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1 (2006) 13, & 2006 Nture Pulishing Group All rights reserved /06 $ Modified vccini virus Ankr protein F1L is novel BH3-domin-inding protein nd cts together with the erly virl protein E3L to lock virus-ssocited poptosis SF Fischer 1, H Ludwig 2, J Holzpfel 1, M Kvnskul 3, L Chen 3, DCS Hung 3, G Sutter 2, M Knese 1 ndghäcker*,1 1 Institute for Medicl Microiology, Technicl University Munich, Germny 2 Pul Ehrlich Institute, Lngen, Germny 3 The Wlter nd Eliz Hll Institute for Medicl Reserch, Melourne, Austrli * Corresponding uthor: G Häcker, Institute for Medicl Microiology, Technicl University Munich, Trogerstr. 9, Munich D-81675, Germny. Tel: þ ; Fx: þ ; E-mil: hcker@lrz.tum.de Received ; revised ; ccepted ; pulished online Edited y A Villunger Astrct Infection with viruses often protects the infected cell ginst externl stimuli to poptosis. Here we explore the lnce of poptosis induction nd inhiition for infection with the modified vccini virus Ankr (MVA), using two MVA mutnts with experimentlly introduced deletions. Deletion of the E3L-gene from MVA trnsformed the virus from n inhiitor to n inducer of poptosis. Nox-deficient mouse emryonic firolsts (MEF) were resistnt to MVA-DE3Linduced poptosis. When the gene encoding F1L ws deleted from MVA, poptosis resulted tht required Bk or Bx. MVA- DF1L-induced poptosis ws locked y Bcl-2. When expressed in HeL cells, F1L locked poptosis induced y forced expression of the BH3-only proteins, Bim, Pum nd Nox. Finlly, iosensor nlysis confirmed direct inding of F1L to BH3 domins. These dt descrie moleculr frmework of how cell responds to MVA infection y undergoing poptosis, nd how the virus locks poptosis y interfering with criticl steps of its signl trnsduction. (2006) 13, doi: /sj.cdd ; pulished online 8 July 2005 Keywords: virus; vccini; poptosis; BH3-domin; F1L; E3L Arevitions: MVA, modified vccini virus Ankr; BH3 domin, Bcl-2 homology domin 3; MEF, mouse emryonic firolsts; CEF, chicken emryonic firolsts Introduction Cell deth y poptosis is common response of humn cells to extrinsic stimuli. It is commonly thought tht one of the purposes of poptosis is the defence ginst noxious stimuli, such s genotoxic influences or infectious gents. In this context, it hs long een ssumed tht poptosis cn serve s defence of humn tissues ginst infecting viruses, nd the hypothesis hs even een put forwrd tht poptosis evolved to comt virl infections. 1,2 Apoptosis occurs upon triggering of specilised signl trnsduction pthwy. A key event in most forms of poptosis is the relese of cytochrome c from the mitochondri into the cytosol, where it initites the ctivtion of cspse proteses. Active cspses then re instrumentl in the ppernce of the typicl morphologicl chnges of poptosis, such s nucler condenstion (for review of the mechnisms of poptosis see Hengrtner 3 ). The ide tht poptosis serves s defence mechnism ginst viruses hs received strong support y the fct tht mny viruses crry genes whose products cn directly interfere with the cell s poptotic pprtus. An erly nd cler exmple of this sitution ws the demonstrtion tht culovirus hs the cpcity to induce poptosis in infected insect cells nd tht, t the sme time, this cpcity is countered y the expression of the virl gene p35. 4 Since then, poptosis inhiitors hve een found in viruses tht infect diverse species, such s Epstein Brr virus, 5 Africn swine fever virus 6 nd vrious culoviruses. 7 In most of these cses, virl inhiitors of poptosis re known cellulr homologues, such s the ntipoptotic protein Bcl-2. Poxviruses hve een found to encode for multitude of proteins tht regulte virus host interctions. 8 Within this virus fmily, especilly vccini virus, the closely relted cowpox virus nd the rit pthogen myxom virus hve een studied nd found to possess genes tht cn interfere with cellulr ctivtion pthwys, such s interferon- nd TNF signlling. It is noteworthy tht importnt host regultory genes re even conserved in the genomes of highly ttenuted vccini virus strins, such s modified vccini virus Ankr (MVA) or NYVAC, cndidte strins for future vccintion protocols. 9,10 One of these regultory proteins, E3L, cn ind doule-strnded (ds) RNA nd inhiit the dsrna-stimulted enzymes, protein kinse R nd RNA-specific denosine deminse. 11,12 A vccini virus deficient in E3L is highly sensitive to the ctivity of type I interferons nd restricted in growth in some cell lines such s HeL ut not in others such s chicken emryonic firolsts (CEF). 13,14 We hve previously generted the mutnt virus MVA-DE3L deficient in E3L coding sequences (open reding frme 050, trnscried leftwrd through nucleotides to in the MVA genome). 15,16 Intriguingly, unlike MVA, MVA-DE3L cused poptosis in infected CEF cells. 15 Furthermore, vccini virus mutnt lcking E3L hs een reported to cuse poptosis in HeL cells. 17 Therefore, the virl erly protein E3L cn e sid to hve n ntipoptotic function in some situtions.

