Myxoma Virus M11L Blocks Apoptosis through ... - Journal of Virology

JOURNAL OF VIROLOGY, Feb. 2006, p. 1140–1151 0022-538X/06/$08.00⫹0 doi:10.1128/JVI.80.3.1140–1151.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 80, No. 3

Myxoma Virus M11L Blocks Apoptosis through Inhibition of Conformational Activation of Bax at the Mitochondria Jin Su,† Gen Wang,†‡ John W. Barrett, Timothy S. Irvine, Xiujuan Gao, and Grant McFadden* Department of Microbiology and Immunology, University of Western Ontario and Robarts Research Institute, London, Ontario, Canada Received 1 September 2005/Accepted 15 November 2005

Many viruses inhibit or retard apoptosis, a strategy that subverts one of the most ancient antiviral mechanisms. M11L, a myxoma virus-encoded antiapoptotic protein, has been previously shown to localize to mitochondria and block apoptosis of virus-infected cells (H. Everett, M. Barry, S. F. Lee, X. J. Sun, K. Graham, J. Stone, R. C. Bleackley, and G. McFadden, J. Exp. Med. 191:1487–1498, 2000; H. Everett, M. Barry, X. Sun, S. F. Lee, C. Frantz, L. G. Berthiaume, G. McFadden, and R. C. Bleackley, J. Exp. Med. 196:1127–1139, 2002; and G. Wang, J. W. Barrett, S. H. Nazarian, H. Everett, X. Gao, C. Bleackley, K. Colwill, M. F. Moran, and G. McFadden, J. Virol. 78:7097–7111, 2004). This protection from apoptosis involves constitutive-forming inhibitory complexes with the peripheral benzodiazepine receptor and Bak on the outer mitochondrial membrane. Here, we extend the study to investigate the interference of M11L with Bax activation during the process of apoptosis. Myxoma virus infection triggers an early apoptotic signal that induces rapid Bax translocation from cytoplasm to mitochondria, despite the existence of various viral antiapoptotic proteins. However, in the presence of M11L, the structural activation of Bax at the mitochondrial membrane, which is characterized by the occurrence of a Bax conformational change, is blocked in both M11L-expressing myxoma-infected cells and M11L-transfected cells under apoptotic stimulation. In addition, inducible binding of M11L to the mitochondrially localized Bax is detected in myxoma virus-infected cells and in M11L/Bax-cotransfected cells as measured by immunoprecipitation and tandem affinity purification analysis, respectively. Importantly, this inducible Bax/M11L interaction is independent of Bak, demonstrated by the complete block of Bax-mediated apoptosis in myxoma-infected cells that lack Bak expression. Our findings reveal that myxoma M11L modulates apoptosis by multiple independent strategies which all contribute to the blockade of apoptosis at the mitochondrial checkpoint. chondrial membrane (OMM), resulting in the release of proapoptogenic factors (1, 13, 28). Although Bak and Bax seem in many circumstances to be functionally equivalent (43, 75), substantial differences in their regulation would be expected from their distinct localization in nonapoptotic cells. Bak resides on the OMM in association with Mcl-1 and Bcl-xL, which occupy the dimerization and killing domain (BH3 domain) of Bak, therefore keeping Bak inactive (76). When activated, Bak is released from Mcl-1 and Bcl-xL and the BH3 domains are displaced for oligomerization, which promotes cell death. Bax, instead, is largely found in the cytoplasm or is loosely attached to OMM as inactive monomers in nonapoptotic somatic cells (77). Although Bax has a hydrophobic groove that could serve as a receptor for a BH3 protein, the groove is occluded by the C-terminal ␣-helical putative transmembrane (TM) domain ␣-helix 9, thus keeping Bax in the cytoplasm in the form of inactive monomers (67). The BH3 domain of Bax, which is essential for its proapoptotic activity, including formation of Bax/Bax homodimers and Bax/ Bcl-2 heterodimers, is masked in the hydrophobic core of the protein as well as in the inactive Bax in cytoplasm (73, 79). Following any one of various cytotoxic signals, Bax is activated and undergoes a series of conformational changes in the N and C termini, leading to Bax translocation to the mitochondria, oligomerization, and integration into the mitochondrial membranes (34, 38, 39, 45, 49, 51, 59, 77). These events all have been implicated in the process of cytochrome c release, al-

Apoptosis is an active death program that contributes to the elimination of damaged, mutated, aged, or virally infected cells (22). A key event in most forms of apoptosis is the change of mitochondrial membrane permeabilization (MMP) and the release of cytochrome c from mitochondria into the cytoplasm, which frequently marks the point of no return in the apoptotic process (78). As one of the critical control points for the life-versus-death decision, MMP is tightly regulated by members of the Bcl-2 family of proteins, which inhibit or promote MMP depending on whether they belong to the anti- or proapoptotic branch of the family, respectively (2, 31, 47). Among the proapoptotic members, the small BH3 (Bcl-2 homology 3)-only proteins including Bad, Bid, Bim, Puma, and Noxa are often responsible for conveying the initial death signal, but the multidomain proteins Bak and/or Bax are required for commitment to cell death via the mitochondrial pathway (11, 43, 75). When activated, Bak and Bax undergo conformational changes to form oligomers and permeabilize the outer mito-

* Corresponding author. Mailing address: Rm 1-33, Siebens Drake Building, 1400 Western Rd., London, Ontario, N6G 2V4 Canada. Phone: (519) 663-3184. Fax: (519) 663-3715. E-mail: mcfadden @robarts.ca. † These authors contributed equally to this work. ‡ Present address: Institute for Nutrisciences and Health, National Research Council of Canada, Charlottetown, Prince Edward Island, C1A 5T1 Canada. 1140

VOL. 80, 2006

though the precise biochemical mechanism by which Bax induces cytochrome c release is still controversial (5, 6, 29, 48, 60, 63). Apoptosis is considered an innate defense mechanism against intracellular pathogen infection, and the mitochondria checkpoint is pivotal to this regulation (9). This concept is supported by the fact that many viruses carry genes whose products can directly interfere with the mitochondrial apoptotic apparatus of the host cell for their own benefit (10, 12, 19, 20, 30, 54). A number of viruses express antiapoptotic proteins that localize to the OMM to prevent or retard induction of MMP by binding to host proapoptotic modulators, allowing the virus to propagate before its host cell dies (8). For example, E1B 19K from adenovirus, a Bcl-2 homolog, interacts with Bax and Bak and blocks Bax and Bak oligomerization to inhibit downstream apoptotic events (53, 65, 66). vMIA, encoded by human cytomegalovirus (CMV), is localized to mitochondria, where it blocks the activity of the adenine nucleotide translocator (ANT), a subunit of the permeability transition port (PTP) complex that spans the inner and outer mitochondrial membranes to induce MMP (25). In addition, vMIA recruits Bax to mitochondria and causes its full membrane insertion as well as its oligomerization and yet suppresses all manifestations of MMP (7, 55). Recently, a new poxvirus antiapoptotic protein, F1L of vaccinia virus, has been identified to localize exclusively in mitochondria (64, 74), thereby inhibiting MMP and the release of cytochrome c, reportedly by direct binding to the BH3 domain of host Bcl-2 members (23) like Bak (74a). Myxoma virus (MV), a member of the Leporipoxvirus genus of poxviruses, is the causative agent of myxomatosis in European rabbits (Oryctolagus cuniculus) (21, 41). MV encodes multiple antiapoptotic proteins, including M-T2, M-T4, M-T5, and M11L, which function to disrupt various classes of proapoptotic signals (18, 62). Among these viral antiapoptotic proteins, M11L, a small protein (166 amino acids) which is important for viral pathogenesis in the rabbit (52), has been found to be capable of functioning in human cells as well as to prevent apoptosis via the mitochondrial pathway (72). This protein possesses a hydrophobic transmembrane (TM) domain in the C terminus which is responsible for localization to the mitochondria, where M11L executes its antiapoptotic function (16). M11L forms an inhibitory complex with the peripheral benzodiazepine receptor (PBR), a component of the PTP complex that promotes MMP (17). Moreover, M11L has been shown to also interact with the proapoptotic protein Bak, a Bcl-2 family member that is constitutively present on mitochondria (72). M11L prevents the release of cytochrome c from mitochondria as well as the dissipation of the mitochondrial membrane potential (D⌿m) induced by the treatment with several apoptotic stimuli, including the PBR ligand protoporphyrin IX (17), and overexpression of Bak (72). In the present study, we extend the investigation and demonstrate that although it does not prevent Bax localization to the mitochondria in response to an apoptotic signal or virus infection, M11L inducibly blocks Bax activation at mitochondria through suppression of Bax conformational change and represents an additional antiapoptotic strategy independent of Bak.

