) cytotoxic effects. In conclusion, it appears that Vpr induces apoptosis through a caspase-independent mitochondrial pathway.     

Human cytomegalovirus infection increases mitochondrial biogenesis

Fibroblasts infected by Human Cytomegalovirus (CMV) undergo a robust increase in mitochondrial biogenesis with a corresponding increase in mitochondrial activity that is partly dependent on the viral anti-apoptotic pUL37x1 protein (vMIA). The increased respiration activity is blocked by the mitochondrial translation inhibitor chloramphenicol, which additionally suppresses viral production. Intriguingly, chloramphenicol and pUL37x1 depletion have different effects on respiration capacity but similar effects on CMV production, suggesting that pUL37x1 promotes viral replication by efficient utilization of new mitochondria. These results argue for a role of pUL37x1 beyond controlling apoptosis.     

Poliovirus induces Bax-dependent cell death mediated by c-Jun NH2-terminal kinase

Poliovirus (PV) is the causal agent of paralytic poliomyelitis, a disease that involves the destruction of motor neurons associated with PV replication. In PV-infected mice, motor neurons die through an apoptotic process. However, mechanisms by which PV induces cell death in neuronal cells remain unclear. Here, we demonstrate that PV infection of neuronal IMR5 cells induces cytochrome c release from mitochondria and loss of mitochondrial transmembrane potential, both of which are evidence of mitochondrial outer membrane permeabilization. PV infection also activates Bax, a proapoptotic member of the Bcl-2 family; this activation involves its conformational change and its redistribution from the cytosol to mitochondria. Neutralization of Bax by vMIA protein expression prevents cytochrome c release, consistent with a contribution of PV-induced Bax activation to mitochondrial outer membrane permeabilization. Interestingly, we also found that c-Jun NH(2)-terminal kinase (JNK) is activated soon after PV infection and that the PV-cell receptor interaction alone is sufficient to induce JNK activation. Moreover, the pharmacological inhibition of JNK by SP600125 inhibits Bax activation and cytochrome c release. This is, to our knowledge, the first demonstration of JNK-mediated Bax-dependent apoptosis in PV-infected cells. Our findings contribute to our understanding of poliomyelitis pathogenesis at the cellular level.     

Cell death suppression by cytomegaloviruses

Cytomegaloviruses (CMVs), a subset of betaherpesviruses, employ multiple strategies to suppress apoptosis in infected cells and thus to delay their death. Human cytomegalovirus (HCMV) encodes at least two proteins that directly interfere with the apoptotic signaling pathways, viral inhibitor of caspase-8-induced apoptosis vICA (pUL36), and mitochondria-localized inhibitor of apoptosis vMIA (pUL37 × 1). vICA associates with pro-caspase-8 and appears to block its recruitment to the death-inducing signaling complex (DISC), a step preceding caspase-8 activation. vMIA binds and sequesters Bax at mitochondria, and interferes with BH3-only-death-factor/Bax-complex-mediated permeabilization of mitochondria. vMIA does not seem to either interact with Bak, a close structural and functional homologue of Bax, or to suppress Bak-mediated permeabilization of mitochondria and Bak-mediated apoptosis. All sequenced betaherpesviruses, including CMVs, encode close homologues of vICA, and those vICA homologues that have been tested, were found to be functional cell death suppressors. Overt sequence homologues of vMIA were found only in the genomes of primate CMVs, but recent observations made with murine CMV (MCMV) indicate that non-primate CMVs may also encode a cell death suppressor functionally resembling vMIA. The exact physiological rolesand relative contributions of vMIA and vICA in suppressing death of CMV-infected cells in vivo have not been elucidated. There is strong evidence that the cell death suppressing function of vMIA is indispensable, and that vICA is dispensable for replication of HCMV. In addition to suppressed caspase-8 activation and sequestered Bax, CMV-infected cells display several other phenomena, less well characterized, that may diminish, directly or indirectly the extent of cell death.     

Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis

Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.     