2 110 MVA F1L inds BH3-domins Besides such virl proteins tht my e involved in signl trnsduction nd indirectly in poptosis inhiition, vccini virus protein ws recently descried tht ppers to hve rod ntipoptotic ctivity ginst experimentl poptotic triggers. 18 This protein, F1L, cn lock poptosis induced y Fs nd y sturosporine, two gents tht use different upstrem pthwys to poptosis. This potent ctivity together with its mitochondril loclistion 18,19 suggests tht F1L directly trgets the cellulr poptotic pthwy nd is specilised inhiitor of poptosis. Interestingly, the open reding frme encoding F1L is highly conserved etween the MVA genome (ORF 029, leftwrd trnscription through nucleotides to , 98% mino-cid identity nd vccini virus strin Copenhgen). 16 Thus, mny viruses cn protect infected cells ginst experimentl insults. It hs, however, not een well explored wht the physiologicl function of virl ntipoptotic proteins is on moleculr level. Antipoptotic proteins re only required if there is poptosis to e inhiited. One hypothesis is tht host cell is le to detect the replicting virus nd responds y undergoing poptosis. This would men tht viruses in fct induce poptosis tht needs to e inhiited to llow virl repliction. In this study we investigte this ide for the infection with MVA nd two mutnt viruses ech lcking one of the genes encoding E3L or F1L. Results Apoptosis induction y MVA-DE3L The mutnt virus MVA-DE3L ws originlly generted to investigte the cellulr response to MVA-sed vccine vectors. When nlysing this response, we noticed tht MVA-DE3L hd lost its ility to grow in CEF, nd tht this filure correlted with the induction of poptosis in CEF cells. 15 To find out whether this poptotic response ws confined to CEF, we first infected HeL humn epithelil cells with MVA or MVA-DE3L. Cells infected with MVA did not undergo poptosis nd showed strong resistnce to poptosis induced y tretment with sturosporine (Figure 1). This ctivity ws lost in MVA-DE3L. Insted, this mutnt virus ctively induced killing of HeL cells, s detected y ssessment of nucler morphology nd y mesurement of effector cspse ctivity (Figure 1 nd c). The poptosisinducing ctivity of MVA-DE3L is thus not restricted to CEF cells. MVA-DE3L ctivtes the mitochondril pthwy nd utilises Nox We next investigted whether MVA-DE3L-induced poptosis proceeded vi the relese of mitochondril cytochrome c. As shown in Figure 2, infection of HeL cells with MVA-DE3L ut not MVA cused the relese of mitochondril cytochrome c, s shown y microscopic nlysis nd y flow cytometry. 20 Cytochrome c relese is the result of the ctivtion of the propoptotic Bcl-2-fmily memers Bx/Bk. Stining for conformtionlly ctivted Bx showed tht infection with MVA-DE3L led to Bx ctivtion (Figure 2). Thus, poptosis induction y MVA-DE3L ctivtes the mitochondril pthwy to poptosis. Bx nd Bk re the most downstrem molecules of the cytochrome c-relese mchinery known. 21 Although they seem to e redundnt in some circumstnces, 22,23 there is cler preference for one over the other in some situtions. 24,25 c Figure 1 Apoptosis induction y MVA-DE3L. () HeL cells were infected with n MOI of of either MVA or MVA-DE3L. At 8 h p.i., some wells were treted with sturosporine (1 mm). After 3 h, cells were extrcted nd cspse ctivity ws mesured y enzyme ssy. () HeL cells were infected s under (). After 15 h, nuclei were stined with Hoechst. Left, representtive res of the plte were photogrphed. Right, quntifiction of nucler poptosis (men/s.d. of triplicte wells). Similr results were otined in three seprte experiments. (c) HeL cells were infected t vrious times with either MVA or MVA-DE3L (MOI ¼ 10 15). All cells were hrvested t the sme time. Dt re men/s.d. from triplicte mesurements of one infected well. Similr results were otined in three seprte experiments. Note the difference in scling etween nd c

3 MVA F1L inds BH3-domins 111 Figure 2 MVA-DE3L induces the relese of cytochrome c nd cuses the ctivtion of Bx. HeL cells grown on coverslips were infected with either MVA or MVA-DE3L (MOI ¼ 10 15). At 10 h p.i., cells were fixed nd stined with MitoTrcker Green FM nd with ntiodies either directed ginst cytochrome c () or ginst conformtionlly ltered Bx (). The ottom pnel in () shows flow cytometric nlysis of cytochrome c content during tretment (lue lines) with sturosporine (control of poptosis induction, 500 nm, 8 h) nd infection with MVA-DE3L or MVA-DF1L s ove (12 h; see elow on the MVA-DF1L virus). Red lines represent controls (for sturosporine, no tretment; for infection with mutnt viruses, controls represent infection with MVA). Similr results were otined in four seprte experiments. Note loss of cytochrome c nd presence of conformtionlly ltered Bx during poptosis The ctivtion of Bx/Bk is the consequence of the ctivtion of memers of the BH3-only group of Bcl-2 protein fmily memers. 21,26 We therefore sought to identify the BH3-only protein required for poptosis induction y MVA-DE3L. Since E3L inds nd proly sequesters dsrna tht is generted during virl repliction, we hypothesised tht poptosis induction y MVA-DE3L ws cused y the ppernce of dsrna nd could e mimicked y tretment of the cells with poly-i:c ( synthetic dsrna-mimetic). The humn kertinocyte cell line HCT ws found to e sensitive to poly-i:c, with out 50 60% of cells undergoing poptosis when treted with 50 mg/ml poly-i:c for 24 h (dt not shown). The expression levels of the BH3-only proteins, Bim nd Pum, did not chnge during poly-i:c-tretment, ut Nox expression ws clerly induced (Figure 3 nd dt not shown). Nox induction y dsrna ws recently lso reported y others. 