Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2
1998; Springer Nature; Volume: 17; Issue: 5 Linguagem: Inglês
10.1093/emboj/17.5.1268
ISSN1460-2075
Autores Tópico(s)RNA Interference and Gene Delivery
ResumoArticle2 March 1998free access Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2 Denis Grandgirard Denis Grandgirard Institute of Medical Microbiology, University of Berne, Friedbuehlstrasse 51, CH-3010 Berne, Switzerland Search for more papers by this author Erwin Studer Erwin Studer Institute of Medical Microbiology, University of Berne, Friedbuehlstrasse 51, CH-3010 Berne, Switzerland Search for more papers by this author Laurent Monney Laurent Monney Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland Search for more papers by this author Tanja Belser Tanja Belser Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland Search for more papers by this author Isabelle Fellay Isabelle Fellay Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland Search for more papers by this author Christoph Borner Christoph Borner Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland Search for more papers by this author Marcel R. Michel Corresponding Author Marcel R. Michel Institute of Medical Microbiology, University of Berne, Friedbuehlstrasse 51, CH-3010 Berne, Switzerland Search for more papers by this author Denis Grandgirard Denis Grandgirard Institute of Medical Microbiology, University of Berne, Friedbuehlstrasse 51, CH-3010 Berne, Switzerland Search for more papers by this author Erwin Studer Erwin Studer Institute of Medical Microbiology, University of Berne, Friedbuehlstrasse 51, CH-3010 Berne, Switzerland Search for more papers by this author Laurent Monney Laurent Monney Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland Search for more papers by this author Tanja Belser Tanja Belser Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland Search for more papers by this author Isabelle Fellay Isabelle Fellay Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland Search for more papers by this author Christoph Borner Christoph Borner Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland Search for more papers by this author Marcel R. Michel Corresponding Author Marcel R. Michel Institute of Medical Microbiology, University of Berne, Friedbuehlstrasse 51, CH-3010 Berne, Switzerland Search for more papers by this author Author Information Denis Grandgirard1, Erwin Studer1, Laurent Monney2, Tanja Belser2, Isabelle Fellay2, Christoph Borner2 and Marcel R. Michel 1 1Institute of Medical Microbiology, University of Berne, Friedbuehlstrasse 51, CH-3010 Berne, Switzerland 2Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1268-1278https://doi.org/10.1093/emboj/17.5.1268 C.Borner and M.R.Michel are joint last authors PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1β-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31↓AGD34↓A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor. Introduction Programmed cell death (apoptosis) plays a crucial role in the proper embryonic development and later life of multicellular organisms because it removes damaged, expended and misplaced cells (Jacobson et al., 1997). When dysregulated, apoptosis contributes to many diverse human diseases that are either caused by too many unwanted cells, for example cancer and autoimmunity, or by insufficient cell numbers, as seen in neurodegeneration and AIDS (Bellamy et al., 1995). Thus, it has become a major challenge to understand the biochemical and molecular events that control apoptosis in the hope that such knowledge may ultimately be used in the treatment of diseases. The apoptotic process can be divided into three phases: initiation, effector and degradation (Kroemer, 1997). The initiation phase comprises death stimulus-specific signalling pathways that converge on a common effector phase whose role it is to execute death by degrading various cellular components (Fraser and Evan, 1996). Crucial