Abstract
22 min readThe 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. 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. Apoptosis is mediated through two main pathways, the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway. The extrinsic pathway is initiated by ligation of a plasma membrane death receptor, which results in a stepwise recruitment of adaptors and initiator caspases (in particular, caspase-8) into the death-inducing signaling complex. In some cells (type I), activation of caspase-8 directly triggers activation of the caspase cascade (1Krammer P.H. Nature. 2000; 407: 789-795Crossref PubMed Scopus (1393) Google Scholar), whereas in other cells (type II), caspase-8 mediates apoptosis only via proteolytic processing of BID (2Li H. Zhu H. Xu C. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3756) Google Scholar, 3Letai A. Bassik M. Walensky L. Sorcinelli M. Weiler S. Korsmeyer S. Cancer Cell. 2002; 2: 183Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar), which in turn leads to mitochondrial membrane permeabilization (MMP) 1The abbreviations used are: MMP, mitochondrial membrane permeabilization; CMV, human cytomegalovirus; CHX, cyclohexamide; GFP, green fluorescent protein; EGFP, enhanced GFP; MEF, murine embryonic fibroblasts, STS, staurosporine; t-Bid, truncated Bid; vMIA, viral mitochondria-localized inhibitor of apoptosis; vMIAt, Myc-tagged vMIA; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; CHAPS, 3-((cholamidopropyl)dimethylammonio)-1-propane sulfonate; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone. 1The abbreviations used are: MMP, mitochondrial membrane permeabilization; CMV, human cytomegalovirus; CHX, cyclohexamide; GFP, green fluorescent protein; EGFP, enhanced GFP; MEF, murine embryonic fibroblasts, STS, staurosporine; t-Bid, truncated Bid; vMIA, viral mitochondria-localized inhibitor of apoptosis; vMIAt, Myc-tagged vMIA; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; CHAPS, 3-((cholamidopropyl)dimethylammonio)-1-propane sulfonate; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone. and consequently to the amplification of the apoptotic signal. The intrinsic pathway is induced by various apoptotic stimuli that converge on mitochondria to trigger MMP (4Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2741) Google Scholar). This permeabilization causes the release of mitochondrial pro-apoptotic factors, which in turn activate the caspase pathway. MMP is tightly controlled, negatively and positively, by the proteins of the Bcl-2 family. Anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1 inhibit MMP, whereas pro-apoptotic Bcl-2 homologues such as Bax, Bak, and BH3-only proteins such as Bid and Bad enhance MMP (3Letai A. Bassik M. Walensky L. Sorcinelli M. Weiler S. Korsmeyer S. Cancer Cell. 2002; 2: 183Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar, 4Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2741) Google Scholar, 5Vander Heiden M.G. Thompson C.B. Nat. Cell Biol. 1999; 1: E209-E216Crossref PubMed Scopus (599) Google Scholar). Recent evidence suggests that many stress signals trigger apoptosis, at least in part, by activation of pro-apoptotic BH3-only proteins (3Letai A. Bassik M. Walensky L. Sorcinelli M. Weiler S. Korsmeyer S. Cancer Cell. 2002; 2: 183Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar). BH3-only proteins seem to induce MMP by two different pathways: either by direct activation of Bax/Bak (e.g. Bid or Bim) or by inhibition of anti-apoptotic activity of Bcl-2/Bcl-XL (e.g. Bad, Bik) (3Letai A. Bassik M. Walensky L. Sorcinelli M. Weiler S. Korsmeyer S. Cancer Cell. 2002; 2: 183Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar, 6Cheng E.H.-Y.A. Wei M.C. Weiler S. Flavell R.A. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; 8: 705-711Abstract Full Text Full Text PDF PubMed Scopus (1406) Google Scholar). Bax is one of the key inducers of MMP (7Knudson C.M. Tung K.S. Tourtellotte W.G. Brown G.A. Korsmeyer S.J. Science. 1995; 270: 96-99Crossref PubMed Scopus (1300) Google Scholar). This protein is predominantly cytosolic in a conformation in which its hydro-phobic α-5/6 helix and its N-terminal helix-1 are hidden within a hydrophilic globular structure. Upon pro-apoptotic signaling, for instance following exposure to t-Bid, Bax undergoes a conformation change that exposes its N terminus and, possibly, its BH3 domain (8Nechushtan A. Smith C.L. Hsu Y.T. Youle R.J. EMBO J. 1999; 18: 2330-2341Crossref PubMed Scopus (622) Google Scholar, 9Suzuki M. Youle R.J. Tjandra N. Cell. 2000; 103: 645-654Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). This allows its translocation and its tight association with the mitochondria, apparently through integration into the outer membrane, forming homo-oligomers and hetero-oligomers with Bak (3Letai A. Bassik M. Walensky L. Sorcinelli M. Weiler S. Korsmeyer S. Cancer Cell. 2002; 2: 183Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar, 10Antonsson B. Montessuit S. Sanchez B. Martinou J.C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 11Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3224) Google Scholar). The mechanism of the association of Bax with the mitochondrial outer membrane during apoptosis is not well understood. Some data suggest that Bax interaction with cardiolipin (a mitochondria-specific lipid) is sufficient to trigger MMP (12Kuwana T. Mackey M.R. Perkins G.A. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Cell. 2002; 111: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar). On the other hand, Bax has been shown to interact (directly or indirectly) with sessile mitochondrial proteins, voltage-dependent anion channel (13Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1898) Google Scholar) and the adenine nucleotide translocase (14Marzo I. Brenner C. Zamzami N. Jürgensmeier J. Susin S.A. Vieira H.L.A. Prévost M.-C. Xie Z. Matsuyama S. Reed J.C. Kroemer G. Science. 1998; 281: 2027-2031Crossref PubMed Scopus (1049) Google Scholar), although these findings are controversial (12Kuwana T. Mackey M.R. Perkins G.A. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Cell. 2002; 111: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar, 15Martinou J.-C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Crossref PubMed Scopus (844) Google Scholar, 16Roucou X. Montessuit S. Antonsson B. Martinou J.C. Biochem. J. 2002; 368: 915-921Crossref PubMed Scopus (168) Google Scholar, 17Zamzami N. Kroemer G. Curr. Biol. 2003; 13: R71-R73Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In any case, most of the available evidence indicates that oligomerization of Bax and its tight association with the outer mitochondrial membrane lead to MMP (12Kuwana T. Mackey M.R. Perkins G.A. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Cell. 2002; 111: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar, 15Martinou J.-C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Crossref PubMed Scopus (844) Google Scholar, 16Roucou X. Montessuit S. Antonsson B. Martinou J.C. Biochem. J. 2002; 368: 915-921Crossref PubMed Scopus (168) Google Scholar, 18Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (961) Google Scholar). Apoptotic elimination of virally infected cells is one of the basic anti-viral responses of multicellular organisms. During evolution, viruses have developed a variety of strategies to suppress the extrinsic and/or the intrinsic pathways. Thus, some viral proteins block the extrinsic pathway by suppressing the activation of initiator caspases such as viral Flice-like inhibitory proteins (FLIPs) encoded by γ-herpesviruses (19Tschopp J. Thome M. Hofmann K. Meinl E. Curr. Opin. Genet. Dev. 1998; 8: 82-87Crossref PubMed Scopus (176) Google Scholar), vICA homologues of β-herpesviruses that associate with and prevent death-inducing signaling complex-dependent activation of pro-caspase-8, (20McCormick A.L. Smith V.L. Chow D. Mocarski E.S. J. Virol. 