27 Intriguingly, Nox ws induced y infection of HeL cells with either MVA or MVA-DE3L to similr extent (Figure 3). This suggests tht the trnscriptionl induction of Nox my not e the sole result of free dsrna, which should e present more undntly in infections with MVA-DE3L thn in infections with MVA, ut my e induced through some other mechnism. To ssess the importnce of Nox for poptosis induction y MVA-DE3L, we infected mouse emryonic firolsts (MEF) with either MVA or MVA-DE3L. As in CEF nd HeL cells, MVA-DE3L cused strong poptotic response in MEF. When MEF from mice deficient in the Nox gene were used, this response ws rogted demonstrting tht Nox crucilly contriutes to poptosis induction y MVA-DE3L (Figure 3c n d). Furthermore, trnsient expression of Bcl-2 y trnsfection locked the induction of poptosis through MVA- DE3L in HeL cells (not shown), consistent with model where Nox is ctivted nd induces poptosis y MVA in the sence of E3L. This rises the question why MVA, which induces Nox to similr extent, does not induce poptosis. We considered the possiility tht MVA-DE3L is defective in the expression of the ntipoptotic protein F1L. Northern lot nlysis showed tht the mrna for F1L is mde t erly times, ut pprently less undntly during infection with MVA-DE3L, s compred to MVA infection (Figure 3e). The most likely model is therefore tht MVA nd MVA-DE3L oth induce the expression of Nox ut only MVA is le to lock the poptosis-inducing ctivity of Nox. It should further e dded tht mere Nox expression is poor poptosis stimulus t lest in HeL cells. When Nox ws induced in tetrcycline-inducile system to levels much higher thn the ones chieved during infection, only pproximtely 10 20% of cells underwent poptosis (Figure 3f nd dt not shown). Therefore, lthough Nox is required, other unknown events must contriute to poptosis induction y MVA-DE3L. Loss of E3L cuses the induction of poptosis in MVAinfected cells, nd E3L my therefore e considered n ntipoptotic protein. However, unlike other virl proteins, E3L does not pper to directly trget the poptotic pthwy, s do for instnce virl Bcl-2 homologues or the culovirl inhiitor of poptosis proteins (IAP). As mentioned ove, the vccini F1L protein hs een found to hve strong ntipoptotic ctivity. 28 This rod inhiition together with its mitochondril loclistion suggested tht F1L ws specilised inhiitor of poptosis tht is expressed during virl infection in order to counter poptosis induction tht my e induced y the virus.

4 112 MVA F1L inds BH3-domins c d e f Figure 3 Nox is induced during infection nd is necessry for poptosis induction y MVA-DE3L. () HCT humn kertinocytes were treted with poly-i:c for 24 h nd nlysed for the expression of Nox nd tuulin (loding control) y Western lotting. Similr results were otined in five experiments. () HeL cells were infected t vrious periods with either MVA or MVA-DE3L (MOI ¼ 10 15). At the time points shown, expression of Nox ws nlysed y Western lotting. In series of five experiments, the levels of expression were similr etween the two viruses, with slight vritions etween individul experiments. (c) MEF generted from WT or from Nox-deficient mice were grown on coverslips nd infected with either MVA or MVA-DE3L.At 15 h p. i., representtive res of the coverslip were photogrphed (left). Right, 300 nuclei were scored visully for poptosis. Dt re men/s.d. of triplicte wells. (d) MEF from WT of Nox-deficient mice were infected with either MVA or MVA-DE3L. At the time points indicted, cspse-3 ctivity ws mesured in cell extrcts y enzyme ssy. Similr results were otined in four seprte experiments. (e) HeL cells were mock infected (U) or infected with either MVA or MVA-DE3L t multiplicity of infection (MOI) of 10. Totl RNA ws isolted t 0, 2, 4, 8 nd 10 h postinfection (h p. i.) nd electrophoreticlly seprted in 1% grose formldehyde gels pplying 1 mg of totl RNA per lne. For loding control riosoml, RNAs were stined with ethidium romide. In MVA-DE3L-infected cells, degrdtion of riosoml RNA ws detectle t 4 h p.i. nd lter during infection. Susequently, RNA ws trnsferred onto positively chrged nylon memrne (Roche Dignostics) vi vcuum lot nd hyridised to rioproes specific for F1L. (f) HeL cells stly crrying tetrcycline-responsive repressor nd Nox under the control of this repressor were incuted for 24 h in the presence or sence of tetrcycline nd sujected to Western lotting for Nox Induction of poptosis y MVA-DF1L If the role of F1L is to lock poptosis cused y the cell s response to MVA infection, mutnt virus lcking F1L would e expected to induce poptosis. MVA hs n F1L-gene tht is very closely relted to vccini F1L. We generted n F1L-deficient MVA mutnt (MVA-DF1L) nd tested it for poptosis induction in HeL cells. As shown in Figure 4 nd, infection with MVA-DF1L cused significnt poptosis, s detected s nucler morphology chnges nd cspse ctivity. (The detected cspse ctivity ws somewht smller thn during poptosis induced y MVA-DE3L (see ove). This could men tht F1L lso locks cspseindependent poptotic events or tht the time course of poptosis induction ws different in the individul cell (compring the two mutnt viruses), leding to different totl cspse ctivity t given time in the popultion.) Apoptosis induction ws ssocited with the ctivtion of Bx nd the relese of mitochondril cytochrome c (Figure 5 nd ; see Figure 2 (ottom pnel) for flow cytometric nlysis of cytochrome c relese). These results indicte tht MVA hs n intrinsic poptosis-inducing cpcity, which is normlly locked y F1L.