molecular players in these phases are interleukin 1β-converting enzyme (ICE)-like cysteine proteases—recently renamed cysteine-aspartate proteases (caspases) (Yuan, 1995; Alnemri et al., 1996). These enzymes are activated from zymogens and cleave substrates by proteolysis at aspartate residues (Martin and Green, 1995), thus forming protease cascades similar to those seen in clotting and complement activation. Whereas most caspases participate in death stimulus-specific signalling, a subgroup founded by caspase-3 (formerly CPP32) controls the common death effector phase (Nicholson et al., 1995; Kumar and Lavin, 1996). Importantly, death effector caspases are the closest homologues of ced-3, a caspase that has previously been shown to be essential for apoptosis in the development of the nematode worm Caenorhabditis elegans (Shaham and Horvitz, 1996). Numerous substrates of the ced-3/caspase-3 subfamily have so far been identified (reviewed in Martin and Green, 1995), among them poly (ADP-ribose) polymerase (PARP), an enzyme implicated in DNA repair (Lazebnik et al., 1994; Orth et al., 1996). The caspase death effector machinery is under negative control by members of the Bcl-2 family (Yang and Korsmeyer, 1996). These are homologues of the C.elegans ced-9 gene product known to suppress apoptosis during nematodal apoptosis (Shaham and Horvitz, 1996). The prototype of this family is Bcl-2, a proto-oncogene product that was originally identified as overexpressed membrane protein in follicular lymphomas carrying a t(14;18) chromosomal translocation (Cleary et al., 1986). Meanwhile, overexpression of Bcl-2 has been shown to delay or block apoptosis induced by numerous, often unrelated physiological and pathological stimuli (Reed, 1994). How Bcl-2 performs this action is about to be revealed. Through its hydrophobic C-terminus, it is anchored to the outer membranes of mitochondria, the endoplasmic reticulum (ER) and the nucleus (Krajewski et al., 1993), where it may form a cation-selective ion channel (Minn et al., 1997; Schendel et al., 1997), block the release of cytochrome c from mitochondria into the cytosol (reviewed in Kroemer, 1997), and attract cytosolic adapter molecules (reviewed in Reed, 1997). This multifunctional action of Bcl-2 probably serves to prevent the activation of the ced-3/caspase-3 subfamily in response to apoptotic stimuli and thus to suppress the death effector machinery (Boulakia et al., 1996; Chinnaiyan et al., 1996; Monney et al., 1996). For its activity Bcl-2 requires four protein domains (BH1–4) that are highly homologous between members of the Bcl-2 family. Among them is a 30 amino acid stretch at the N-terminus of Bcl-2, called BH4 (Borner et al., 1994). There are many examples of viruses that kill cells apoptotically. In most cases, apoptosis is a defence mechanism beneficial for the host because it curtails the infectious cycle and prevents neighbouring cells from being infected with progeny virions (Vaux et al., 1994). However, pathological situations of viral infections exist. At one extreme, the host defence system is circumvented by the presence of anti-apoptotic proteins that are either endogenous to host cells (for example Bcl-2; Levine et al., 1993) or brought into the cells by viruses [viral latent membrane protein-1 (LMP-1) of Epstein–Barr virus, E1B of adenovirus; Hendersen et al., 1991; Rao et al., 1992]. In these cases, host cells survive viral infections and persistently produce progeny virions (viral persistence). At the other extreme, viruses provoke pathological lesions because they benefit from the anti-apoptotic properties of the host cells for reproduction but then kill these cells by overcoming their survival potential at a later stage (Ubol et al., 1994). Our particular interest is in unveiling the molecular mechanisms that govern the survival and death of cells infected by the alphaviruses Sindbis (SIN) and Semliki Forest virus (SFV). Alphaviruses are