M11L BLOCKS Bax AT MITOCHONDRIA

1141

MATERIALS AND METHODS Antibodies and reagents. Monoclonal antibodies (MAb) detecting Bax conformational change (clone 6A7) and detecting ␤-actin as loading control for immunoblot as well as anti-Flag (M2) were from Sigma-Aldrich Inc. (St. Louis, MO). Mouse MAb against a peptide epitope derived from the hemagglutinin (HA) protein of human influenza virus (clone 12CA5) was obtained from Roche Diagnostics (Mannheim, Germany). MAb detecting full-length caspase 3 as well as the cleaved fragments (19 and 17 kDa) were from Cell Signaling Technology (clone 8G10; Beverly, MA), and MAb detecting poly (ADP-ribose) polymerase (PARP) and the cleavage product was from BD Biosciences Clontech (Palo Alto, CA). MAb detecting cytochrome oxidase subunit IV (COX IV; Molecular Probes, Eugene, OR) was used for a mitochondrial loading control. Polyclonal antibodies against Bax (recognizing both inactive and active Bax) were from Santa Cruz Biotechnology (clone N20; Santa Cruz, CA) and from Upstate Biotechnology (clone NT; Lake Placid, NY), respectively. Polyclonal antibody detecting Bak (clone NT) was from Upstate Biotechnology as well. Polyclonal antibody against M11L has been previously described (52). Horseradish peroxidase-coupled goat anti-mouse and goat anti-rabbit secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA). A rabbit peroxidase–anti-peroxidase antiserum (anti-PAP; Sigma-Aldrich Inc.) recognizing the protein A sequence of the tandem affinity purification (TAP) tag was used for monitoring TAP tag expression. MitoTracker Red, Oregon Green 514-conjugated goat anti-mouse, and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies were purchased from Molecular Probes, Invitrogen Canada, Inc. (Burlington, Ontario, Canada). Tumor necrosis factor alpha (TNF-␣) was purchased from Medicorp (Montreal, Canada), and CHAPS [3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate] was obtained from Calbiochem (San Diego, CA). A complete protease inhibitor cocktail (Roche Diagnostics) was included in all lysis buffers. Protein A- and G-agarose beads were from Upstate Biotechnology. Cells and viruses. Human osteosarcoma (HOS) cells, renal cancer 786-0 cells, HEK293 cells (kindly provided by Jin Q. Cheng from the University of South Florida), and HEK293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% (vol/vol) fetal bovine serum (FBS). Simian virus 40-transformed mouse embryonic fibroblasts derived from wild-type (wtMEF) and Bak knockout mice (MEF Bak⫺/⫺), kindly provided by S. J. Korsmeyer (Harvard Medical School, Boston, MA) (75), were grown in the same medium as that described above. HEK-Neo and HEK-M11L transfectants (72) were cultured in RPMI 1640 supplemented with 10% FBS and 500 ␮g/ml G418. BGMK monkey kidney cells were grown in DMEM supplemented with 10% newborn calf serum. Recombinant MV viruses used in this study included vMyxlac (a wild-type control) and vMyxM11LKO (a mutant MV that failed to express M11L due to a targeted gene disruption within the open reading frame M011L [75]). Infections and measurement of viral growth. Viral infections were carried out at a multiplicity of infection (MOI) of 5, unless stated specifically. To measure the multistep viral growth in HOS and 786-0 cells, cells were incubated with 0.1 MOI of MV for 1 h at 37°C followed by three washes to get rid of the unbound viruses. The infected cells were harvested at 0, 12, 24, 48, 72, and 96 h postinfection, processed by three cycles of freeze/thaw, and titrated in BGMK cells with serial dilutions. To measure the single-step viral growth in wtMEF and MEFBak⫺/⫺ cells, cells were infected with 5 MOI of MV as described above, harvested at 0, 10, 20, 30, 40, and 50 h postinfection, and titrated in BGMK cells. Immunoblot for detecting apoptosis. Cells were harvested and lysed in 1% NP-40 buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2 mM EDTA, pH 8.0, 10% glycerol, and 1% NP-40) containing a cocktail of protease inhibitors. The cell lysate was cleared by centrifugation at 15,000 ⫻ g for 30 min at 4°C. Supernatant proteins (50 ␮g) were resolved by 15% (for caspase 3) or 8% (for PARP) denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20, the membrane was incubated with the desired primary antibody overnight at 4°C. The membrane was then washed and treated with appropriate secondary antibody for 1 h at room temperature, and the immunoreactive bands were visualized using the Western Lightning enhanced chemiluminescence kit from PerkinElmer Life Sciences, Inc. (Boston, MA). Membranes were also stripped and reprobed with anti-␤-actin antibody to confirm the equal loading of the proteins. Immunoprecipitation and immunoblot for detecting Bax and M11L interaction. Cells infected with MV at 5 MOI were harvested 24 h postinfection and lysed in 1% NP-40 buffer or 2% CHAPS lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 2% CHAPS) containing a cocktail of protease inhibitors. The lysate was centrifuged at 15,000 ⫻ g for 30 min at 4°C,

1142

SU ET AL.

and the supernatant was collected for the assays. Immunoprecipitation was performed with 2 ␮g of anti-Bax (clone NT) at 4°C overnight. Protein A-agarose beads (50 ␮l) were then added, and the incubation was continued for 4 h at 4°C. The immunoprecipitated complexes with agarose beads were washed four times with the lysis buffer containing 0.5% sodium deoxycholate and subjected to SDS–15% PAGE followed by immunoblotting with anti-M11L antibody. Construction of TAP tag vectors and TAP-tagged protein purification. The N-terminal TAP dual-epitope tags (58) were provided by J. Walkenhorst (Cellzome GmbH and European Molecular Biology Laboratory, Heidelberg, Germany). An N-TAP cassette was subcloned into pcDNA3 resulting in pcDNA3.4N-TAP fusion vector (72), which was used for further subcloning of M11L and human Bax (pcDNA3.4-N-TAP-M11L and pcDNA3.4-N-TAP-Bax, respectively). Logarithmic-phase HEK293T cells were seeded in 100-mm-diameter tissue culture dishes (5 ⫻ 106 cells/dish) in fresh medium the day before transfection. Cells were transiently transfected or cotransfected with 20 ␮g of each of the expression plasmids using a calcium phosphate mammalian transfection kit (BD Clontech) according to the manufacturer. Cells expressing the TAP-tagged M11L protein or pcDNA3-M11L plus N-TAP-Bax were lysed in 2% CHAPS or 1% NP-40 lysis buffer 24 h after transfection. Following centrifugation at 15,000 ⫻ g for 30 min at 4°C, the supernatant was recovered and added to rabbit immunoglobulin G (IgG) agarose beads (Sigma-Aldrich, Inc.), which were then rotated for 2 h at 4°C to allow the binding of the fusion protein. The beads were washed three times with wash buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% CHAPS or 0.1% NP-40) and resuspended in 1 ml of tobacco etch virus (TEV) cleavage buffer (10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol) containing 200 U of TEV protease (Invitrogen). The samples were mixed by inversion at room temperature for 2 h to cleave the tagged protein. Calmodulin binding buffer (10 mM ␤-mercaptoethanol, 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl2, and 0.1% CHAPS or 0.1% NP-40) was added to the eluate together with calmodulin affinity resin (Stratagene, La Jolla, CA) and mixed for 1 h at 4°C to allow binding to the beads. Following three washes with calmodulin binding buffer, the samples were eluted with calmodulin elution buffer (10 mM ␤-mercaptoethanol, 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM EGTA, and 0.1% CHAPS or 0.1% NP-40). The eluted purified product was concentrated to an appropriate volume through a NANOSEP 10K OMEGA protein concentrator (Pall Gelman Laboratory, Ann Arbor, MI) and resolved by denaturing SDS-PAGE followed by immunoblotting with the proper antibodies. Construction of Flag-M11L and HA-Bax vectors and immunoprecipitation of Flag-M11L/HA-Bax complex in transfected cells. Flag-M11L was constructed as described previously (72). Briefly, M11L was cloned into pFlag-CMV2 vector (kindly provided by Jin Q. Cheng, University of South Florida), resulting in an N-terminally Flag-tagged M11L construct (pFlag-CMV2-M11L). Human Bax was kindly provided by Ping Q. Dou (Wayne State University) and subcloned into pcDNA3-HA to generate an N-terminally HA-tagged Bax construct (pcDNA3-HA-Bax). HEK293T cells were cotransfected with 20 ␮g of FlagM11L and HA-Bax by calcium phosphate precipitation as described elsewhere (72). Cells were collected 24 h posttransfection and lysed in 2% CHAPS lysis buffer. The cell lysate was cleared by centrifugation at 15,000 ⫻ g for 30 min at 4°C, and supernatant was incubated overnight at 4°C with 2 ␮g anti-Flag or anti-HA MAb, respectively. Protein G-agarose beads (50 ␮l) were then added to each sample, and the incubation was continued for 4 h at 4°C. The immunoprecipitated complexes with agarose beads were washed four times with the lysis buffer containing 0.5% sodium deoxycholate and a cocktail of protease inhibitors and subjected to SDS–15% PAGE followed by immunoblotting using anti-HA or anti-Flag MAb, respectively. Immunoprecipitation and immunoblot for detecting Bax conformational change. Two anti-Bax antibodies were used in this assay. Clone 6A7 recognizes an epitope in the N terminus which is normally hidden in inactive Bax and exposed when Bax undergoes conformational change for apoptosis induction (36). Therefore, 6A7 does not bind to inactive Bax. Clone N20 instead recognizes an exposed epitope and hence reacts with both inactive and active forms of Bax. HOS, 786-0, HEK293, wtMEF, or MEF Bak⫺/⫺ cells infected with MV at 5 MOI for 24 h or HEK-Neo and HEK-M11L stimulated with 10 ng/ml of TNF-␣ for 24 h were collected and lysed in 2% CHAPS buffer containing a cocktail of protease inhibitors. The cell lysate was cleared by centrifugation at 15,000 ⫻ g for 30 min at 4°C. Thereafter, aliquots of equal amounts of supernatant proteins (1 mg/ml), determined by Bradford dye binding (Protein Assay; Bio-Rad Laboratories, Hercules, CA) using a spectrophotometer, was incubated overnight at 4°C with 2 ␮g anti-Bax MAb 6A7. Protein G-agarose beads (50 ␮l) were then added to each sample, and the incubation was continued for 4 h at 4°C. The immunoprecipitated complexes with agarose beads were washed four times with 1% CHAPS wash buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 1.5 mM MgCl2,