5 MVA F1L inds BH3-domins 113 Figure 4 Apoptosis induction y MVA-DF1L. () HeL cells grown on coverslips were infected with either MVA or with MVA-DF1L (MOI ¼ 10 15). At 15 h p.i., nuclei were stined with Hoechst. Left, representtive re of the coverslip is shown. Right, 300 nuclei per coverslip were scored visully for poptosis. Dt re men/s.d. of triplicte wells. Dt re tken from the sme experiment shown in Figure 1. () HeL cells were infected with MVA or with MVA-DF1L for the time periods shown. Cspse-3 ctivity ws mesured in cell extrcts y enzyme ssy Anlysis of poptosis induction y MVA-DF1L We used MVA-DF1L to nlyse the pthwy to poptosis tht is triggered y MVA infection. First, we tested whether the cellulr nti poptosis protein Bcl-2 ws le to inhiit MVA- DF1L-induced poptosis. HeL cells were trnsiently trnsfected with vector driving Bcl-2 expression nd infected with MVA-DF1L. As shown in Figure 6, expression of Bcl-2 inhiited poptosis induction y MVA-DF1L, indicting tht MVA ctivted poptosis in wy tht could e locked y Bcl-2. This suggested tht F1L hs moleculr function similr to tht of Bcl-2. How Bcl-2 works on moleculr level is still not completely cler, ut its function is emedded in the wider Bcl- 2 fmily of proteins. Within this fmily, three sufmilies re commonly recognised. Bcl-2 itself nd its close reltives (such s Bcl-x L, Bcl-w, A1) re inhiitors of poptosis. Bx nd Bk re the most downstrem molecules known in the pthwy, nd ctivtion of these proteins is linked to the relese of cytochrome c. Upstrem of Bx nd Bk lies the fmily of propoptotic Bcl-2 Homology Domin-3-only (BH3-only) proteins (such s Bim, Pum, Nox; 8 re known t present). An poptotic stimulus ctivtes one or severl BH3-only proteins, which then cuse ctivtion of Bx nd/or Bk. Bcl-2 nd Bcl-2-like molecules cn ind ctive BH3-only proteins. Bcl-2-like proteins my thus function y sequestering ctive BH3-only proteins. Alterntively, BH3-only proteins my ct y displcing Bcl-2-like molecules nd therey llowing utoctivtion of Bx/Bk. High-level expression of BH3-only proteins induces poptosis, which cn e locked y coexpression of Bcl-2. F1L ws found to hve the sme cpcity: like Bcl-2, F1L inhiited Figure 5 MVA-DF1L induces cytochrome c relese nd ctivtion of Bx. HeL cells grown on coverslips were infected with either MVA or MVA-DF1L (MOI ¼ 10 15). At 10 h p.i., cells were fixed nd stined with MitoTrcker Green FM nd with ntiodies either directed ginst cytochrome c () or ginst ctive Bx (). Similr results were otined in four seprte experiments. Dt in re tken from the sme experiment s the one shown in Figure 2 poptosis induced y the BH3-only proteins Bim, Pum nd Nox when coexpressed in HeL cells (Figure 6). Like Bcl-2, F1L must therefore ct y locking process downstrem of BH3-only protein ctivtion. Note tht the induction of poptosis y trnsient trnsfection of Nox is much higher thn during tetrcycline-regulted expression (Figure 3f), proly either ecuse of higher expression levels or ecuse of stress ssocited with the trnsient trnsfection. We next nlysed the contriution of Bx nd Bk to poptosis induced y MVA-DF1L. MEF lcking either or oth of these proteins were infected with MVA or MVA-DF1L nd

6 114 MVA F1L inds BH3-domins c Figure 6 F1L functions in Bx/Bk-dependent mnner downstrem of BH3- only protein ctivtion. () HeL cells were trnsiently cotrnsfected with n expression vector for -glctosidse nd either the empty vector s control (white rs) or the sme vector driving the expression of humn Bcl-2 (lck rs). At 24 h fter trnsfection, cells were infected either with MVA or with MVA- DF1L s indicted. After 15 h, cultures were fixed, stined for -glctosidse expression, nd lue cells were scored for cell deth. 48,49 In ll, 900 cells per well were counted, nd dt re represented s men/s.d. of results from three independent trnsfection/infections. () HeL cells were trnsiently cotrnsfected with -glctosidse expression vector nd expression vectors for Bim, Pum or Nox together with expression vectors for Bcl-2, F1L or the empty vector s indicted. After15 h, cells were fixed nd lue cells were scored for cell deth. One out of three experiments with very similr results is shown. (c) MEF generted from mice deficient for Bk nd/or Bk s indicted were infected with either MVA or MVA-DF1L. After12 h, cspse-3ctivity in cell extrcts ws mesured y enzyme ssy. Dt re men/s.d. of triplicte mesurements from one extrct. Similr results were otined in four experiments poptosis