2003; 77: 631-641Crossref PubMed Scopus (118) Google Scholar), CrmA of poxvirus, a serpin that inhibits the enzymatic activity of caspase-8 (21Zhou Q. Snipas S. Orth K. Muzio M. Dixit V.M. Salvesen G.S. J. Biol. Chem. 1997; 272: 7797-7800Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar), and the Serp-2 serpin of myxoma virus (22Messud-Petit F. Gelfi J. Delverdier M. Amardeilh M. Py R. Sutter G. Bertagnoli S. J. Virol. 1998; 72: 7830-7839Crossref PubMed Google Scholar). Other viruses encode inhibitors of the intrinsic pathway in particular structural and functional Bcl-2 homologues. Examples of structural homologues of Bcl-2 include KSbcl-2, a Bcl-2 homologue from human herpesvirus (HHV8), which resembles Bcl-2 and Bcl-xL in its three-dimensional structure and, likely, in its function (23Huang Q. Petros A. Virgin H. Fesik S. Olejniczak E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3428-3433Crossref PubMed Scopus (107) Google Scholar). Another cell death suppressor BHRF1, encoded by Epstein-Barr virus, shares some homologies with Bcl-2, but its mechanism of cell death suppression remains unclear (24Huang Q. Petros A. Virgin H. Fesik S. Olejniczak E. J. Mol. Biol. 2003; 332: 1123-1130Crossref PubMed Scopus (76) Google Scholar). The E1B19K cell death suppressor of adenovirus shares only a modest amino acid sequence homology with Bcl-2 and appears to suppress cell death via its interaction with Bax and Bak, preventing their oligomerization (25Sundararajan R. Cuconati A. D. N. E. W. J. Biol. Chem. 2001; 276: 45120-45127Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Sundararajan R. E. W. J. Virol. 2001; 75: 7506-7516Crossref PubMed Scopus (79) Google Scholar). Finally, a number of viruses intercept both the intrinsic and the extrinsic pathways. This applies to Myxoma virus, which encodes both M11L (27Everett H. Barry M. Sun X. Lee S.F. Frantz C. Berthiaume L.G. McFadden G. Bleackley R.C. J. Exp. Med. 2002; 196: 1127-1139Crossref PubMed Scopus (89) Google Scholar) and Serp-2 (22Messud-Petit F. Gelfi J. Delverdier M. Amardeilh M. Py R. Sutter G. Bertagnoli S. J. Virol. 1998; 72: 7830-7839Crossref PubMed Google Scholar) and the cytomegalovirus with viral mitochondria-localized inhibitor of apoptosis (vMIA) (28McCormick A. Skaletskaya A. Barry P. Mocarski E. Goldmacher V. Virology. 2003; 316: 221-233Crossref PubMed Scopus (114) Google Scholar) and vICA (20McCormick A.L. Smith V.L. Chow D. Mocarski E.S. J. Virol. 2003; 77: 631-641Crossref PubMed Scopus (118) Google Scholar). The vMIA is encoded by exon 1 of the immediate early UL37 gene of human cytomegalovirus (CMV). Although vMIA shares no sequence homology with Bcl-2, it has functional similarities with Bcl-2: it localizes at mitochondria, inhibits MMP, and is a potent cell death suppressor (29Goldmacher V.S. Bartle L.M. Skletskaya S. Dionne C.A. Kedersha N.L. Vater C.A. Han J.W. Lutz R.J. Watanabe S. McFarland E.D.C. Kieff E.D. Mocarski E.S. Chittenden T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12536-12541Crossref PubMed Scopus (361) Google Scholar, 30Vieira H.L. Belzacq A.-S. Haouzi D. Bernassola F. Cohen I. Jacotot E. Ferri K.F. Hamel E.H. Bartle L.M. Melino G. Brenner C. Goldmacher V. Kroemer G. Oncogene. 2001; 20: 4305-4316Crossref PubMed Scopus (228) Google Scholar, 31Belzacq A.S. El Hamel C. Vieira H.L.A. Cohen I. Haouzi D. Metivier D. Marchetti P. Goldmacher V. Brenner C. Kroemer G. Oncogene. 