J. VIROL. 1 mM EDTA, 1% CHAPS, 0.5% sodium deoxycholate, and a cocktail of protease inhibitors) and subjected to SDS–15% PAGE followed by immunoblotting using polyclonal anti-Bax N20. Detection of the IgG light chain was used as an indicator for equal loading of the samples. Subcellular fractionation for localization of Bax. Both cytosolic and heavy membrane (HM) fractions (including mitochondria) were isolated at 4°C by using an established protocol with some modifications (72). Briefly, cells were washed twice in cold phosphate-buffered saline (PBS) buffer, resuspended in a hypotonic buffer containing 20 mM HEPES-KOH (pH 7.5), 1.5 mM MgCl2, 5 mM KCl, 1 mM dithiothreitol, and a cocktail of protease inhibitors, and incubated on ice for 10 min. The cells were sheared four times through a 30-gauge 1/2-inch needle fitted on a 1-ml syringe, and the lysate was centrifuged at 1,000 ⫻ g for 10 min at 4°C. The supernatant was collected and centrifuged again under the same conditions. The resulting supernatant was then centrifuged at 14,000 ⫻ g for 30 min at 4°C, followed by collection of both the supernatant and pellet fractions. The pellet was washed twice with the mitochondria washing buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl, pH 7.5, 1 mM EDTA, and a protease inhibitor cocktail). The pellet was resuspended in the mitochondria lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, and 0.5% NP-40, supplemented with a cocktail of protease inhibitors), and this represented the HM fraction including mitochondria. The supernatant was further ultracentrifuged at 100,000 ⫻ g for 60 min at 4°C, and the resulting supernatant was collected as the cytosolic fraction. The cytosolic fraction was concentrated to an appropriate volume by a NANOSEP 10K OMEGA protein concentrator (Pall Gelman Laboratory). Twenty micrograms of protein from the HM or cytosolic fraction was resolved by SDS–15% PAGE and blotted with anti-Bax (NT) antibody. Immunofluorescent staining and confocal microscopy. Cells were cultured on coverslips (Fisher Scientific Company, Ottawa, Ontario, Canada) overnight followed by infection with MV for 24 h. The infected cells were then washed briefly with PBS and fixed with 100% prechilled methanol for 10 min. Thereafter, the fixed cells were washed with PBS and blocked with PBS containing 2% bovine serum albumin (Sigma-Aldrich Inc.) for 1 h at room temperature. After staining with MitoTracker Red for 1 h, cells were washed and incubated with anti-Bax antibody (NT or 6A7; 1:1,000 dilution) for 1 h followed by a further incubation with the secondary antibody at 1:1,000 dilution (Alexa Fluor 488-conjugated goat anti-rabbit for clone NT or Oregon Green 514-conjugated goat anti-mouse for clone 6A7) for 1 h at room temperature. The coverslips were washed with PBS, mounted with VECTASHIELD Mounting Medium (Vector Laboratories Canada Inc., Burlington, Ontario, Canada), and examined under a laser scanning microscope (LSM) 510 (Carl Zeiss Inc., Jena, Germany).

RESULTS Myxoma M11L inducibly interacts with Bax. M11L is an early gene product expressed by MV, and its synthesis could be observed in MV-infected permissive human cancer cells as early as 4 h postinfection (Fig. 1A) but not in cells infected with vMyxM11LKO, similar to what is observed for infected rabbit cells (26). It has been shown previously that M11L localizes to mitochondria and blocks apoptosis (16, 17, 72). In an effort to identify the binding partners of M11L in human tumor cells, we reported the constitutive interaction between M11L and Bak and noticed that M11L also can bind to Bax under certain inducible conditions but not with the other Bcl-2 family members tested, including Bad, Bid, and Bcl-2 (72). Hence, we sought to investigate the interaction between M11L and Bax in MV-infected cells by immunoprecipitation assays. Human osteosarcoma (HOS) cells and renal cancer 786-0 cells were permissive for both wild-type MV (vMyxlac) and the M11L knockout virus (vMyxM11LKO) (Fig. 1B), although a lower replication level (nearly a log) of vMyxM11LKO was observed in both cells. These cell lines were used to study the potential interaction and interference of M11L with endogenous Bax. To rule out the possibility that the use of any specific detergent contained in lysis buffer may affect M11L/Bax protein binding, MV-infected cells were lysed in either 1% NP-40 buffer or 2% CHAPS buffer 24 h postinfection, and the immune complexes

VOL. 80, 2006

FIG. 1. Infection of wild-type myxoma virus (vMyxlac) (lanes 1 through 6) and M11L knockout myxoma virus (vMyxM11LKO) (lanes 7 through 12) in human tumor cells. (A) Early expression of M11L in HOS cells infected with vMyxlac at 5 MOI, detected by immunoblotting with anti-M11L antibody. Myxoma viral protein MT-7 was used as a control for viral protein expression. (B) Virus replication and spread of vMyxlac and vMyxM11LKO in HOS and 786-0 cells. Cells were infected at 0.1 MOI and titrated in BGMK cells. The numbers depicted the averages of three experiments, and bars represent the standard deviations.

in cell lysate were precipitated with anti-Bax (NT) antibody followed by immunoblotting with anti-M11L antibody. M11L formed an immunocomplex with endogenous Bax in vMyxlacinfected 786-0 cells under the condition of 2% CHAPS lysis buffer (Fig. 2A). The same result was obtained when 1% NP-40 lysis buffer was used (data not shown), indicating that lysis buffer containing NP-40 or CHAPS had no effect on this binding in MV-infected cells. Complexes between M11L and Bax were detected repeatedly in other tested cells that were permissive for vMyxlac, including HOS and HEK293, and also in uninfected HEK-M11L transfectants (stably expressing M11L) following TNF-␣ stimulation (data not shown). To further confirm the interaction between M11L and Bax, TAP analysis was performed using either TAP-M11L as bait to pull down Bax or TAP-Bax as bait to pull down M11L. In uninfected cells transfected with TAP-M11L, Bax copurified with the TAPM11L eluate only when NP-40 lysis buffer was used (Fig. 2B, image i) but not when CHAPS lysis buffer was used (data not shown). This result is consistent with what was reported earlier (72). However, in cells cotransfected with TAP-Bax and M11L plasmids, M11L was detected to be copurified with the TAPBax eluate in the presence of CHAPS in the lysis buffer (Fig. 2C, image i). For the TAP-M11L bait, controls indicate expression of the 20-kDa TAP alone, the intact 35-kDa TAPM11L fusion protein in cell lysate prior to the TAP procedure, and recovery of the 17-kDa TEV cleaved M11L (M11L-CBP) after the TAP procedure as shown in Fig. 2B, images iii and ii, respectively. For the TAP-Bax bait, expression of M11L and the intact 40-kDa TAP-Bax fusion protein in cell lysate prior to the TAP procedure are shown in Fig. 2C, images ii and iii, respectively. In agreement with these results, complexes of M11L