Cytomegalovirus proteins vMIA and m38.5 link mitochondrial morphogenesis to Bcl-2 family proteins

Apoptosis is a host defense mechanism against viruses that can be subverted by viral gene products. Human cytomegalovirus encodes viral mitochondria-localized inhibitor of apoptosis (vMIA; also known as pUL37x1), which is targeted to mitochondria and functions as a potent cell death suppressor by binding to and inhibiting proapoptotic Bcl-2 family members Bax and Bak. vMIA expression also dramatically alters mitochondrial morphology, causing the fragmentation of these organelles. A potential ortholog of vMIA, m38.5, which was identified in murine cytomegalovirus, has been shown to localize to mitochondria and protect against chemically induced apoptosis by unknown mechanisms. Despite sharing negligible homology with vMIA and no region detectably corresponding to the vMIA Bax-binding domain, we find that m38.5, like vMIA, binds to Bax and recruits Bax to mitochondria. Interestingly, m38.5 and vMIA appear to block Bax downstream of translocation to mitochondria and after an initial stage of Bax conformational change. In contrast to vMIA, m38.5 neither binds to Bak nor causes mitochondrial fragmentation. Consistently with Bax-selective inactivation by m38.5, m38.5 fragments mitochondria in Bak knockout (KO) cells and protects Bak KO cells from apoptosis better than Bax KO cells. Thus, vMIA and m38.5 share some, but not all, features of apoptosis regulation through Bcl-2 family interaction and allow the dissection of Bax translocation into discrete steps.     

The murine cytomegalovirus cell death suppressor m38.5 binds Bax and blocks Bax-mediated mitochondrial outer membrane permeabilization

Apoptosis is increasingly implicated as an early line of defense against viral infections. Viruses have devised numerous strategies to delay apoptosis of infected cells. Many viruses encode cell death suppressors that target mitochondrial apoptotic signaling pathway, indicating the importance of this pathway in the anti-viral response. Human and primate cytomegaloviruses encode the viral mitochondria-localized inhibitor of apoptosis vMIA, but no overt homologue of vMIA was identified in any non-primate cytomegalovirus. Here we report that m38.5 protein encoded by murine cytomegalovirus, which is unrelated to vMIA in its amino acid sequence, delays death receptor ligation-induced cell death, and that m38.5 associates with Bax, recruits it to mitochondria, and blocks Bax-mediated but not Bak-mediated mitochondrial outer membrane permeabilization. Thus, primate and murine cytomegaloviruses have evolved non-homologous but functionally similar cell death suppressors selectively targeting the Bax-mediated branch of the mitochondrial apoptotic signaling pathway, indicating the importance of this branch in the response of diverse host organisms against cytomegalovirus infections.     

Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization

Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.     

Mitochondrial cell death suppressors carried by human and murine cytomegalovirus confer resistance to proteasome inhibitor-induced apoptosis

Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses. We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation, the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell death.     

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.     

Disruption of mitochondrial networks by the human cytomegalovirus UL37 gene product viral mitochondrion-localized inhibitor of apoptosis

By 24 h after infection with human cytomegalovirus, the reticular mitochondrial network characteristic of uninfected fibroblasts was disrupted as mitochondria became punctate and dispersed. These alterations were associated with expression of the immediate-early (alpha) antiapoptotic UL37x1 gene product viral mitochondrion-localized inhibitor of apoptosis (vMIA). Similar alterations in mitochondrial morphology were induced directly by vMIA in transfected cells. A 68-amino-acid antiapoptotic derivative of vMIA containing the mitochondrial localization and antiapoptotic domains also induced disruption, whereas a mutant lacking the antiapoptotic domain failed to cause disruption. These data suggest that the fission and/or fusion process that normally controls mitochondrial networks is altered by vMIA. Mitochondrial fission has been implicated in the induction of apoptosis and vMIA-mediated inhibition of apoptosis may occur subsequent to this event.     

Contribution of GADD45 family members to cell death suppression by cellular Bcl-xL and cytomegalovirus vMIA

Mammalian cells and viruses encode inhibitors of programmed cell death that localize to mitochondria and suppress apoptosis initiated by a wide variety of inducers. Mutagenesis was used to probe the role of a predicted alpha-helical region within the hydrophobic antiapoptotic domain (AAD) of cytomegalovirus vMIA, the UL37x1 gene product. This region was found to be essential for cell death suppression activity. A screen for proteins that interacted with the AAD of functional vMIA but that failed to interact with mutants identified growth arrest and DNA damage 45 (GADD45alpha), a cell cycle regulatory protein activated by genotoxic stress, as a candidate cellular binding partner. GADD45alpha interaction required the AAD alpha-helical character that also dictated GADD45alpha-mediated enhancement of death suppression. vMIA mutants that failed to interact with GADD45alpha were completely nonfunctional in cell death suppression, and any of the three GADD45 family members (GADD45alpha, GADD45beta/MyD118, or GADD45gamma/OIG37/CR6/GRP17) was able to cooperate with vMIA; however, none influenced cell death when introduced into cells alone. GADD45alpha was found to increase vMIA protein levels comparably to treatment with protease inhibitors MG132 and ALLN. Targeted short interfering RNA knockdown of all three GADD45 family members maximally reduced vMIA activity, and this reduction was abrogated by additional GADD45alpha. Interestingly, GADD45 family members were also able to bind and enhance cell death suppression by Bcl-xL, a member of the Bcl-2 family of cell death suppressors, suggesting a direct cooperative link between apoptosis and the proteins that regulate the DNA damage response.     