ws monitored y mesuring cspse ctivity. MVA-DF1L-induced poptosis ws rogted in cells lcking oth Bx nd Bk. Cells lcking only Bk showed strong reduction in their poptotic response, while cells tht lcked only Bx were protected only to moderte degree (Figure 6c). This indictes tht the poptotic pthwy ctivted y MVA preferentilly ctivtes Bk, ut cn lso, to lesser degree, work vi the ctivtion of Bx. MEF deficient in the BH3-only proteins, Bim, Bid, Pum, Nox, Bik or Bmf, were lso tested for their ility to respond with poptosis to infection with MVA-DF1L. All of them underwent poptosis like the wild-type MEF (dt not shown). The most likely explntion for this finding is tht MVA-DF1Linfection triggers more thn one BH3-only protein, nd tht the sence of one of them is insufficient to lock poptosis. Our results with the MVA mutnts suggest tht they trigger poptosis y Bcl-2-dependent mechnism. Since the killing is rogted y the sence of the essentil poptosis meditors Bx/Bk (Figure 6c) nd infection y the cytotoxic mutnts drive Bx ctivtion (Figures 2 nd 5), n ttrctive model for F1L function is direct sequestrtion of these molecules. However, unlike certin other ntipoptotic virl proteins, such s EBV BHRF-1, which shre sequence nd structurl homology 29 with mmmlin Bcl-2, F1L does not hve ny ovious sequence homology to the wider Bcl-2 fmily, or indeed ny other protein. When F1L ws nlysed using FUGUE, progrmme for recognizing distnt homologues y sequence-structure comprison nd incorporting BLAST serches, 30 only F1L homologues from other orthopoxviruses were identified. The next est mtch fell elow sttisticl significnce. However, the ntipoptotic proteins expressed y humn cytomeglovirus vmia nd tht expressed y myxom virus M11L sequester Bx 31 nd Bk, 32 respectively. To test this hypothesis for F1L-function, we initilly ssessed the cpcity of purified recominnt F1L expressed in Escherichi coli to ind Bcl-2 homology domin (BH3 domin) peptides since these regions medite the killing ctivity of the propoptotic BH3-only proteins s well s Bx/Bk. 33,34 These in vitro inding ssys were performed using the Bicore opticl iosensor, which cn sensitively nd ccurtely determine inding ffinities s we hve recently undertken for mmmlin Bcl-2 fmily proteins. 35 Strikingly, we found tht F1LDC20, form tht lcks its hydrophoic C-terminus, inds BimBH3 vidly with dissocition equilirium constnt (K D ) of 80 nm (Figure 7 nd ). This significnt inding ws confirmed in solution competition ssys (Figure 7c) s the IC50, the concentrtion of free Bim peptide required to reduce F1LDC20 inding to the immoilised BimBH3 peptide y hlf, ws 75 nm, which pproximtes the K D. F1LDC20 could lso ind peptides spnning the BH3 regions of Bx nd Bk, ut not sutle point mutnt of this, lthough t lower ffinities (B1 mm) consistent with results otined using isotherml clorimetry (dt not shown). In greement with our oservtion tht F1L functions in mnner kin to mmmlin prosurvivl Bcl-2, F1L cn ind BH3 regions. Thus, F1L despite hving no significnt primry sequence similrity to mmmlin prosurvivl Bcl-2 proteins, proly possesses groove trgeted for BH3 inding. 36,37 By impliction, F1L my ct to sequester BH3-contining

7 MVA F1L inds BH3-domins 115 Figure 7 F1L inding to selected BH3 domin peptides. () Recominnt F1L DC20 ws injected onto sensorchips with Bim wt BH3 (solid line) or mutnt Bim 4E BH3 (dotted line) peptides immoilised. To otin the specific inding, the seline response with 4E BH3 ws sutrcted from tht with wt BH3. () Biosensor responses when nm ws injected over the BimBH3 sensorchip. (c) BH3 peptides used in the solution competition ssys. The BH3 region contins four hydrophoic residues (h1 h4) required for intercting with the prosurvivl proteins. The competitor peptides were derived from humn proteins except for mouse Bim. Sequences were ligned s descried. 