1143

and Bax were also detected in the Flag-CMV2-M11L- and pcDNA3-HA-Bax-cotransfected HEK293T cells by immunoprecipitation with anti-HA antibody followed by immunoblotting with anti-Flag antibody and, vice versa, immunoprecipitation with anti-Flag followed by immunoblotting with anti-HA when CHAPS lysis buffer was used (Fig. 2D). Taken together, we conclude that M11L enters into complexes with Bax not only in MV-infected cells but also in cells where transfected M11L is transiently expressed, thus indicating that no additional viral factors are required to mediate the binding between Bax and M11L. Bax conformational change is inhibited in the presence of M11L. To investigate any biological functions of Bax that might be blocked by interaction with M11L, we first focused on the Bax protein conformational change which is essential for initiating apoptosis following Bax activation (6, 14, 50, 68). HOS cells infected with vMyxlac or vMyxM11LKO were collected 24 h postinfection and lysed in 2% CHAPS buffer, which does not alter the conformational status of Bax (5, 6, 35). Equal amounts of lysate proteins were immunoprecipitated by anti-Bax 6A7 to pull down conformationally active Bax followed by immunoblotting with anti-Bax antibody N20 (which recognizes a common region in both native and active Bax). During the course of an MV infection, Bax conformational change was detected with 6A7 antibody in the cells infected with vMyxM11LKO at 6 h postinfection and the amount of Bax*/19kD increased dramatically over time (Fig. 3A, lanes 5 to 7). In contrast, no Bax conformational change was detected over the course of 24 h in cells infected with vMyxlac, in which M11L was present (Fig. 3A, lanes 2 to 4). This result demonstrated that M11L blocks some aspect of Bax conformational change in the MV-infected host cells. This inhibition of Bax conformational change was also detected in HEK-M11L transfectants, which stably express M11L (Fig. 3B). Control cells (HEK-Neo) and HEK-M11L were stimulated with 10 ng/ml of TNF-␣ for 24 h and then collected and lysed in 2% CHAPS buffer followed by immunoprecipitation with 6A7 antibody as described above. HEK-Neo cells underwent apoptosis as expected in the absence of M11L (72) with the detection of the anticipated change in Bax conformation (Fig. 3B, lanes 1 to 2). However, a dramatic inhibition of this Bax conformational change was observed in HEK-M11L in the presence of constitutively expressed M11L (Fig. 3B, lanes 3 and 4). These findings suggest that M11L is capable of inducible binding to Bax and inhibiting Bax conformational change in addition to the previously identified mechanisms that feature M11L constitutive binding to PBR and Bak at mitochondria (17, 72). It is known that Bax normally resides in the cytoplasm but undergoes conformational change in response to apoptotic stimuli accompanied by translocation to the mitochondria to promote apoptosis. To extend our findings and to ascertain in which cellular compartment this conformational change of Bax occurs, we performed immunofluorescent staining of Bax on cells infected with vMyxlac or vMyxM11LKO. Bax recognized by conformation-dependent 6A7 antibody was visualized by staining with Oregon Green 514-conjugated anti-mouse antibody (green color in Fig. 4A, D, and G), and mitochondria were stained with MitoTracker Red (red color in Fig. 4B, E, and H). The merged yellow color indicates the presence of conformationally changed activated Bax colocalized with mitochondria

1144

SU ET AL.

J. VIROL.

FIG. 2. Myxoma M11L interacts inducibly with human Bax. (A) Interaction of M11L with endogenous Bax in MV-infected cells, as detected by immunoprecipitation assays. 786–0 Cells were infected with vMyxlac (5 MOI) for 24 h, and cell lysates prepared with CHAPS buffer were immunoprecipitated with anti-Bax (NT) antibody followed by immunoblotting with anti-M11L. Lane 1, mock immunoprecipitation; lane 2, immunoprecipitation of vMyxlac-infected cell lysate; lane 3, mock-infected cell lysate; lane 4, vMyxlac-infected cell lysate. a*, antibody. (B) Bax was detected as a binding partner of M11L in transfected cells using NP-40 lysis buffer, with pcDNA3.4-N-TAP-M11L as bait. HEK293T cells were transfected with pcDNA3.4-N-TAP vector alone (lanes 1, 3, and 5) or with N-TAP-M11L (lanes 2, 4, and 6). The eluted product was blotted with anti-Bax (NT) (i) and with anti-M11L to show the recovery of the TEV-cleaved M11L, M11L-CBP (ii). N-TAP-M11L bait expression and TAP alone in HEK293T cell lysate prior to the TAP procedure is shown in image iii, blotted with anti-PAP to detect the TAP tag. (C) M11L was detected as a binding partner of Bax using pcDNA3.4-N-TAP-Bax as bait, with CHAPS lysis buffer. HEK293T cells were cotransfected with pcDNA3-M11L plus pcDNA3.4-N-TAP vector (lanes 1, 3, and 5) or with pcDNA3-M11L plus N-TAP-Bax (lanes 2, 4, and 6). (i) Eluted product was blotted with anti-M11L to reveal M11L that copurified with N-TAP-Bax. (ii) Cell lysate was blotted with anti-M11L to show the expression of M11L prior to the TAP procedure. (iii) Cell lysate was also blotted with anti-PAP to show the expression of N-TAP-Bax bait and control TAP alone in the cells. (D) Flag-M11L interacts with HA-Bax in the presence of CHAPS lysis buffer. HEK293T cells were cotransfected with Flag-CMV2-M11L and pcDNA3-HA-Bax, and immunoprecipitation was performed with anti-HA (12CA5) (lane 1 and 2) or with anti-Flag (M2) (lane 3 and 4) followed by immunoblotting with anti-Flag (lane 1 and 2) or with anti-HA (lane 3 and 4). WB, Western blot; IP, immunoprecipitation; IgG(L), IgG light chain.

(Fig. 4C, F, and I). This activated Bax recognized by 6A7 could only be detected in cells infected with vMyxM11LKO (Fig. 4G to I) but was not detected in mock- or vMyxlac-infected cells. In addition, this result demonstrated that this Bax conformational change, as detected by 6A7, only occurred at the mitochondria, not in the non-mitochondria compartment, such as cytoplasm, in MV-infected cells (Fig. 4I). Our finding that the 6A7 antibody detects only a Bax conformational change at the

mitochondria, not in the cytoplasm, is consistent with the observation by others under different experimental settings (40, 50). Translocation of Bax to mitochondria is triggered by myxoma infection and is not blocked by M11L. Bax-induced apoptosis involved two key events, the translocation of Bax to mitochondria and then a conformational change that activates Bax (67, 69). To determine whether M11L could block Bax translocation to the mitochondria and hence prevent Bax con-

VOL. 80, 2006

FIG. 3. Bax conformational change in myxoma virus-infected cells is inhibited in the presence of M11L. (A) Bax conformational change (indicated by Bax*/19kD) was detected by immunoprecipitation with 6A7 antibody in HOS cells infected with vMyxM11LKO (lanes 5 to 7) but not infected with vMyxlac (lanes 2 to 4). Lane 1, mock-infected cells. (B) M11L blocks Bax conformational change induced by TNF-␣ in HEK293 cells that stably express M11L. Following stimulation with 10 ng/ml of TNF-␣, Bax conformational change can be detected in the control HEK-Neo cells (in the absence of M11L) 24 h poststimulation (lane 2) but not in the HEK-M11L cells which stably express M11L (lane 4). Lane 1, untreated HEK-Neo cells; lane 3, untreated HEKM11L cells. The IgG light chain [IgG(L)] was used as an indicator for equal loading of the samples.

formational change, we harvested and fractionated cells infected with MV (vMyxlac or vMyxM11L). Cytosolic and mitochondrial fractions were separated by centrifugation, and equal amounts of proteins were resolved by SDS–15% PAGE followed by immunoblotting with anti-Bax (NT) antibody. As shown in Fig. 5A (lane 1), a low level of Bax was detected in the mitochondrial fraction from mock-infected cells. This may represent a small fraction of loosely attached inactive Bax on the mitochondrial membrane. In contrast, regardless of whether cells were infected with vMyxlac or vMyxM11LKO, we detected a significantly larger amount of Bax in the mitochondrial fractions than in mock-infected cells (Fig. 5A, lanes 2 and 3). Accordingly, the amount of Bax in cytosolic fractions from both vMyxlac- and vMyxM11L-infected cells was notably decreased in comparison to that in the mock-infected cells (lanes 4 to 6). This suggests that Bax translocates from the cytoplasm to the mitochondria in MV-infected cells despite the expression of M11L. This was further confirmed by immunofluorescent staining of the vMyxlac- or vMyxM11LKO-infected cells. Bax was stained with anti-Bax NT (recognizing both native and activated Bax) and visualized by Alexa Fluor 488-conjugated anti-rabbit antibody (green color in Fig. 5B, images I, IV, and VII), and mitochondria were stained with MitoTracker Red (red color in Fig. 5B, images II, V, and VIII). The merged yellow color indicates the colocalization of Bax and mitochondria (Fig. 5B, images III, VI, and IX). A low level of Bax could be seen on mitochondria in the mock-infected cells, whereas in the MV-infected cells, both in the presence (vMyxlac infection) and the absence (vMyxM11LKO infection) of M11L, a significant amount of Bax can be detected at the mitochondria, indicating again that some Bax has translocated to the mito-


