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Originally published In Press as doi:10.1074/jbc.M510662200 on April 11, 2006

J. Biol. Chem., Vol. 281, Issue 24, 16296-16304, June 16, 2006
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Coxsackievirus Protein 2BC Blocks Host Cell Apoptosis by Inhibiting Caspase-3*

Michael A. Salako1, Michael J. Carter, and George E. N. Kass2

From the School of Biomedical and Molecular Sciences, University of Surrey, Guildford, GU2 7XH, United Kingdom

Received for publication, September 29, 2005 , and in revised form, March 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Virus infection may induce host cell death by apoptosis, but some DNA viruses are capable of preventing this process. RNA viruses were thought not to display anti-apoptotic activities, as their spread appears to benefit from a rapid induction of cell death. Here, we report an antiapoptotic activity in the Picornavirus Coxsackievirus B4 (CVB4). CVB4 infection of HeLa cells induced negligible apoptosis over a period of 10 h. However, infected cells developed resistance to drug-induced apoptosis using staurosporine and actinomycin D and to death receptor-induced apoptosis using tumor necrosis factor-related apoptosis-inducing ligand. Despite this resistance, the apoptotic machinery was nonetheless fully activated in these drug-treated infected cells because the levels of pro-caspase-3 processing to its active form were similar to control cells. However, the DEVDase (Asp-Glu-Val-Asp protease) activity of the processed caspase was significantly inhibited in the virus-infected staurosporine-treated cells compared with drug treatment alone. Likewise, extracts of CVB4-infected cells suppressed recombinant caspase-3 activity in vitro. Immunoprecipitation of activated caspase-3 from radiolabeled virus-infected cells revealed the co-precipitation of a 48-kDa protein that was tentatively identified as viral protein 2BC. Recombinant caspase-3 was found to co-precipitate with virus protein 2BC. Finally, when protein 2BC was expressed in HeLa cells, both staurosporine-induced apoptosis and in vitro caspase-3 DEVDase activity were significantly reduced. Taken together these data imply that CVB4 infection suppresses apoptosis through virus protein 2BC associating with caspase-3 and inhibiting its function. Thus, 2BC is the first reported RNA virus inhibitor of apoptosis protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is a mechanism used by metazoan organisms including mammals to eliminate cells that are no longer required or have become potentially dangerous to the host as a result of mutation, failure of elimination, or infection by a virus (1, 2). Two main pathways have been identified that activate the apoptotic machinery (3–7). The first pathway operates through so-called death receptors. Members of this family of transmembrane receptors include tumor necrosis factor (TNF) receptor I, CD95 (also known as Fas or APO-1), and TNF-related apoptosis-inducing ligand (TRAIL)3 receptors; activation by their corresponding physiological ligands induces the formation of a complex referred to as the death-inducing signaling complex. Although different in composition between the different death receptors, the death-inducing signaling complex includes adaptor proteins such as FADD or TRADD and cysteine proteases called caspases such as caspase-8. Caspases belong to a family of cysteine proteases comprising at least 12 isoforms in human cells and are located primarily in the cytosol where they exist as inactive zymogens (procaspases), which require proteolytic cleavage for activation. Caspases cleave their target proteins at residues next to Asp, and those involved in apoptosis are organized in a hierarchical structure composed of initiator and executioner caspases that provides great potential for signal amplification (8). The aggregation of the death receptor during activation provides a molecular platform allowing the death-inducing signaling complex to bring initiator procaspases-8 into close proximity and, thus, promote their dimerization and subsequent activation through autocatalytic proteolysis (9). Active caspase-8 proteolytically processes and activates the executioner procaspases-3 and -7 that in turn are thought to be responsible for the apoptotic demise of the cell. This apoptosis pathway controlled by the death receptor is often referred to as the extrinsic pathway and is responsible for the elimination of unwanted cells during development, immune system development, and immunosurveillance.

The second pathway activating apoptosis in mammalian cells is called the intrinsic pathway and involves the mitochondria (3, 4). Stress signals emerging from exposure to cytotoxic drugs, irradiation, genomic damage, but also cross-talk from the extrinsic pathway leads to an increase in mitochondrial outer membrane permeability that is sufficient to allow the release of cytochrome c. This then assembles with Apaf-1 to form a large proapoptotic platform structure known as the apoptosome that also includes the initiator caspase-9. Again, close proximity of several pro-caspase-9 molecules promotes their activation by dimerization and autocatalytic processing (9), and once more this is followed by downstream proteolytic activation of executioner caspases-3 and -7.

Both these routes of induction are tightly regulated by a remarkable array of cellular regulatory proteins such as the inhibitors of apoptosis proteins (IAPs) (10), FLIPs (11), and Bcl-2 family members (12) that control both the activation and the activity of various caspases. Interestingly, many of the cellular regulators of apoptosis were discovered to have structural or functional viral homologues (13–15).