2001; 20: 7579-7587Crossref PubMed Scopus (176) Google Scholar). Two domains of vMIA are necessary and, together, sufficient for its cell death-suppressing activity, its N-terminal domain (amino acids 5-34), and a segment between amino acids 117 and 147 (32Hayajneh W.A. Colberg-Oley A.M. Skaleskaya A. Bartle L.M. Lesperance M.M. Contopoulos-Ionnidis D.G. Kedersha N.L. Goldmacher V.S. Virology. 2001; 279: 233-240Crossref PubMed Scopus (86) Google Scholar). The N-terminal domain targets vMIA to mitochondria, whereas the function of the second domain as well as the molecular mechanism of cell death suppression by vMIA until now remained unknown. Here we report that vMIA inhibits apoptosis through a unique mechanism distinct from that of any previously characterized cell death suppressor-targeting MMP. We show that vMIA, through an association with Bax, recruits Bax to mitochondria, inducing its oligomerization and tight association with mitochondrial outer membrane while inhibiting its proapoptotic function. Our findings thus reveal a yet undescribed viral strategy for interfering with the mitochondrial apoptotic signaling pathway. Cell Lines, Culture Conditions, and Transfection/Infection—HeLa and BJAB cells were stably transfected with UL37 exon 1 (vMIA), a deletion mutant of vMIA (32Hayajneh W.A. Colberg-Oley A.M. Skaleskaya A. Bartle L.M. Lesperance M.M. Contopoulos-Ionnidis D.G. Kedersha N.L. Goldmacher V.S. Virology. 2001; 279: 233-240Crossref PubMed Scopus (86) Google Scholar), Bcl-2, or the empty vector, pcDNA3 (neo), as described elsewhere (29Goldmacher V.S. Bartle L.M. Skletskaya S. Dionne C.A. Kedersha N.L. Vater C.A. Han J.W. Lutz R.J. Watanabe S. McFarland E.D.C. Kieff E.D. Mocarski E.S. Chittenden T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12536-12541Crossref PubMed Scopus (361) Google Scholar). Murine embryonic fibroblasts (MEF) were a generous gift of stanley Korsmeyer (Harvard University, Boston, MA), and human colorectal carcinoma HCT116/Bax+/- and HCT116/Bax+/- otherwise isogenic cells were a generous gift of Bernd Vogelstein (The John Hopkins Onclogy Center, Baltimore, MD). The MRC-5 human normal fibroblasts and CMV strain AD169varATCC were purchased from the American Type Culture Collection. CMV-infected cells were fixed at 24 h after infection for immunofluorescence analysis. Cells were cultured in Dulbecco's modified Eagle's medium and RPMI 1640 (PAA laboratories), respectively, supplemented with 10% fetal calf serum, 1 mm sodium pyruvate, 10 mm HEPES, and 100 units/ml penicillin/streptomycin at 37 °C under 5% CO2. Transient transfections of HeLa cells, MEFs, and HCT116 cells with vectors for Myc-tagged vMIA or its deletion mutants (32Hayajneh W.A. Colberg-Oley A.M. Skaleskaya A. Bartle L.M. Lesperance M.M. Contopoulos-Ionnidis D.G. Kedersha N.L. Goldmacher V.S. Virology. 2001; 279: 233-240Crossref PubMed Scopus (86) Google Scholar), a Bax-GFP fusion gene (kindly provided by Dr. Shigemi Matsuyama (33Sawada M. Hayes P. Matsuyama S. Nat. Cell Biol. 2003; 4: 352-357Crossref Scopus (135) Google Scholar)), pEGFP only and pEGFP-Bid (Clontech), vMIA mutants (32Hayajneh W.A. Colberg-Oley A.M. Skaleskaya A. Bartle L.M. Lesperance M.M. Contopoulos-Ionnidis D.G. Kedersha N.L. Goldmacher V.S. Virology. 2001; 279: 233-240Crossref PubMed Scopus (86) Google Scholar), or an empty vector, pcDNA3.1, were performed using the LipofectAMINE 2000 reagent (Invitrogen). Immunofluorescence and Confocal Microscopy—Cells grown on coverslips were fixed with paraformaldehyde (4% w/v) and permeabilized with 0.1% SDS in phosphate-buffered saline (34Castedo M. Ferri K.F. Blanco J. Roumier T. Larochette N. Barretina J. Amendola A. Nardacci R. Metivier D. Este J.A. Piacentini M. Kroemer G. J. Exp. Med. 2001; 194: 1097-1110Crossref PubMed Scopus (139) Google Scholar, 35Castedo M. Ferri K. Roumier T. Metivier D. Zamzami N. Kroemer G. J. Immunol. Methods. 