1145

chondria in response to viral infection despite the existence of M11L in the vMyxlac-infected cells. In addition, a fraction of the total Bax was still detectable in the nonmitochondrial compartment (presumably the cytoplasm) in the infected cells, which is in agreement with the finding from immunoblotting of the cytosolic and mitochondrial fractions (Fig. 5A) that only a subset of Bax translocated from cytoplasm to mitochondria in response to viral infection. These results indicate that the Bax conformational change detected by the 6A7 antibody is a later event following Bax translocation to mitochondria and that M11L can specifically block this posttranslocational change of Bax conformation. M11L blocks Bax activity in the absence of Bak. Bax and Bak can have overlapping functions under certain conditions when promoting apoptosis (43, 75). Previously we have shown that M11L blocks apoptosis that is Bak induced and mitochondria dependent (72). To clarify whether the inhibition of Bax activity by M11L might require the presence of Bak, we infected MEF cells lacking Bak expression (MEF Bak⫺/⫺) with MV and then examined the antiapoptotic properties of M11L. vMyxlac-infected MEF Bak⫺/⫺ cells failed to exhibit detectable apoptosis as measured by induced caspase 3 or PARP cleavage, indicating that MEF Bak⫺/⫺ cells were protected from MV-induced apoptosis, whereas vMyxM11LKO infection induced detectable caspase 3 and PARP cleavage as early as 8 h postinfection in the absence of M11L (Fig. 6A and B). To further confirm the Bak-independent interaction of M11L and Bax, MEF Bak⫺/⫺ cells were infected with MV and immunoprecipitation assays were performed as described above. M11L interacted directly with Bax in the vMyxlac-infected MEF Bak⫺/⫺ cells (Fig. 7A, lanes 1 and 2). Immunoblotting controls for the expression of M11L in the cell lysate before immunoprecipitation and the expression of Bax and the absence of Bak were also confirmed (Fig. 7A, lanes 3 to 6). Together, these results indicate that there is a direct interaction between M11L and Bax, and this interaction does not require the involvement of Bak. Furthermore, immunoprecipitation with conformation-dependent anti-Bax 6A7 antibody was also performed on the infected wtMEF and MEF Bak⫺/⫺ cells using 2% CHAPS lysis buffer as described above. Bax conformational change, detected in the vMyxM11LKO-infected MEF Bak⫺/⫺ cells, was blocked by M11L in the vMyxlac-infected MEF Bak⫺/⫺ cells, which is the same as in the wtMEF-infected cells (Fig. 7B). This confirms that the process of M11L binding to Bax, thus preventing Bax conformational change on mitochondria, is independent of Bak. Both vMyxlac and vMyxM11LKO viruses were permissive in wtMEF and MEF Bak⫺/⫺ cells, indicated by the growth curves (Fig. 7C), but a lower replication level of vMyxM11LKO was observed in both wtMEF and MEF Bak⫺/⫺ cells, consistent with the results from MV-infected human tumor cell lines (see Fig. 1B). In summary, our results demonstrate that myxoma M11L can block mitochondria-mediated apoptosis by inducibly arresting the activation of mitochondria-translocalized Bax and thereby maintaining Bax in a conformation that is unfavorable to the progression of apoptosis. In addition, this block to Bax conformational activation at the mitochondria is independent of Bak.

1146

SU ET AL.

J. VIROL.

FIG. 4. Mitochondrial localization of conformationally activated Bax in myxoma-infected cells. Bax that is personalized by 6A7 antibody was stained with Oregon Green 514 (green), and the mitochondria were stained with MitoTracker Red (red). The merged color (yellow) indicates colocalization of 6A7-reactive Bax on mitochondria. (A to C) Staining in mock HOS cells; (D to F) staining in HOS cells infected with vMyxlac; (G to I) staining in HOS cells infected with vMyxM11LKO virus.

DISCUSSION In the present study, we demonstrate that myxoma M11L inducibly interacts with a subset of Bax that translocalizes to mitochondria in response to virus infection or a proapoptotic stimuli and blocks the Bax protein conformational change which is required for apoptosis initiation. This block to apoptosis, by interfering with Bax conformational activation, requires no involvement of Bak to which M11L can also form a constitutive inhibitory complex at the mitochondria (72). This reveals an independent antiapoptotic mechanism for M11Lmediated protection from apoptosis, thereby adding Bax to the list of M11L binding partners at the mitochondria, together with Bak and PBR (17, 72). Mitochondria transduce key signals during the apoptotic response and thus function as authentic killer organelles (9). Members of the Bcl-2 family are key regulators of apoptosis via the mitochondrial pathway (2, 31). In addition, the multidomain proapoptotic Bcl-2 members Bax and Bak are the most downstream activator molecules known to date for the cytochrome c release machinery (56). Hence, it is not surprising that mitochondria can be directly targeted for modulating ap-

optosis by numerous viral proteins, many of which share homology with Bcl-2 members (10, 12, 19, 20, 30, 54). These viral proteins often antagonize the effects of cellular proapoptotic Bcl-2 family proteins, especially Bax and Bak (12). For example, Epstein-Barr virus (EBV) codes for BHRF1, a Bcl-2 homolog which colocalizes to OMM with Bcl-2 and suppresses MMP and apoptosis (32, 33). Another EBV-coded Bcl-2 homolog, BALF-1, is able to interact with Bax and Bak to inhibit apoptosis (46). Vaccinia virus encodes an antiapoptotic Bcl-2related protein, F1L, that localizes exclusively in mitochondria leading by its C-terminal hydrophobic domain, which is also responsible for the antiapoptotic function (64, 74). Recently, direct binding of F1L expressed by modified vaccinia virus Ankara to host BH3 domains has been reported (23). The viral antiapoptotic protein vMIA from human CMV does not have any sequence similarity with any Bcl-2 family member or other known host proteins, but nevertheless it localizes to mitochondria, where it blocks the activity of ANT, a component of the proapoptotic PTP complex (25). In addition, vMIA recruits Bax from cytoplasm to mitochondria and suppresses MMP (7, 55). Similarly, the infection of the murine cytomegalovirus

VOL. 80, 2006


1147

FIG. 5. Myxoma virus infection triggers Bax translocation to mitochondria. HOS cells were infected at 5 MOI and analyzed at 24 h postinfection. (A) Mitochondrial (lanes 1 to 3) and cytosolic (lanes 4 to 6) fractions were separated and resolved by SDS–15% PAGE followed by blotting with anti-Bax (NT), which recognizes both inactive and active Bax. Lanes 1 and 4, mock-infected cells; lanes 2 and 5, cells infected with vMyxlac; and lanes 3 and 6, cells infected with vMyxM11LKO. Loading controls blotted with anti-COXIV were used for the mitochondrial fractions, and loading controls blotted with anti-␤-actin were used for the cytosolic fractions. (B) Immunofluorescent staining of Bax and mitochondria colocalization. Bax that is recognized by anti-Bax (NT) was stained with Alexa Fluor 488 (green), and mitochondria were stained with MitoTracker Red. The merged color (yellow) indicates colocalization of Bax on mitochondria. Images I to III depict the staining in mock-infected HOS cells, images IV to VI depict staining in HOS cells infected with vMyxlac, and images VII to IX depict staining in HOS cells infected with vMyxM11LKO virus.

could also render the cells apoptosis resistant, which is associated with blocking of Bax activity on mitochondria (3). Myxoma M11L possesses a putative BH3-like domain (72) in which at least two residues (Leu, amino acid position 78 in Bak or 63 in Bax, and Asp, position 83 in Bak or 68 in Bax) are

highly conserved in the Bcl-2 family and have been shown to be required for heterodimerization with Bcl-xL (61). In addition, analysis of the protein sequence of M11L indicates that the charge distribution of the 26-amino-acid transmembrane domain shares some similarity with the Bcl-2 signal anchor se-

1148

SU ET AL.