RNA viruses that can be severe pathogens for a broad range of mammals, including humans (reviewed in Strauss and Strauss, 1994). Following endocytotic uptake by the host cell the viral RNA is delivered to the cytoplasm where first non-structural and then structural proteins are synthesized. The capsid protein is a serine protease that cleaves itself from the nascent structural polyprotein and contributes to the dramatic shut-off of host cell protein synthesis early after infection (reviewed in Strauss and Strauss, 1994; Favre et al., 1996). This allows the production of high titres of progeny virions. Depending on cell type and viral strain, the host cell undergoes apoptosis or develops persistent infection (Levine et al., 1991). While apoptosis involves the activation of caspase-3 and PARP cleavage by so far unknown viral factors (Ubol et al., 1996), viral persistence appears to be due to the overexpression of Bcl-2 or other survival factors that block these activations (Levine et al., 1993). During the study of the molecular mechanisms underlying viral persistence in Bcl-2-overexpressing cells, we discovered that alphaviruses can actively break this persistence by inducing the cleavage and inactivation of Bcl-2 in a caspase-dependent manner. To our knowledge this is the first time that an apoptotic stimulus such as alphaviruses is shown to exploit caspase proteases to destroy the death-protective action of a survival factor. Results Cells overexpressing Bcl-2 are rapidly killed by infection with SIN or SFV The infection of subconfluent rat 6 (R6) embryo fibroblasts with SFV [multiplicity of infection (m.o.i.) 30] leads to a time-dependent loss of cell adherence (Figure 1A and B) and viability (Figure 2A). To examine whether viability loss could be blocked by the survival factor Bcl-2, we overexpressed this protein in R6 cells (R6-Bcl-2). Unexpectedly, R6-Bcl-2 cells were similarly sensitive to killing by SFV as control counterparts (Figure 1C and D and Figure 2A). This was also true for vector control and Bcl-2-overexpressing U937 monocytes and L929 fibroblasts as well as for the respective pairs of R6 cells infected with SIN (Figure 2). To ensure that Bcl-2 was functional as a survival factor in R6 cells, control and Bcl-2-overexpressing cells were treated with 50 mM NH4Cl. This agent provoked a loss of control R6 cell viability that was markedly delayed by Bcl-2 overexpression (Figure 2C). Similar death-protection by Bcl-2 has been reported in R6, U937 and L929 cells exposed to numerous other agents that induce apoptosis (Borner, 1996; Monney et al., 1996; Olivier et al., 1997). Thus, in spite of being capable of conferring cell death resistance to other stimuli, Bcl-2 did not provide significant protection from death induced by alphavirus infection. Figure 1.Phase-contrast microscopy of R6 and R6-Bcl-2 cells following infection with SFV at a m.o.i. of 30. (A) Mock-infected R6 control cells; (B) SFV-infected R6 control cells; (C) mock-infected R6-Bcl-2 cells; (D) SFV-infected R6-Bcl-2 cells; (E) mock-infected R6-Bcl-2 cells treated with Z-VAD; (F) SFV-infected R6-Bcl-2 cells treated with Z-VAD. All pictures taken at 48 h post-infection. Download figure Download PowerPoint Figure 2.Cell viability determined by trypan blue exclusion of (A) SFV-infected R6 control and Bcl-2 cells, (B) SIN-infected R6 control and Bcl-2 cells, (C) NH4Cl-treated R6 control and Bcl-2 cells, and SFV-infected U937 (D) and L929 (E) control and Bcl-2 cells. Error bars indicate the SEM. Download figure Download PowerPoint The death of control and Bcl-2-overexpressing cells induced by SFV exhibits the characteristic hallmarks of apoptosis To determine the kind of cell death induced by alphaviruses, we analysed the genomic DNA of control R6 and R6-Bcl-2 cells infected with 30 m.o.i. of SFV. As shown in Figure 3, both cell types exhibited a marked, time-dependent cleavage of their DNA into nucleosome-sized fragments, a hallmark of