51 The IC50 (nm) for the indicted interctions re shown on the right nd re from representtive experiments (NB, no inding) propoptotic proteins, ut precise moleculr mechnism(s) y which it promotes cell survivl wit further studies. Discussion In this study, we investigted two MVA mutnts with regrd to their potentil to ctivte the poptotic pthwy. It is commonly ccepted tht viruses crry ntipoptotic genes (some of which hve een well chrcterised), nd it hs een demonstrted on mny occsions tht virl infection cn protect the infected cell ginst experimentl poptotic insults. The role of virl ntipoptotic proteins for the virl infection, however, is fr less well documented. The fct tht virus cn lock poptosis suggests tht there is concomitnt propoptotic ctivity tht needs to e locked y the virus. This most likely mens tht the virl infection in fct induces poptosis or, from the cell s perspective, tht the cell notices the replicting virus nd undergoes poptosis. Our dt provide strong support for this model. In the sence of E3L, MVA-infection induces poptosis tht criticlly involves Nox. Expression of E3L y cotrnsfection filed to lock poptosis upon forced expression of Nox (not shown). It is therefore more likely tht E3L cts upstrem of the ctivtion of Nox. However, the moleculr pthwy is not s strightforwrd s ppers to e t first glnce. Since E3L is known to ind dsrna we tested induction of BH3-only proteins through dsrna nd found tht expression of Nox ws induced (this ws very recently lso reported y others 27 lthough nother group recently reported findings suggesting tht dsrna induces poptosis not through Nox ut through the formtion of cspse-8-ctivting protein complex 38,39 ). Indeed, Nox expression ws induced upon infection with MVA-DE3L. However, the expression ws not very high nd, surprisingly, ws very similr in cells infected with MVA (where no poptosis ws induced). Bsed on results in Nox / cells, Nox clerly contriuted to poptosis induction through MVA-DE3L. Why, then, did MVA not lso induce poptosis? One fctor tht is of likely relevnce is the finding tht F1L expression my e reduced in infection with MVA-DE3L s compred to MVA. As shown ove, strong F1L expression protects ginst poptosis induced y overexpression of Nox, suggesting tht F1L, if present t high enough levels, would prevent poptosis through MVA-DE3L. The oserved reduction in F1L is therefore proly relevnt. A second possiility is suggested y dt indicting tht Nox my e required, ut on its own would not e enough for poptosis. Nox expression ws found to e insufficient to induce poptosis on its own ut le to cooperte in poptosis induction with concomitnt expression of Bd, proly ecuse of its limited inding cpcity to some ut not ll ntipoptotic Bcl-2-like proteins. 35 It hs further een suggested tht the mjority of known BH3-only proteins (nd Nox mongst them) fil to ctivte Bx directly ut ct to sensitise the system to the propoptotic ctivities of the BH3-only proteins Bim nd tbid. 40 In ddition, our own dt presented here show tht experimentlly induced high-level expression of Nox only wekly cuses poptosis. Therefore, it ppers likely tht loss of E3L leds to the induction of Nox nd lso provides second trigger (perhps the ctivtion of nother BH3-only protein), which then collorte to induce poptosis during infection with MVA-DE3L. Our dt suggest tht poptosis induced y MVA-DE3L is indirect, possily vi the reduction in F1L expression. F1L, on the other hnd, ppers to e true ntipoptotic protein in tht it loctes to mitochondri, the site of cytochrome c relese 18,19, nd directly trgets components of the core poptosis mchinery of this study. Apoptosis induction y MVA-DF1L required Bk nd/or Bx. The only known wy to ctivte these molecules is vi the ctivtion of BH3-only

8 116 MVA F1L inds BH3-domins proteins. The finding tht loss of ny one of the six investigted BH3-only proteins filed to protect the cell ginst MVA-DF1L therefore suggests tht more thn one BH3-only protein is ctivted (or tht other, perhps unidentified BH3-only proteins contriute). Bcl-2-expression ws le to cover for the loss of F1L, further indicting tht oth proteins t lest ct in the sme pthwy. Bx/Bk re the most downstrem proteins known prior to cytochrome c relese, nd ctive Bx is sufficient to induce the relese of molecules from synthetic liposomes. 41 BH3-only proteins re required to ctivte Bx proly in most if not ll instnces of Bcl-2-controlled poptosis. F1L ws le to lock poptosis induced y overexpression of the BH3-only proteins Bim, Pum nd Nox. Furthermore, evidence for direct inding of F1L to BH3-contining propoptotic proteins ws found. Therefore, F1L either inds nd sequesters BH3-only proteins or inds to Bk/Bx precluding their ctivtion. Future work will hve to clrify wht the direct trgets of F1L in vivo re. Tken together, this study shows tht n infected cell is le to detect the presence of infecting MVA nd to rect to this infection y ctivting the poptotic pthwy. Understnding this reltionship will llow us to pprecite n importnt spect of the infectious iology of viruses. Mterils