FIG. 6. M11L blocks apoptosis in MEF Bak⫺/⫺ cells. MEF Bak⫺/⫺ cells were infected with vMyxlac or vMyxM11LKO at 5 MOI, and cells were collected at various times. Caspase 3 and PARP cleavage were used as apoptotic markers. (A) Apoptotic progression was detected in MEF Bak⫺/⫺ cells infected with vMyxM11LKO, but not vMyxlac, probed for caspase 3 cleavage. (B) PARP cleavage. Lanes 1 and 7, mock-infected cells; lanes 2 to 6, vMyxlac-infected cells; lanes 8 to 12, vMyxM11LKO-infected cells. ␤-actin was used as a loading control.

quence and is required for targeting to mitochondria (16). This virus-encoded antiapoptotic protein selectively resides at the OMM, where it promotes the survival of the host cell and guarantees the propagation of the virus (16). Significantly, M11L can inhibit the apoptotic cascade at the mitochondria by multiple independent strategies involving constitutive complexes with PBR (17) and Bak (72), and now we report its ability to form inducible complexes with Bax. M11L binds inducibly to Bax in response to apoptosis stimulation, and this interaction requires no other MV protein (Fig. 2) or cellular Bak (Fig. 7A). This inducible M11L-Bax interaction would be expected to occur at the mitochondria due to the localization of M11L, hence suggesting that cytosolic Bax must first translocate to the mitochondria before the actual binding event. Immunoprecipitation with anti-Bax clone NT (recognizing both inactive and conformationally active Bax) performed on the cytosolic fraction of vMyxlac-infected cells failed to copurify any detectable M11L, which rules out the possibility of significant binding of M11L and Bax in the cytoplasm (data not shown). In MV-infected cells, translocation of Bax from the cytoplasm to the mitochondria appears to be an inducible cellular response to viral infection, and this is supported by the fact that similar levels of Bax translocation occurs in the vMyxM11LKO-infected cells compared to the vMyxlac-infected cells (Fig. 5). This is in contrast to the active recruitment of Bax by CMV vMIA (7, 55). It has been estimated that translocation of only 20% of cellular Bax from the cytoplasm to the OMM is sufficient to trigger apoptosis (4). Although the percentage of translocalized Bax in our experimental system cannot be calculated precisely, an estimate can be made by comparing the amount of cytosolic Bax in MV-infected cells with that in mock-infected cells (Fig. 5). Cytosolic Bax was decreased significantly in MV-infected cells in comparison with that in mock-infected cells. It was noted that a low level of Bax at mitochondria in the mock-infected cells was detected by

J. VIROL.

immunoblotting of the mitochondrial fraction (Fig. 5A). This is not likely to be due to any contamination from the cytosolic fraction, because this colocalization of Bax on mitochondria was visualized by immunostaining with confocal microscopy as well (Fig. 5B). However, this small portion of Bax probably represents the portion of inactive monomers that loosely attached to mitochondrial membrane but does not convey a proapoptotic signal (77). This is supported by the findings that neither N-terminal conformational change of Bax (Fig. 3 and 4) nor standard apoptotic markers (caspase 3 or PARP cleavage; Fig. 6) could be detected in mock-infected cells. Apparently, activated and OMM translocalized Bax in apoptotic cells is in a conformation different from that of Bax that is loosely attached to the mitochondria in unstimulated cells, and this difference seems to be critical for Bax binding to M11L. This would explain the observation that Bax inducibly enters into a complex with M11L only in the presence of apoptotic stimuli which could activate Bax, such as virus infection (Fig. 2A and 7A), TNF-␣ stimulation (data not shown), transfection, or the overexpression of Bax (Fig. 2C and D). For example, in TAPM11L-transfected cells, with no overexpression of Bax, M11L could still coprecipitate endogenous Bax in the presence of NP-40 (Fig. 2B) but not CHAPS lysis buffer (data not shown). Our interpretation for this finding is that, unlike CHAPS, the nonionic detergent NP-40 may promote certain conformational changes of Bax (36), after which the Bax fraction already loosely attached to the OMM and in close proximity to M11L may gain the opportunity to form a complex with M11L during cell lysis. Despite the observation that similar amounts of cytosolic Bax translocalized to the mitochondria following MV infection, vMyxlac-infected cells are protected from apoptosis, whereas vMyxM11LKO-infected cells induce apoptosis rapidly. The difference resides in the fact that Bax undergoes further conformational change at the OMM in the vMyxM11LKO-infected cells but fails to do so in the vMyxlacinfected cells where M11L prevents this further activation of Bax (Fig. 3 and 4). It has been observed in epithelial cells during anoikis (i.e., inhibition of adhesion) that Bax translocalization does not, per se, commit cells to apoptosis (24). The point beyond which cells are committed to apoptosis is when Bax forms large perimitochondrial clusters in the OMM and cytochrome c is subsequently released (70). A recent study (4) has suggested that Bax permeabilizes mitochondrial membranes via discrete steps, beginning with the integration of Bax monomers into OMM with insertion of ␣-helixes 5, 6, and 9 followed by a conformational change at the N terminus (recognized by 6A7 antibody), resulting in oligomerization. Ongoing oligomerization then reorganizes the bilayer of the membrane to form large holes, through which a host of apoptosis-inducing factors from the inter membrane space, including cytochrome c, AIF, SMAC/Diablo, Endo G, Bit, and Omi/HtrA2, can be released, and apoptosis progresses to the point of no return (15, 27, 37, 42, 57, 68, 71). Consistent with these recent findings on Bax activation, our results verify that Bax translocation alone does not trigger apoptosis, because when M11L is also present at the OMM, Bax-induced apoptosis is blocked. The further conformational change of Bax at the N terminus, following Bax localization to the mitochondria, is thus the crucial step toward apoptosis. Our results suggest that M11L blocks

VOL. 80, 2006


1149

FIG. 7. M11L prevents Bax conformational change through M11L-Bax interaction in myxoma-infected MEF Bak⫺/⫺ cells. MEF Bak⫺/⫺ cells were infected with vMyxlac or vMyxM11LKO at 5 MOI for 24 h. (A) Interaction of M11L with Bax was detected by immunoprecipitation with anti-Bax (NT) followed by immunoblotting with anti-M11L (lane 1, mock-infected cells; lane 2, vMyxlac-infected cells). M11L expression in cell lysate was confirmed (lane 3, mock-infected cells; lane 4 vMyxlac-infected cells). Bax and Bak expression in cell lysate was also confirmed (lane 5, mock-infected cells; lane 6, vMyxlac-infected cells) using anti-Bax (NT) and anti-Bak (NT), respectively. (B) Bax conformational change is inhibited by M11L in the absence of Bak. Wild-type MEF (wtMEF; lanes 1 to 3) and MEF Bak⫺/⫺ cells (lanes 4 to 6) were infected with vMyxlac or vMyxM11LKO at 5 MOI for 24 h. Bax conformational change (indicated as Bax*/19kD) was detected by immunoprecipitation with anti-Bax 6A7 followed by immunoblotting with anti-Bax N20 in both wtMEF and MEF Bak⫺/⫺ cells infected with vMyxM11LKO myxoma virus but not with vMyxlac. Lanes 1 and 4, mock-infected cells; lanes 2 and 5, vMyxlac-infected cells; lanes 3 and 6, vMyxM11LKO-infected cells. (C) Replication of vMyxlac and vMyxM11LKO in wtMEF and MEF Bak⫺/⫺ cells. Cells were infected at 5 MOI and then titrated in BGMK cells. The numbers depict the averages of three experiments, and bars represent the standard deviations. WB, Western blot; IP, immunoprecipitation; IgG(L), IgG light chain.

this late-stage transition in the Bax protein conformational status, thereby maintaining Bax in an unfavorable conformation for apoptosis progression (Fig. 3 and 4). Although the protein sequence of M11L contains a putative BH3-like domain with homology to the Bcl-2 family, especially Bax, Bak, and Bcl-xL (72), it is not yet clear whether binding of Bax and/or Bak is mediated by this domain. Whether M11L is capable of binding to the Bax which has already undergone conformational change on mitochondria is yet to be determined. Although immunoprecipitation with 6A7 failed to copurify any detectable M11L from vMyxlac-infected cells or HEK-M11L stably transfected cells stimulated with TNF-␣ (data not shown), this could be due to the small amount of

6A7-reacting Bax generated in the presence of M11L, as shown in Fig. 3. This remains to be further investigated in other experimental systems. Based on our results, we propose a molecular model in which the MV-infected cell reacts quickly to the virus infection and induces a death signal in the attempt to initiate apoptosis. This proapoptotic stimulus normally triggers Bax activation in cytoplasm, exposing the Bax C-terminal transmembrane domain resulting in translocation of Bax to mitochondria (Fig. 5). Mitochondrial membrane-inserted Bax then undergoes further conformational change in the N terminus which permits oligomerization and development of mitochondrial membrane permeabilization and release of the proapoptotic factors from

1150

SU ET AL.

J. VIROL.

the intermembrane space, such as cytochrome c. This scenario likely applies to the cells infected with vMyxM11LKO, as we have shown in our studies that these cells begin to undergo apoptosis several hours postinfection (Fig. 6). However, in the presence of M11L, translocalized Bax is arrested by binding with M11L at the mitochondria, and this complex then blocks Bax from achieving the final conformational transition required for the execution of apoptosis (Fig. 3 and 4). Hence, the apoptotic process is halted (Fig. 6) and premature cell death is prevented or retarded sufficiently to guarantee optimal viral propagation. It would be reasonable to speculate that the higher replication level seen with the vMyxlac virus in both human tumor cells and MEFs, in comparison with the growth curves of vMyxM11LKO virus (Fig. 1B and 7C), is a sum of benefit from the activity of M11L to restrain the apoptotic progress in the infected cells by blocking PBR, Bak, and activated Bax at the mitochondria. In some MV-infected cells, such as rabbit T lymphocytes, this advantage is even more extreme, and infection with vMyxM11LKO virus is fully nonpermissive (44). Taken together, our findings reveal a novel self-sufficient antiapoptotic mechanism utilized by M11L through the inducible interaction and inhibition of activated Bax at the mitochondria. This provides evidence that myxoma M11L modulates apoptosis at the mitochondrial level by multiple strategies which all contribute to the blockade of apoptosis to benefit viral replication. The identification of these antiapoptotic mechanisms exploited by viruses will hopefully provide additional avenues for pharmacologic intervention to either accelerate apoptosis in tumor cells or inhibit apoptosis in diseases such as stroke and neurological trauma.