apoptosis (Duvall and Wyllie, 1986). This fragmentation was initially delayed by 4 h in R6-Bcl-2 cells as compared with control R6 cells but occurred as efficiently in both cell types at later time points of infection (Figure 3A and B). Again, the inability of Bcl-2 to prevent DNA fragmentation was a peculiarity of viral infection, since Bcl-2 potently interfered with the degradation when induced by 50 mM NH4Cl (Figure 3C) or other apoptotic agents (Borner, 1996; Monney et al., 1996; Olivier et al., 1997). Figure 3.DNA fragmentation in response to SFV infection. R6 control cells (Ctrl, A) and R6-Bcl-2 cells (Bcl-2, B) were infected with SFV and DNA fragmentation was determined by 1% agarose gel electrophoresis at serial time points after infection as described in Materials and methods. (C) DNA laddering of R6 and R6-Bcl-2 cells stressed with 50 mM NH4Cl for 48 h. m, mock-infected. Download figure Download PowerPoint Infection of SFV results in caspase-3 activation and PARP cleavage that cannot be inhibited by Bcl-2 overexpression It has been reported previously that apoptosis induced by viral infection is preceded by the activation of the death protease caspase-3 and cleavage of its major substrate PARP (Ubol et al., 1996). Moreover, Bcl-2 is known to prevent caspase-3 activation in response to apoptotic stimuli (Boulakia et al., 1996; Chinnaiyan et al., 1996; Monney et al., 1996). We therefore examined whether SFV infection triggered caspase-3 activation in our cells and whether Bcl-2 was somehow defective in interfering with the caspase activation process in this particular case. As shown in Figure 4A, infection of control R6 cells with SFV leads to a time-dependent cleavage of the 32 kDa pro-caspase-3 zymogen into the 17 kDa active protease. Similar kinetics of activation were noted upon SFV infection of control U937 cells (Figure 4B). In addition, simultaneously with caspase-3 maturation, the latter cells displayed a cleavage of the 116 kDa caspase-3 substrate PARP into the 85 kDa fragment (Figure 4B), typically seen in apoptotic cells (Lazebnik et al., 1994; Orth et al., 1996). As found previously for DNA fragmentation, R6-Bcl-2 cells exhibited a delay in caspase-3 activation by 4 h as compared with the respective vector control cells (compare Figure 3A and B and Figure 4). Bcl-2 also delayed caspase-3 activation and PARP cleavage in U937 cells (Figure 4B). However, neither of these processes could be prevented by Bcl-2 overexpression (Figure 4). By contrast, in both R6 and U937 cells, Bcl-2 markedly intervened with caspase-3 activation and PARP cleavage induced by NH4Cl (Figure 4A and unpublished data) and other apoptotic agents (Monney et al., 1996 and unpublished data). These results indicate that although Bcl-2 is functional in preventing caspase-3 activation in response to various apoptotic stimuli, it is defective in fully exerting this activity in cells infected with alphaviruses. Figure 4.Time course of the activation of caspase-3 and cleavage of PARP following infection with SFV. (A) Anti-caspase-3 immunoblots from total extracts of R6 control (Ctrl) and R6-Bcl-2 (Bcl-2) cells. Caspase-3 activation was also examined by stressing R6 control and Bcl-2 cells with 50 mM NH4Cl for 48 h. (B) Anti-caspase-3 and anti-PARP immunoblots from total extracts of U937 control (Ctrl) and Bcl-2 (Bcl-2) cells. m, mock-infected. Download figure Download PowerPoint Apoptosis induced by SFV and SIN in Bcl-2-overexpressing cells is preceded by proteolytic cleavage of Bcl-2 To identify the deficiency in the death-protective action of Bcl-2 in alphavirus-infected cells we monitored the presence and/or integrity of the Bcl-2 protein by Western blot analysis. Bcl-2 is highly expressed as a 26 kDa protein in both R6-Bcl-2 and U937-Bcl-2 cells (Figure 5). Infection of these cells with SFV led to a time-dependent cleavage of the 26 kDa Bcl-2 resulting in a 23 kDa protein (Figure 5A and D). The beginning of the cleavage