nd Methods Cells nd viruses HeL cells (humn cervicl denocrcinom cell line) were otined from the Americn Type Culture Collection (ATCC) nd HCt cells (humn kertinocyte cell line) were otined from the Germn Cncer Reserch Centre (DKFZ, Heidelerg, Germny). Mouse emryonic firolsts deficient in Nox, Pum, Bim or Bik (kindly provided y A Strsser, The Wlter nd Eliz Hll Institute of Medicl Reserch, Melourne, Austrli, immortlised y C. Borner, Alert-Ludwigs-University Freiurg, Germny), Bid (S Korsmeyer, Hrvrd Medicl School, Boston, USA) were used. Bx þ / Bk /, Bx / Bk þ /, Bx þ / þ Bk þ / nd Bx / Bk / MEF were derived from E14 C57Bl/6 mice nd immortlised (t pssge 2 4) with SV40 lrge T ntigen. Culture of ll cells ws crried out in DMEM supplemented with 10% FCS. Vccini virus MVA (cloned isolte F6 t 582nd CEF pssge), 42 nd MVA-DE3L 15 were routinely propgted nd titrted y vccini virusspecific immunostining on BHK-21 cells to determine the numers of infectious units (IU) per milliliter. Vccini virus MVA (cloned isolte II new ) 42,43 nd MVA-DF1L were mplified nd titrted on CEF. In experiments using MVA-DE3L, the MVA isolte F6 ws used s control, in experiments with MVA-DF1L, the control ws MVA isolte II new. MVA DNA sequences flnking the F1L gene (MVA029L nucleotides , GenBnk ccession No. U94848) were mplified y polymerse chin rection (PCR) using genomic MVA (isolte F6) DNA s templte. The resulting PCR frgments were inserted into the plsmid pdk1l 44 to otin the F1L deletion plsmid pdf1l. For genertion of F1L deletion mutnt virus, monolyers of confluent CEF cells grown in six-well tissue-culture pltes were infected with MVA isolte II new t multiplicity of infection (MOI) of 0.01 IU per cell. At 90 min fter infection cells from one well were trnsfected with 2 mg plsmid DNA of pdf1l using FUGENE (Roche Dignostics) s recommended y the mnufcturer. At 48 h fter infection the cells nd medium were hrvested, freeze thwed three times nd homogenized in cup sonictor (Sonopuls HD 200, Bndelin, Germny). From this mteril MVA mutnt virus ws isolted following previously descried methodology. 44 Briefly, 10-fold seril dilutions (10 1 to 10 4 ) of the hrvested mteril in medium were used to infect suconfluent monolyers of RK-13 cells grown in six-well tissue-culture pltes. After 3 dys incution t 371C, single foci of infected RK-13 cells were picked nd processed y freeze thwing nd soniction for nother infection of RK-13 cell monolyers. After elimintion of prentl MVA during pssge on RK-13 cells, 10-fold seril dilutions (10 1 to 10 6 ) of the recominnt viruses were used for infection of suconfluent CEF cells grown in six-well tissueculture pltes. Well-seprted foci of infected CEF cells were hrvested to isolte K1L-negtive mutnt viruses. Virl DNA from cloned MVA isoltes ws routinely nlysed y PCR s descried previously. 44 Detils of PCR nd the cloning procedure re ville from the uthors upon request. Induction nd detection of poptosis Host cells ( /well in 12-well pltes seeded the dy efore) were either infected or not, treted with sturosporine (1 mm, Sigm) nd poptosis ws detected s descried previously. 45 Briefly, for detection of nucler poptosis, cells were stined with 20 mm Hoechst (Sigm) for 30 min nd nucler morphology ws ssessed under fluorescence microscope. At lest 300 nuclei per smple were scored. For detection of cspse-3-like ctivity, cells were lysed in NP-40 lysis uffer nd 45 triplictes of liquots were dded to fluorogenic peptide contining cspse-3-recognition sequence (DEVD-AMC, 10 mm, Bchem, Heidelerg, Germny) in ssy uffer contining BSA nd Hepes. Free AMC ws mesured fter 1 h of incution t 371C nd vlues re presented s ritrry reltive fluorescence units (men7s.d. of triplicte rections). After tretment with poly-i:c (50 mg/ml) for 24 h, proes were hrvested nd sujected to Western lot nlysis for detection of Nox s descried elow. Microscopy, immunocytochemistry nd flow cytometry HeL cells were grown on glss coverslips, infected or left uninfected. Cells were fixed with 2% formlin for 30 min, stined with mouse nticytochrome c ma (Becton Dickinson) nd Cy3-lelled nti-mouse ntiserum in PBS-contining 1% FCS nd 1% sponin. For the detection of ctive Bx, cells were stined with ntictive Bx ma (6A7, Upstte Biotechnology, 46 ) nd Cy3-lelled nti-rit ntiserum (Dinov). Then, cells were stined with MitoTrcker Green FM (Moleculr Proes). Pictures were otined with Zeiss lser scnning microscope. For detection of cytochrome c relese y flow cytometry, cells were stined nd nlysed s descried. 