2002; 265: 39-47Crossref PubMed Scopus (242) Google Scholar). Cells were then stained for the detection of cytochrome c (monoclonal antibody 6H2.B4 from Pharmingen) and the c-Myc epitope of tagged vMIA (monoclonal antibody 9E10, Santa Cruz Biotechnology). To detect subcellular localization of Bax, cells were stained with either monoclonal anti-Bax (6A7, Pharmingen), rabbit polyclonal anti-Bax antiserum (N20, Santa Cruz Biotechnology), or rabbit polyclonal vMIA (29Goldmacher V.S. Bartle L.M. Skletskaya S. Dionne C.A. Kedersha N.L. Vater C.A. Han J.W. Lutz R.J. Watanabe S. McFarland E.D.C. Kieff E.D. Mocarski E.S. Chittenden T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12536-12541Crossref PubMed Scopus (361) Google Scholar). Epifluorescence analyses were performed using a Leica microscope (DMIRE2), and confocal analyses were performed using an LSM 510 Zeiss microscope equipped with a ×63 objective (oil immersion). Isolation of Mitochondria—Cells resuspended in a homogenization buffer (300 mm sucrose, 10 mm TES, 300 μm EGTA) supplemented with a mixture of protease inhibitors (Roche Applied Science) were disrupted by cavitation (PAR system; 150 p.s.i., 2 × 15 min) and centrifuged at 900 × g for 15 min twice to remove nuclei and unbroken cells. The supernatant was subsequently centrifuged at 9000 × g for 15 min, and the pellet was used as a fraction enriched in mitochondria ("mitochondrial fraction"). The supernatant was further centrifuged at 100,000 × g for 1 h to yield the cytosolic cell fraction. Preparation of Mitochondrial Extracts—The mitochondrial fraction was resuspended in the homogenization buffer supplemented with 2% CHAPS (Sigma), incubated on ice for 1 h, sonicated, and centrifuged at 100,000 × g for 30 min. The supernatant ("soluble mitochondrial fraction") was then recovered. Protein concentrations were determined using the Bio-Rad DC protein assay. Detection of Tight Association of Bax with Mitochondrial Membranes—The mitochondrial fraction was resuspended in 100 mm Na2CO3, pH 12, to a final protein concentration of 1 mg/ml and incubated on ice for 20 min. Subsequently, the sample was centrifuged at 100,000 × g for 1 h. The supernatant, which contained loosely attached proteins, was recovered, and CHAPS was added to a final concentration of 2%. The pellet, which contained tightly membrane-associated proteins, was suspended in the homogenization buffer containing 2% CHAPS, incubated on ice for 1 h, sonicated, and centrifuged at 100,000 × g for 30 min. Gel Filtration Analysis—Gel filtrations were performed at 4 °C on a Superdex 200 column (16/60 and 10/30, Amersham Biosciences), as described before (10Antonsson B. Montessuit S. Sanchez B. Martinou J.C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar). Briefly, the column was equilibrated in 25 mm HEPES-NaOH, 300 mm NaCl, 200 μm dithiothreitol, 2% (w/v) CHAPS, pH 7.5, and run at a flow rate of 1 ml/min. The column was calibrated with gel filtration standard proteins from Amersham Biosciences. Fractions of 2 ml were collected. The proteins present in these fractions were precipitated with trichloroacetic acid (10%) in water and subjected to immunoblot analysis. Cell-free Assay of Bax Incorporation into Mitochondria—The association of Bax or vMIA with purified rat liver mitochondria was determined as described (36Cartron P.F. Moreau C. Oliver E. Maya C. Meflah K. Vallette F.M. FEBS Lett. 2002; 512: 95-100Crossref PubMed Scopus (59) Google Scholar). Briefly, proteins labeled with [35S]Met (Amersham Biosciences) were synthesized from cDNA using the TnT-coupled transcription/translation system (Promega). Labeled proteins were incubated with mitochondrial fractions at 37 °C for 1 h in homogenization buffer. Radiolabeled proteins bound to the mitochondria were recovered in the pellet after centrifugation of the incubation mixture for 10 min at 4 °C at 8000 × g. [35S]Met-labeled vMIA associated with isolated mitochondria was subjected to SDS-PAGE followed by quantitation