coincided approximately with the detection of caspase-3 activation and PARP cleavage (16–24 h post-infection) and preceded morphological signs of apoptosis. Similar results were obtained with R6-Bcl-2 infected with SIN (Figure 5C) and L929-Bcl-2 infected with SFV (unpublished data), indicating that the cleavage of Bcl-2 was neither host cell- nor virus-type specific. Importantly, Bcl-2 was not cleaved at any time point following exposure of Bcl-2-overexpressing R6, U937 or L929 cells to NH4Cl (unpublished data) or other apoptotic agents (Borner, 1996; Monney et al., 1996; Olivier et al., 1997). These findings indicate that infection of eukaryotic cells with alphaviruses, but no other as yet known apoptotic stimuli, activates a protease that cleaves Bcl-2 into a 23 kDa product. Figure 5.Time course of the cleavage of Bcl-2 following infection with SFV and SIN, respectively, revealed by immunoblots of total cellular extracts. (A) SFV-infected R6 and R6-Bcl-2 cells using an antibody directed against the N-terminal amino acid residues 41–54 of Bcl-2 (27-6). (B) SFV-infected R6 and R6-Bcl-2 cells using an antibody directed against the N-terminal amino acid residues 20–34 of Bcl-2 (Ab-2). (C) SIN-infected R6 and R6-Bcl-2 cells using the 27-6 antibody. (D) SFV-infected U937 and U937-Bcl-2 cells using the 27-6 antibody. Download figure Download PowerPoint Cleavage of Bcl-2 occurs at membranes and also affects the heterodimeric Bcl-2 partner Bax Previous subcellular and immunofluorescence analyses have shown that Bcl-2 mainly localizes to the ER/nuclear membranes in R6-Bcl-2 cells (Krajewski et al., 1993 and unpublished data). Consistent with this notion, the 26 kDa Bcl-2 was detected in a nuclear cell extract from which it could be entirely extracted with Nonidet P-40 (NP-40) (Figure 6). This was also the case for the 23 kDa proteolytic fragment of Bcl-2 in SFV-infected cells (Figure 6), indicating that both wild-type and cleaved Bcl-2 were integral membrane proteins on nuclear membranes and that the protease in question probably acted on these membranes. Immunofluorescence analysis of infected R6-Bcl-2 cells confirmed that Bcl-2 did not alter its nuclear/ER membrane localization upon cleavage (unpublished data). Figure 6.The truncated 23 kDa Bcl-2 remains membrane bound. The nuclear pellets obtained 48 h post-infection of SFV-infected R6-Bcl-2 cells were treated (+) or not (−) with NP-40 (see Materials and methods) and the supernatants (sup) and pellets (pellet) obtained after centrifugation were subjected to anti-Bcl-2 (27-6) immunoblot analysis. Download figure Download PowerPoint Bax is a pro-apoptotic Bcl-2 homologue that heterodimerizes with Bcl-2. It can be co-immunoprecipitated with Bcl-2 from cell extracts (Oltvai et al., 1993) and co-localizes with Bcl-2 on mitochondria (Zha et al., 1996; Rosse et al., 1998), ER and nuclei in intact cells (our unpublished data). In agreement with the notion that a virus-induced protease acted on Bcl-2, endogenous 21 kDa Bax was found to be cleaved into a 18 kDa fragment with similar kinetics to that of Bcl-2 following infection of R6-Bcl-2 cells with SFV (Figure 7A and B). A similar cleavage of Bax has recently been reported in lymphatic cells following treatment with cytotoxic agents (Thomas et al., 1996). Cleavage of Bax was also noted in SFV-infected L929-Bcl-2 (our unpublished data) and SIN-infected R6-Bcl-2 cells (Figure 7C), but not in R6-Bcl-2 cells treated with NH4Cl (unpublished data) or other apoptotic agents (Borner, 1996; Monney et al., 1996; Olivier et al., 1997). Since Bax cleavage occurred as efficiently in infected vector control and Bcl-2 overexpressing cells (Figure 7), the responsible protease was not attracted to Bax because of Bcl-2 overexpression but by an as yet unknown alphavirus-induced mechanism. Importantly, alphavirus-induced proteolytic cleavage of both Bcl-2 and Bax appeared to be specific, because there was no bulk degradation of total protein