8. 9. 10.

11.

12. 13. 14.

15.

16.

17.

18. 19. 20. 21. 22.

ACKNOWLEDGMENTS In the greatest respect to the memory of Stanley J. Korsmeyer, we thank him for originally supplying the wtMEF and MEF Bak⫺/⫺ cells for this work and for his numerous contributions to the field. We also thank Ping Q. Dou (Wayne State University, Detroit, MI) for the gift of Bax plasmid and his helpful suggestions and Honggang Wang (H. Lee Moffitt Cancer Center and Research Institute, Tampa, Fla.) for valuable discussion. This work was supported by the Canadian Institutes of Health Research (CIHR). Jin Su has received career support from the Ontario HIV Treatment Network (OHTN), Canada. Grant McFadden holds a Canada Research Chair in Molecular Virology and a Howard Hughes Medical Institute (HHMI) International Scholarship.

23.

24.

25.

26.

REFERENCES 1. Adams, J. M. 2003. Ways of dying: multiple pathways of apoptosis. Genes Dev. 17:2481–2495. 2. Adams, J. M., and S. Cory. 2001. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci. 26:61–66. 3. Andoniou, C. E., D. M. Andrews, M. Manzur, P. Ricciardi-Castagnoli, and M. A. Degli-Esposti. 2004. A novel checkpoint in the Bcl-2-regulated apoptotic pathway revealed by murine cytomegalovirus infection of dendritic cells. J. Cell Biol. 166:827–837. 4. Annis, M. G., E. L. Soucie, P. J. Dlugosz, J. A. Cruz-Aguado, L. Z. Penn, B. Leber, and D. W. Andrews. 2005. Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J. 24: 2096–2103. 5. Antonsson, B., S. Montessuit, S. Lauper, R. Eskes, and J. C. Martinou. 2000. Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem. J. 345(Part 2):271–278. 6. Antonsson, B., S. Montessuit, B. Sanchez, and J.-C. Martinou. 2001. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J. Biol. Chem. 276:11615–11623. 7. Arnoult, D., L. M. Bartle, A. Skaletskaya, D. Poncet, N. Zamzami, P. U.

27. 28. 29.

30. 31.

32.

33.

34.

Park, J. Sharpe, R. J. Youle, and V. S. Goldmacher. 2004. Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria. Proc. Natl. Acad. Sci. USA 101:7988–7993. Benedict, C. A., P. S. Norris, and C. F. Ware. 2002. To kill or be killed: viral evasion of apoptosis. Nat. Immunol. 3:1013–1018. Blanke, S. R. 2005. Micro-managing the executioner: pathogen targeting of mitochondria. Trends Microbiol. 13:64–71. Boya, P., A. L. Pauleau, D. Poncet, R. A. Gonzalez-Polo, N. Zamzami, and G. Kroemer. 2004. Viral proteins targeting mitochondria: controlling cell death. Biochim. Biophys. Acta 1659:178–189. Cheng, E. H., M. C. Wei, S. Weiler, R. A. Flavell, T. W. Mak, T. Lindsten, and S. J. Korsmeyer. 2001. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX-and BAK-mediated mitochondrial apoptosis. Mol. Cell 8:705–711. Cuconati, A., and E. White. 2002. Viral homologs of BCL-2 role of apoptosis in the regulation of virus infection. Genes Dev. 16:2465–2478. Danial, N. N., and S. J. Korsmeyer. 2004. Cell death: critical control points. Cell 116:205–219. Desagher, S., A. Osen-Sand, A. Nichols, R. Eskes, S. Montessuit, S. Lauper, K. Maundrell, B. Antonsson, and J. C. Martinou. 1999. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144:891–901. Du, C., M. Fang, Y. Li, L. Li, and X. Wang. 2000. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibitions. Cell 102:33–42. Everett, H., M. Barry, S. F. Lee, X. J. Sun, K. Graham, J. Stone, R. C. Bleackley, and G. McFadden. 2000. M11L: a novel mitochondria-localized protein of myxoma virus that blocks apoptosis in infected leukocytes. J. Exp. Med. 191:1487–1498. Everett, H., M. Barry, X. Sun, S. F. Lee, C. Frantz, L. G. Berthiaume, G. McFadden, and R. C. Bleackley. 2002. The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore. J. Exp. Med. 196:1127–1139. Everett, H., and G. McFadden. 2002. Poxviruses and apoptosis: a time to die. Curr. Opin. Microbiol.5:395–402. Everett, H., and G. McFadden. 2001. Viral proteins and the mitochondrial apoptotic checkpoint. Cytokine Growth Factor Rev. 12:181–188. Everett, H., and G. McFadden. 2001. Viruses and apoptosis: meddling with mitochondria. Virology 288:1–7. Fenner, F., and F. N. Ratcliffe. 1965. Myxomatosis. Cambridge University Press, Cambridge, United Kingdom. Ferri, K. F., and G. Kroemer. 2001. Organelle-specific initiation of cell death pathways. Nat. Cell Biol. 3:255–263. Fischer, S. F., H. Ludwig, J. Holzapfel, M. Kvansakul, L. Chen, D. C. Huang, G. Sutter, M. Knese, and G. Hacker. 2005. Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis. Cell Death Differ. 13:109–118. Gilmore, A. P., A. D. Metcalfe, L. H. Romer, and C. H. Streuli. 2000. Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J. Cell Biol. 149:431– 446. Goldmacher, V. S., L. M. Bartle, A. Skaletskaya, C. A. Dionne, N. L. Kedersha, C. A. Vater, J. Han, R. J. Lutz, S. Watanabe, E. D. McFarland, E. D. Kieff, E. S. Mocarski, and T. Chittenden. 1999. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc. Natl. Acad. Sci. USA 96:12536–12541. Graham, K. A., A. Opgenorth, C. Upton, and G. McFadden. 1992. Myxoma virus M11L ORF encodes a protein for which cell surface localization is critical for manifestation of viral virulence. Virology 191:112–124. Green, D. R. 1998. Apoptotic pathways: the roads to ruin. Cell 94:695–698. Green, D. R., and G. Kroemer. 2004. The pathophysiology of mitochondrial cell death. Science 305:626–629. Gross, A., J. Jockel, M. C. Wei, and S. J. Korsmeyer. 1998. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17:3878–3885. Hardwick, J. M., and D. S. Bellows. 2003. Viral versus cellular BCL-2 proteins. Cell Death Differ. 10(Suppl. 1):S68–S76. Harris, M. H., and C. B. Thompson. 2000. The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability. Cell Death Differ. 7:1182–1191. Henderson, S., D. Huen, M. Rowe, C. Dawson, G. Johnson, and A. Rickinson. 1993. Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc. Natl. Acad. Sci. USA 90:8479–8483. Hickish, T., D. Robertson, P. Clarke, M. Hill, F. di Stefano, C. Clarke, and D. Cunningham. 1994. Ultrastructural localization of BHRF1: an EpsteinBarr virus gene product which has homology with bcl-2. Cancer Res. 54: 2808–2811. Hsu, Y. T., K. G. Wolter, and R. J. Youle. 1997. Cytosol-to-membrane

VOL. 80, 2006

35.

36. 37.

38.

39.

40.

41. 42. 43.

44.

45.

46.

47. 48.

49.

50.

51.

52.

53.

54.

55.