of the radioactivity in a PhosphorImager (Amersham Biosciences) using the IPLab program (Signal Analytics, Vienna, VA). Cell-free Association of vMIA Bax Δ2-37 and Bax Δ2-20—His-tagged Bax (4 fmol) was incubated overnight with 10 μl of nickel-nitrilotriacetic acid agarose beads (Qiagen) at 4 °C. The reaction mix was centrifuged at 15,000 × g for 10 min at 4 °C, and the pellet was resuspended in 10 μl of LB buffer (8 m urea, 100 mm NaP04, 10 mm Tris-Cl, pH 8) in the presence of in vitro translated [35S]Met-labeled vMIA (4 fmol) and the incubated for 2 h at 37 °C, and the beads were then washed in LB buffer. Proteins bound to the beads were eluted from the beads with 250 μm imidazole (20 min, 4 °C) and subjected to SDS-PAGE and autoradiography. Immunoprecipitation—BJAB cell clones stably transfected with vMIA, a vMIA deletion mutant, or an empty vector were used in these experiments. Cells were lysed in lysis buffer (150 mm NaCl, 50 mm Tris HCl, pH 7.4, 5 mm EDTA, supplemented with either 1% Triton X-100 or 2% CHAPS) in the presence of protease inhibitors for 30 min on ice and then centrifuged (5 min at 1000 × g) to remove nuclei and cell debris, and the supernatants were rotated overnight at 4 °C with the 9E10 anti-Myc antibody covalently bound to Affi-prep10 beads (29Goldmacher V.S. Bartle L.M. Skletskaya S. Dionne C.A. Kedersha N.L. Vater C.A. Han J.W. Lutz R.J. Watanabe S. McFarland E.D.C. Kieff E.D. Mocarski E.S. Chittenden T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12536-12541Crossref PubMed Scopus (361) Google Scholar). The beads were then washed in lysis buffer, and proteins were eluted from beads in non-reducing NuPAGE (Invitrogen) sample buffer and then separated by SDS-PAGE (Invitrogen) under reducing conditions. Cell lysates were also run as controls. The gels were then analyzed by a standard immunoblot protocol using anti-Bax antibody (N20, Santa Cruz Biotechnology) and the ECL (enhanced chemiluminescence) detection system (Amersham Biosciences). Bax Is Predominantly Localized at Mitochondria of Nonapoptotic vMIA Cells—We examined the localization of Bax in two clones (clones 3 and 8) of HeLa cells stably transfected with vMIA by immunofluorescence with the anti-Bax N20 antibody. A representative experiment with HeLa/vMIA#3 and the control HeLa/pcDNA3 (Neo) cells stably transfected with the empty vector is shown in Fig. 1A. In control HeLa/pcDNA3 cells, Bax was diffusely distributed and did not co-localize with cytochrome c, consistent with cytoplasmic localization of Bax. In the HeLa clones constitutively expressing vMIA, Bax largely co-localized with both cytochrome c (Fig. 1A) and vMIA (not shown), two proteins that are localized at mitochondria (20McCormick A.L. Smith V.L. Chow D. Mocarski E.S. J. Virol. 2003; 77: 631-641Crossref PubMed Scopus (118) Google Scholar, 29Goldmacher V.S. Bartle L.M. Skletskaya S. Dionne C.A. Kedersha N.L. Vater C.A. Han J.W. Lutz R.J. Watanabe S. McFarland E.D.C. Kieff E.D. Mocarski E.S. Chittenden T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12536-12541Crossref PubMed Scopus (361) Google Scholar, 32Hayajneh W.A. Colberg-Oley A.M. Skaleskaya A. Bartle L.M. Lesperance M.M. Contopoulos-Ionnidis D.G. Kedersha N.L. Goldmacher V.S. Virology. 2001; 279: 233-240Crossref PubMed Scopus (86) Google Scholar, 37Daugas E. Susin S.A. Zamzami N. Ferri K. Irinopoulos T. Larochette N. Prevost M.C. Leber B. Andrews D. Penninger J. Kroemer G. FASEB J. 2000; 14: 729-739Crossref PubMed Scopus (696) Google Scholar). The finding that Bax was predominantly localized at mitochondria in HeLa/vMIA cells was unexpected since relocation of Bax to mitochondria is considered to constitute an early step in apoptosis (38Wolter K.G. Hsu Y.