in response to virus infection as judged from protein analysis on SDS–PAGE (unpublished data). Figure 7.Time course of the cleavage of Bax following infection with SFV and SIN, revealed by immunoblots of total cellular extracts. (A) SFV-infected R6 and R6-Bcl-2 cells using an antibody directed against amino acid residues 43–61 of Bax (Pharmingen). (B) SFV-infected R6 and R6-Bcl-2 cells using an antibody directed against the N-terminal amino acid residues 1–21 of Bax (UBI). (C) SIN-infected R6 and R6-Bcl-2 cells using the antibody UBI. Download figure Download PowerPoint Both Bcl-2 and Bax are cleaved at their N-termini during virus-induced apoptosis Next, we determined the cleavage sites of Bcl-2 and Bax by antibody epitope mapping. Both Bcl-2 and Bax have been shown to insert into membranes via their C-terminal, hydrophobic amino acid sequence. Because the cleaved 23 kDa Bcl-2 (Figure 6) and 18 kDa Bax (unpublished data) were still membrane-bound, we assumed that cleavage occurred at their N-termini. We therefore searched for antibodies that reacted with different epitopes in the N-terminal parts of Bcl-2 and Bax. As expected, whereas the polyclonal anti-Bcl-2 antibody 27-6 directed against residues 41–54 of Bcl-2 detected the proteolytic 23 kDa fragment (Figure 5A, C and D), no reactivity of this fragment was seen with the anti-Bcl-2 antibody Ab-2, recognizing amino acids 20–34 (Figure 5B). This indicates that the 23 kDa Bcl-2 was devoid of the N-terminus including the BH4 domain. As shown before in neurones (Borner et al., 1994), a Bcl-2 mutant lacking the BH4 was incapable of protecting R6 cells from apoptosis induced by staurosporine (Figure 8), suggesting that the 23 kDa cleaved Bcl-2 is most likely inactive as a cell survival factor in SFV- and SIN-infected R6-Bcl-2 cells. As with Bcl-2, Bax was cleaved at its N-terminus in both control R6 and R6-Bcl-2 cells infected with SFV. While an antibody reactive against residues 43–61 of Bax detected the 18 kDa Bax fragment (Figure 7A), this was not the case with an antibody towards the first 21 amino acids of the N-terminus (Figure 7B and C). Since it is not yet known whether endogenous Bax plays an active role in toxin- or virus-induced apoptosis, we cannot determine whether the 18 kDa Bax fragment is an active or inactive species. Because this fragment has retained the BH3 region known to be crucial for the pro-apoptotic activity of Bax when overexpressed (Chittenden et al., 1995), it is possible that cleavage of Bax in virus-infected cells does not abrogate its potential cytotoxic activity. Figure 8.Time course of the viability of R6, R6-Bcl-2(wt) and mutant R6-Bcl-2ΔBH4 cells, as determined by trypan blue exclusion. Cells were treated with 1 μM staurosporine. Download figure Download PowerPoint Alphavirus-induced cleavage of Bcl-2 and apoptosis are prevented by the caspase inhibitor Z-VAD Calculation of the molecular mass of the proteolytic fragments and antibody epitope mapping revealed that a protein domain between approximately amino acids 30 and 40 of mouse Bcl-2 served as cleavage site for proteolysis in response to SFV and SIN infection. The sequence YEWDAGDADAAPL of this region contains three putative target sites for caspases, one for caspase-1 (YEWD31↓A), one for caspase-3 (DAGD34↓A) and one for another caspase (GDAD36↓A) (Figure 9). Since at least caspase-3 (but also maybe other caspases) was activated in response to SFV and SIN infection (Figure 4), we tested whether Bcl-2 cleavage was caspase-mediated and could be prevented by caspase inhibition. As anticipated, treatment of R6-Bcl-2 cells with 100 μM benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD) 12 h before and during infection with SFV (see Materials and methods) completely blocked Bcl-2 cleavage (Figure 10C). Bcl-2 appeared to be functional in these cells because activation of caspase-3 was barely detected following SFV infection (Figure 10B) and the cells were partially