20 Briefly, cells were fixed in 2% formldehyde, wshed consecutively in PBS, BPS/BSA 0.5% nd PBS/BSA/Sponin 0.5%. Cells were stined with nticytochrome c ma in PBS/BSA/Sponin, followed y stining with FITC-lelled got ntimouse A (Dinov). Cells were nlysed in Becton Dickinson FACSCliur. Western lot nlysis Cells were hrvested nd detergent extrcts were prepred y lysis of cells in 50 ml Triton uffer (1% Triton X-100, 0.05 M Pipes-NOH, 0.05 M Hepes ph 7.0, 2 mm MgCl 2, 1 mm EDTA, 10 mm DTT nd protese inhiitors (Roche)) for 30 min on ice. After centrifugtion t 2000 gt41cfor 10 min, loding uffer ws dded nd lystes were run

9 MVA F1L inds BH3-domins 117 on 12% polycrylmide gel. Proteins were trnsferred to nitrocellulose memrnes, which were consecutively proed with ntiodies specific for Nox (Alexis) or tuulin (Sigm). Proteins were visulized y chemiluminescence detection system (Perkin-Elmer Lifescience, Boston, MA, USA). Trnsfection of cells Cells were trnsfected using ethylene imine polymer solution (Fluk 47 )s descried previously. 48 Briefly, HeL cells were trnsfected with 1.5 mg of control vector pef, pef-bcl-2-expression vector or F1L-expression vector together with 1.5 mg Bim-, Pum- or Nox-expression vector (kindly provided y Andres Villunger, Innsruck) nd 0.5 mg of vector driving the expression of E. coli -glctosidse (CMV-LcZ). 48 The F1Lexpression vector ws generted y PCR mplifiction of the F1L ORF from genomic MVA DNA, followed y su-cloning into the pegfp-c2- expression vector (Clontech). For trnsfection of cells y electroportion ( cells t 240 V, 960 mf), 15 mg of control vector (pef) or pef-bcl- 2-expression vector together with 5 mg CMV-LcZ ws used. At 24 h fter trnsfection, cells were infected or left uninfected for 12 h. Cells were then stined for -glctosidse ctivity nd lue cells were viewed under microscope nd scored live or ded using morphologicl criteri. 48,49 Northern lot nlysis HeL cells cultured in 35 mm-dimeter dishes were mock infected or infected with MVA or MVA-DE3L using n MOI of 10. Following 30 min dsorption t 371C, virus inocul were replced y prewrmed tissue culture medium (DMEM with 2% FCS). After 0, 2, 4, 8 nd 10 h, totl RNA of mock infected, MVA nd MVA-DE3L-infected cells ws isolted with TRIzol regent (Invitrogen) following the mnufcturer s instructions. Qulity of RNA ws exmined y electrophoresis in 1% grose formldehyde gels nd ethidiumromide stining. Susequently, RNA ws trnsferred onto positively chrged nylon memrne (Roche Dignostics) vi vcuum lot. For synthesis of rioproes for detection of MVA- F1L trnscript, PCR using virl DNA s templte nd primer pir HL68 (5 0 - ATG TAG ATG GTA TAG TAC AGG-3 0 )/HL69 (5 0 -CTA ATA CGA CTC ACT ATA GGG AGA ATT ATC TGG TGG TGA AAT GTC-3 0 ) ws employed. Reverse primer contined T7 RNA polymerse promoter recognition sequence (underlined). Digoxigenin (DIG)-lelled rioproes were otined y in vitro trnscription with T7 RNA polymerse (Roche Dignostics) using the PCR-generted DNA frgment s templte. In vitro RNA lelling, hyridistion nd signl detection were crried out ccording to the mnufcturer s instructions (DIG RNA lelling kit nd detection chemicls, Roche Dignostics), pplying 681C for prehyridistion, hyridistion nd high stringency wsh. Recominnt protein production HexHIS-tgged F1LDC20 ws expressed using the pet Duet vector (Novgen) in E. coli BL21 DE3 plyss cells. Following induction of protein expression, the cell lyste ws homogenised, clered nd the protein purified on HiTrp (Amershm)-chelting column chrged with nickel. The protein ws eluted in 50 mm Tris ph 8.0, 150 mm NCl nd 5 mm 2- mercptoethnol, 250 mm imidzole nd sujected to gel-filtrtion chromtogrphy in 20 mm Tris ph 8.0, 150 mm NCl nd 5 mm 2- mercptoethnol using Superdex 200 column; it eluted s single pek. Affinity mesurements nd solution competition ssys Affinity mesurements were performed t room temperture on Bicore 3000 iosensor s previously descried. 50 All the peptides used were supplied y Mimotopes, Austrli, nd their sequences re indicted in Figure 7c. Isotherml clorimetry dt were collected on VP-ITC (MicroCl) with F1LDC20 diluted in 20 mm Tris ph 8.0, 150 mm NCl nd 5 mm 2-mercptoethnol to finl concentrtion of 10 mm. Dt were otined t 251C nd nlysed using the MicroCl Origin softwre. Acknowledgements We thnk M Evngelist for excellent technicl ssistnce. 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