-T. Smith C.L. Nechushtan A. Xi X.G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1562) Google Scholar) followed by MMP. Instead, vMIA-expressing HeLa cells are resistant to apoptosis due to the ability of vMIA to prevent MMP (29Goldmacher V.S. Bartle L.M. Skletskaya S. Dionne C.A. Kedersha N.L. Vater C.A. Han J.W. Lutz R.J. Watanabe S. McFarland E.D.C. Kieff E.D. Mocarski E.S. Chittenden T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12536-12541Crossref PubMed Scopus (361) Google Scholar). To find out whether vMIA-induced recruitment of Bax to mitochondria is limited to HeLa cells or is a general phenomenon, we examined localization of Bax in MEF transiently transfected with vMIA/pcDNA3 (or with pcDNA3 as a control) and in MRC5 normal human fibroblasts infected with the AD169varATCC strain of human cytomegalovirus. We observed in both experimental systems that ectopic expression of vMIA induces relocation of Bax to mitochondria (Fig. 1, B and C), whereas in control MEFs (Fig. 1B) and non-infected MRC5 fibroblasts (data not shown), Bax is diffusely distributed. To study the kinetics of vMIA expression and Bax relocation to mitochondria, we examined the time course of both vMIA and Bax staining in transiently transfected HeLa cells. As shown in Fig. 1D, 2 h after transfection in the absence of vMIA protein, Bax shows a diffuse distribution in the cytoplasm, whereas from 4 h after transfection to 17 h, we noted that even low expression level of vMIA (at 4 h) was sufficient to trigger significant relocation of Bax to mitochondria. Next, we determined whether exposure of vMIA-expressing cells to various apoptotic stimuli induced apoptosis. HeLa/vMIA#3, were exposed to staurosporin (STS) and analyzed by immunofluorescence confocal microscopy for the intracellular distribution of Bax and cytochrome c. Representative images of the distribution of Bax (Fig. 2A, red fluorescence) and cytochrome c (Fig. 2A, green fluorescence) in HeLa/vMIA#3 cells (vMIA) and HeLa/pcDNA3 cells (Neo) that have or have not been exposed to STS are shown in Fig. 2A. In non-treated Neo cells, Bax staining is diffuse and is not colocalized with cytochrome c. Following the exposure of Neo cells to STS, Bax distribution becomes punctate and co-localized with cytochrome c (Fig. 2A, yellow) in mitochondria, which induces cytochrome c release (Fig. 2A, inset). In HeLa/vMIA#3 cells, Bax is constitutively co-localized with cytochrome c in granular structures irrespective of whether the cells were or were not exposed to STS (Fig. 2A). Cells were then exposed to STS, cis-platin, oligomycin + carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or anti-CD-95 + CHX. To quantitate the percentage of cells undergoing apoptosis, cytochrome c staining and chromatin condensation were examined either 3 or 16 h later. In vMIA-expressing cells, both cytochrome c release and chromatin condensation were blocked (Fig. 2B). Anti-apoptotic effects of vMIA were subsequently tested in transiently transfected cells (vMIAt) in comparison with vMIA#3. As shown in Fig. 2, C and D, the transient expression of vMIA was effective in inhibiting both cytochrome c release and chromatin condensation induced by CD-95 ligation. The subcellular distribution of Bax in HeLa/Neo and HeLa/vMIA that had or had not been exposed to STS was also determined by cell fractionation. As shown in Fig. 2E, exposure of HeLa/Neo cells to STS resulted in redistribution of Bax from the cytosol to the mitochondria/heavy membrane-enriched fraction and release of cytochrome c, in accord with previous observations (10Antonsson B. Montessuit S. Sanchez B. Martinou J.C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar). In contrast, both Bax and cytochrome c predominantly associated with the mitochondria-enriched fraction of vMIA cells that had or had not been treated with STS. One important observation made in these experiments was that in vMIA-expressin
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