protected from virus-induced apoptosis (Figure 10, compare Figure 1D and F). However, since Z-VAD also delayed caspase-3 activation and apoptosis in control R6 cells (Figure 10B), it was impossible to distinguish whether the protective effect of Z-VAD was due to the lack of caspase-3 activation or to the maintenance of functional Bcl-2. This is in agreement with previous reports showing that the death-protective effects of Bcl-2 and Z-VAD are often non-additive because they probably target similar molecules (Boulakia et al., 1996; Chinnaiyan et al., 1996; Monney et al., 1996). Importantly, the effect of Z-VAD on R6 cells was not due to an interference with viral infection, replication and/or progeny production as similar amounts of viral structural proteins and titres of infectious progeny virions were made in the presence or absence of Z-VAD after SFV infection (unpublished data). These findings indicate that R6-Bcl-2 cells may be susceptible to SFV- or SIN-induced apoptosis because the viruses have developed ways to activate a Z-VAD-sensitive caspase(s) that directly or indirectly cleave(s) and inactivate(s) Bcl-2. Figure 9.Putative caspase cleavage sites in Bcl-2 and Bax showing the epitopes of all antibodies described in this study. Download figure Download PowerPoint Figure 10.Effect of Z-VAD on SFV-induced apoptosis and Bcl-2 cleavage in R6 and R6-Bcl-2 cells. (A) R6 and R6-Bcl-2 cells were either incubated or not with 100 μM Z-VAD for 12 h prior to and during infection with SFV and viability was determined by trypan blue exclusion. (B) Anti-caspase-3 immunoblots of total extracts of SFV-infected R6 control and Bcl-2 cells treated (+) or not (−) with Z-VAD. (C) Anti-Bcl-2 (27-6) immunoblots of total extracts of SFV-infected R6 control and Bcl-2 cells treated with Z-VAD. m, mock-infected. Download figure Download PowerPoint Mutation of Asp31, Asp34 and Asp36 to glutamic acid in the BH4 region prevents cleavage of Bcl-2 and abrogates apoptosis To confirm that alphavirus infection inactivates Bcl-2 by caspase cleavage within the amino acid region YEWDAGDADAAPL, we changed the caspase target residues Asp31, Asp34 and Asp36 to glutamic acid by site-directed mutagenesis. The mutant Bcl-2 (D31E, D34E, D36E) protein had the same molecular mass (26 kDa) and could be stably overexpressed in R6 cells at similar levels to wild-type Bcl-2 in the R6-Bcl-2 cell line (Figures 5 and 11). However, in contrast to R6-Bcl-2 cells, R6 clones overexpressing the mutant Bcl-2 were resistant to alphavirus-induced apoptosis and did not exhibit cleavage of the mutant Bcl-2 protein at any time following alphavirus infection (Figures 1, 5 and 11). These data clearly show that alphaviruses are capable of killing Bcl-2-overexpressing host cells by a caspase-mediated proteolytic inactivation of the survival factor Bcl-2. Figure 11.Effect of caspase cleavage site-mutated Bcl-2 on alphavirus-induced apoptosis and Bcl-2 cleavage. Following infection with SFV (30 m.o.i.), R6 cells overexpressing the mutant Bcl-2 (D31E, D34E, D36E) were tested for cell viability by trypan blue exclusion (A) and Bcl-2 cleavage by anti-Bcl-2 immunoblot analysis of total cellular extracts (B). The epitope for the anti-Bcl-2 antibody used (27-6) was entirely maintained in the Bcl-2 mutant, i.e. cleaved Bcl-2 should be detected by the antibody. Download figure Download PowerPoint Discussion The present study shows that the alphaviruses SFV and SIN can kill three different host cell lines by apoptosis in spite of the fact that the cells overexpress Bcl-2. This is because the viruses activate a caspase-dependent pathway to cleave Bcl-2 into a protein which is devoid of its N-terminus and thus inactive in conferring cell death protection. Consistent with this notion, cells overexpressing a Bcl-2 variant mutated at three putative caspase cleavage sites do not exhibit Bcl-2 cleavage and are protected against al
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