56.

redistribution of Bax and Bcl-X(L) during apoptosis. Proc. Natl. Acad. Sci. USA 94:3668–3672. Hsu, Y. T., and R. J. Youle. 1998. Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J. Biol. Chem. 273:10777–10783. Hsu, Y. T., and R. J. Youle. 1997. Nonionic detergents induce dimerization among members of the Bcl-2 family. J. Biol. Chem. 272:13829–13834. Jan, Y., M. Matter, J. T. Pai, Y. L. Chen, J. Pilch, M. Komatsu, E. Ong, M. Fukuda, and E. Ruoslahti. 2004. A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors. Cell 116: 751–762. Jia, L., M. G. Macey, Y. Yin, A. C. Newland, and S. M. Kelsey. 1999. Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis. Blood 93:2353–2359. Jia, L., Y. Patwari, S. M. Srinivasula, A. C. Newland, T. Fernandes-Alnemri, E. S. Alnemri, and S. M. Kelsey. 2001. Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis. Oncogene 20: 4817–4826. Karbowski, M., Y. J. Lee, B. Gaume, S. Y. Jeong, S. Frank, A. Nechushtan, A. Santel, M. Fuller, C. L. Smith, and R. J. Youle. 2002. Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. J. Cell Biol. 159:931–938. Kerr, P., and G. McFadden. 2002. Immune responses to myxoma virus. Viral Immunol. 15:229–246. Li, L. Y., X. Luo, and X. Wang. 2001. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412:95–99. Lindsten, T., A. J. Ross, A. King, W. X. Zong, J. C. Rathmell, H. A. Shiels, E. Ulrich, K. G. Waymire, P. Mahar, K. Frauwrith, Y. Chen, M. Wei, V. M. Eng, D. M. Adelman, M. C. Simon, A. Ma, J. A. Golden, and G. Evan. 2000. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol. Cell 6:1389– 1399. Macen, J. L., K. A. Graham, S. F. Lee, M. Schreiber, L. K. Boshkov, and G. McFadden. 1996. Expression of the myxoma virus tumor necrosis factor receptor homologue (T2) and M11L genes is required to prevent virusinduced apoptosis in infected rabbit T lymphocytes. Virology 218:232–237. Makin, G. W., B. M. Corfe, G. J. Griffiths, A. Thistlethwaite, J. A. Hickman, and C. Dive. 2001. Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate. EMBO J. 20:6306–6315. Marshall, W. L., C. Yim, E. Gustafson, T. Graf, D. R. Sage, K. Hanify, L. Williams, J. Fingeroth, and R. W. Finberg. 1999. Epstein-Barr virus encodes a novel homolog of bcl-2 oncogene that inhibits apoptosis and associates with Bax and Bak. J. Virol. 73:5181–5185. Martinou, J.-C., and D. R. Green. 2001. Breaking the mitochondrial barrier. Nat. Rev. 2:63–67. Marzo, I., C. Brenner, N. Zamzami, J. M. Jurgensmeier, S. A. Susin, H. L. Vieira, M. C. Prevost, Z. Xie, S. Matsuyama, J. C. Reed, and G. Kroemer. 1998. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281:2027–2031. Murphy, K. M., U. N. Streips, and R. B. Lock. 2000. Bcl-2 inhibits a Fasinduced conformational change in the Bax N terminus and Bax mitochondrial translocation. J. Biol. Chem. 275:17225–17228. Nechushtan, A., C. L. Smith, Y. T. Hsu, and R. J. Youle. 1999. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J. 18:2330–2341. Nechushtan, A., C. L. Smith, I. Lamensdorf, S. H. Yoon, and R. J. Youle. 2001. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J. Cell Biol. 153:1265–1276. Opgenorth, A., K. Graham, N. Nation, D. Strayer, and G. McFadden. 1992. Deletion analysis of two tandemly arranged virulence genes in myxoma virus, M11L and myxoma growth factor. J. Virol. 66:4720–4731. Perez, D., and E. White. 2000. TNF-alpha signals apoptosis through a biddependent conformational change in Bax that is inhibited by E1B 19K. Mol. Cell 6:53–63. Polster, B. M., J. Pevsner, and J. M. Hardwick. 2004. Viral Bcl-2 homologs and their role in virus replication and associated diseases. Biochim. Biophys. Acta 1644:211–227. Poncet, D., N. Larochette, A. L. Pauleau, P. Boya, A. A. Jalil, P. F. Cartron, F. Vallette, C. Schnebelen, L. M. Bartle, A. Skaletskaya, D. Boutolleau, J. C. Martinou, V. S. Goldmacher, G. Kroemer, and N. Zamzami. 2004. An anti-apoptotic viral protein that recruits Bax to mitochondria. J. Biol. Chem. 279:22605–22614. Puthalakath, H., and A. Strasser. 2002. Keeping killers on a tight leash:


1151

transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ. 9:505–512. 57. Reed, J. C. 2000. Mechanisms of apoptosis. Am. J. Pathol. 157:1415–1430. 58. Rigaut, G., A. Shevchenko, B. Rutz, M. Wilm, and B. Seraphin. 1999. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17:1030–1032. 59. Ruffolo, S. C., D. G. Breckenridge, M. Nguyen, J. S. Goping, A. Gross, S. J. Korsmeyer, H. Li, J. Yuan, and G. C. Shore. 2000. BID-dependent and BID-independent pathways for BAX insertion into mitochondria. Cell Death Differ. 7:1101–1108. 60. Saito, M., S. J. Korsmeyer, and P. H. Schlesinger. 2000. BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat. Cell Biol. 2:553–555. 61. Sattler, M., H. Liang, D. Nettesheim, R. P. Meadows, J. E. Harlan, M. Eberstadt, H. S. Yoon, S. B. Shuker, B. S. Chang, A. J. Minn, C. B. Thompson, and S. W. Fesik. 1997. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 275:983–986. 62. Seet, B. T., J. B. Johnston, C. R. Brunetti, J. W. Barrett, H. Everett, C. Cameron, J. Sypula, S. H. Nazarian, A. Lucas, and G. McFadden. 2003. Poxviruses and immune evasion. Annu. Rev. Immunol. 21:377–423. 63. Shimizu, S., Y. Matsuoka, Y. Shinohara, Y. Yoneda, and Y. Tsujimoto. 2001. Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells. J. Cell Biol. 152:237–250. 64. Stewart, T. L., S. T. Wasilenko, and M. Barry. 2005. Vaccinia virus F1L protein is a tail-anchored protein that functions at the mitochondria to inhibit apoptosis. J. Virol. 79:1084–1098. 65. Sundararajan, R., A. Cuconati, D. Nelson, and E. White. 2001. Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K. J. Biol. Chem. 276:45120–45127. 66. Sundararajan, R., and E. White. 2001. E1B 19K blocks Bax oligomerization and tumor necrosis factor alpha-mediated apoptosis. J. Virol. 75:7506–7516. 67. Suzuki, M., R. J. Youle, and N. Tjandra. 2000. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103:645–654. 68. Suzuki, Y., Y. Imai, H. Nakayama, K. Takahashi, K. Takio, and R. Takahashi. 2001. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol. Cell 8:613–621. 69. Tan, Y. J., W. Beerheide, and A. E. Ting. 1999. Biophysical characterization of the oligomeric state of Bax and its complex formation with Bcl-XL. Biochem. Biophys. Res. Commun. 255:334–339. 70. Valentijn, A. J., A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore. 2003. Spatial and temporal changes in Bax subcellular localization during anoikis. J. Cell Biol. 162:599–612. 71. Verhagen, A. M., P. G. Ekert, M. Pakush, J. Silke, L. M. Connolly, G. E. Reid, R. L. Moritz, R. J. Simpson, and D. L. Vaux. 2000. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonising IAP proteins. Cell 86:147–157. 72. Wang, G., J. W. Barrett, S. H. Nazarian, H. Everett, X. Gao, C. Bleackley, K. Colwill, M. F. Moran, and G. McFadden. 2004. Myxoma virus M11L prevents apoptosis through constitutive interaction with Bak. J. Virol. 78:7097– 7111. 73. Wang, K., A. Gross, G. Waksman, and S. J. Korsmeyer. 1998. Mutagenesis of the BH3 domain of BAX identifies residues critical for dimerization and killing. Mol. Cell. Biol. 18:6083–6089. 74. Wasilenko, S. T., T. L. Stewart, A. F. Meyers, and M. Barry. 2003. Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis. Proc. Natl. Acad. Sci. USA 100:14345–14350. 74a.Wasilenko, S. T., T. L. Banadyga, D. Bond, and M. Barry. 2005. The vaccinia virus F1L protein interacts with the proapoptotic protein Bak and inhibits Bak activation. J. Virol. 79:14031–14043. 75. Wei, M. C., W.-X. Zong, E. H.-Y. Cheng, T. Lindsten, V. Panoutsakopoulou, A. J. Ross, K. A. Roth, G. R. MacGregor, C. B. Thompson, and S. J. Korsmeyer. 2001. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292:727–730. 76. Willis, S. N., L. Chen, G. Dewson, A. Wei, E. Naik, J. I. Fletcher, J. M. Adams, and D. C. Huang. 2005. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev. 19:1294–1305. 77. Yamaguchi, H., and H. G. Wang. 2002. Bcl-XL protects BimEL-induced Bax conformational change and cytochrome C release independent of interacting with Bax or BimEL. J. Biol. Chem. 277:41604–41612. 78. Zamzami, N., and G. Kroemer. 2003. Apoptosis: mitochondrial membrane permeabilization - the (w)hole story? Curr. Biol. 13:R71–R73. 79. Zha, H., C. Aime-Sempe, T. Sato, and J. C. Reed. 1996. Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J. Biol. Chem. 271:7440–7444.