Neurotensin receptor binding and neurotensin-induced growth signaling in prostate cancer PC3 cells are sensitive to metabolic stress

Neurotensin (NT) stimulates the proliferation of prostate cancer PC3 cells, which express high levels of its G protein-coupled receptor NTS1. To shed light on mechanisms that might serve to coordinate mitogenic responses to metabolic status, we studied the effects of metabolic inhibitors on NTS1 function. We also related these effects to cellular ATP levels and to the activation of AMP-activated protein kinase (AMPK). Glycolytic and mitochondrial inhibitors, at concentrations that reduced cellular ATP levels, altered NT binding to the cells, inhibited NT-induced inositol phosphate formation, and inhibited NT-induced DNA synthesis. For eight of the nine inhibitors, the potencies to alter NT receptor function correlated to the potencies to decrease cellular ATP levels. In keeping with its known role to oppose metabolic stress, AMPK was activated by the metabolic inhibitors. Accordingly, the AMPK activator AICAR elevated cellular ATP levels and produced effects on NTS1 function that were opposite to those for the metabolic inhibitors. These results indicate that metabolic stress inhibited NTS1 function by a mechanism that involved a fall in cellular ATP levels and that was opposed by activation of AMPK. In a broader context, these findings are compatible with the idea that one means by which cells might coordinate mitogenic signaling to metabolic status could involve changes in growth factor receptor function.     

Antibiotic anisomycin induces cell cycle arrest and apoptosis through inhibiting mitochondrial biogenesis in osteosarcoma

The anti-cancer activities of antibiotic anisomycin have been demonstrated in kidney, colon and ovarian cancers whereas its underlying mechanisms are not well elucidated. In this work, we investigated whether anisomycin is effective in sensitizes osteosarcoma cell response to chemotherapy. We show that anisomycin inhibits proliferation via inducing osteosarcoma cell arrest at G2/M phase, accompanied by the increased levels of mitotic marker cyclin B and the decreased levels of Rb and E2F-1. Anisomycin also induces apoptosis in a caspase-dependent manner in osteosarcoma cells. Importantly, anisomycin is less effective in normal control NIH3T3 cells compared to osteosarcoma cells. In addition, anisomycin inhibits osteosarcoma growth in xenograft mouse model and enhances the inhibitory effects of doxorubicin in osteosarcoma in vitro and in vivo. Mechanistically, anisomycin targets mitochondrial biogenesis in osteosarcoma as shown by the decreased mitochondrial membrane potential, suppressed mitochondrial respiration via decreasing complex I activity, reduced ATP production. Furthermore, mitochondrial biogenesis stimulator acetyl-L-Carnitine (ALCAR) significantly rescues the inhibitory effects of anisomycin in osteosarcoma cells. Our work demonstrates that anisomycin is active against osteosarcoma cells and the molecular mechanism of its action is the inhibition of mitochondrial biogenesis.     

Glutamate dehydrogenase requirement for apoptosis induced by aristolochic acid in renal tubular epithelial cells

Ingestion of aristolochic acids (AA) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adduct formation, is well-documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. Epithelial cell death is a critical characteristic of these pathological conditions. To elucidate the mechanisms of AA-induced cytotoxicity, we explored AA-interacting proteins in tubular epithelial cells (TEC). We found that AA interacts with a mitochondrial enzyme glutamate dehydrogenase (GDH) and moderately inhibits its activity. We report that AA induces cell death in GDH-knockdown TEC preferentially via non-apoptotic means, whereas in GDH-positive cells, death was executed by both the non-apoptotic and apoptotic mechanisms. Apoptosis is an energy-reliant process and demands higher adenosine 5′-triphosphate (ATP) consumption than does the non-apoptotic cell death. We found that, after AAI treatment, the ATP depletion is more pronounced in GDH-knockdown cells. When we reduced ATP in TEC cells by inhibition of glycolysis and mitochondrial respiration, cell death mode switched from apoptosis and necrosis to necrosis only. In addition, in cells incubated at low glucose and no glutamine conditions, oxaloacetate and pyruvate reduced AAI-induced apoptosis our data suggest that AAI-GDH interactions in TEC are critical for the induction of apoptosis by direct inhibition of GDH activity. AA binding may also induce changes in GDH conformation and promote interactions with other molecules or impair signaling by GDH metabolic products, leading to apoptosis.