However, in contrast to large and slow replicating DNA viruses, where anti-apoptotic viral proteins are thought to benefit the invading organism by preventing premature host cell death, RNA viruses replicate much more efficiently, and their spread appears to benefit from a rapid induction of cell death. The genomes of RNA viruses were, therefore, thought not to encode proteins with anti-apoptotic activity. This assumption, however, seems likely to be incorrect at least in the case of some enteroviruses. Enteroviruses are nonenveloped cytolytic positive-stranded RNA viruses of the Picornaviridae family. They are capable of rapid and cytopathic replication. The cytopathic effects (CPE) induced share characteristics with necrosis (16, 17). However, in some cell types or environments or when virus replication is inhibited, the virus-triggered cell destruction adopts the apoptotic path, as extensively documented for poliovirus (18–22) and Coxsackievirus B (23–27). The molecular mechanism responsible for the switch between the two forms of cell death is still poorly understood, but poliovirus has been shown to induce an antiapoptotic activity under conditions of permissive replication and infection that would favor necrosis-like CPE over cell death by apoptosis. Under these conditions the virus opposes the development of actinomycin D-, cycloheximide-, or sorbitol-induced apoptosis (19, 28). The identity of the viral protein(s) responsible for this effect and the mechanisms by which they might act remain unknown although the cellular target has been suggested to be located downstream of cytochrome c release (29).

The aim of this work was to investigate whether the closely related Enterovirus, Coxsackievirus B4 (CVB4), is also capable of inducing an antiapoptotic activity in human cells and to define the mechanism of any such activity. Generalized CVB infection is very common, particularly in young children. Although usually silent, it has been associated with several diseases, including severe acute human myocarditis, dilated myocardiopathy, chronic pancreatitis, and aseptic meningitis and encephalitis (25, 30–32). Like other enteroviruses, the CVB4 genome is single positive-stranded RNA of 7.5 kilobases containing one large open reading frame that is translated into a precursor polyprotein that is subsequently processed by proteases to release the functional virus proteins (33). The polyprotein is divided into three functional regions; four capsid proteins, VP1-VP4, are encoded within the P1 region of the genome, whereas the nonstructural proteins involved in replication are encoded within the P2 and P3 regions. Processing of the P2 and P3 regions generates the mature products termed proteins 2Apro, 2B, 2C, 3B, 3A, 3Cpro, and 3Dpol and also some partially processed forms, 2BC, 3AB, and 3CDpro, that are believed to have distinct functions.

Here we report that CVB4 infection of HeLa cells confers resistance to induction of apoptosis by a variety of agents, including staurosporine, actinomycin D, and TRAIL. The protection from apoptosis was not mediated by a failure of apoptotic machinery activation but was caused by a binding of the 2BC protein to caspase-3 that resulted in the inhibition of the activity of this execution caspase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—Staurosporine and soluble human recombinant TRAIL (SuperKillerTRAIL) were purchased from Alexis Corp. Ltd (Nottingham, UK), and actinomycin D and protein A-Sepharose were obtained from Sigma-Aldrich. Complete Protease Inhibitor Mixture Tablets and T7 Cap Scribe were purchased from Roche Applied Science. N-Acetyl-Asp-Glu-Val-Asp-aminofluoromethylcoumarin was obtained from Bachem (Bubendorf, Switzerland). An annexin V-FITC (fluorescein isothiocyanate) kit was from Merck, and 4',6-diamidino-2-phenylindole (DAPI) was from Molecular Probes (Leiden, NL). Phycoerythrin-conjugated anti-caspase-3 (active form) (clone C92–605) and polyclonal anti-active caspase-3 (catalog #557035) were purchased from BD Pharmingen. All secondary horseradish peroxidase-conjugated antibodies were bought from Dako Ltd (Cambridge, UK). All restriction enzymes, nuclease-free water, pGEM-3Z, and luciferase DNA template containing a T7 promoter were from Promega (Southampton, UK), and TransMessenger transfection reagent was purchased from Qiagen (Crawley, UK). L-[35S]Methionine (>37 TBq/mmol) was obtained from Amersham Biosciences. All cell culture media and supplements were bought from Invitrogen. All other reagents were of analytical grade. Recombinant active human caspase-3 was a gift from Dr. Don Nicholson (Merck Frosst).

Cell Culture and Infection with CVB4—HeLa and buffalo green monkey kidney cell lines were obtained from the European Collection of Animal Cell Cultures, Porton Down, UK. Both cell lines were grown at 37 °C in minimal essential medium (MEM) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), and nonessential amino acids in an humidified incubator with 5% carbon dioxide, 95% air. CVB4 was propagated in buffalo green monkey kidney cells and stored at –80 °C. The virus titer was routinely determined by plaque assay in buffalo green monkey kidney cells. Subconfluent cultures of HeLa cells were infected with CBV4 at a multiplicity of infection of 15 in growth medium containing 2% (v/v) heat-inactivated fetal bovine serum by allowing the virus to adsorb for 1 h at 37 °C in a humidified atmosphere of 5% CO2 with gentle rocking at 10-min intervals. The inoculum was then removed, fresh growth medium containing 2% (v/v) fetal bovine serum was added, and the cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 to allow the virus to grow as required. Mock infection of cultures was carried out in the same fashion but in the absence of virus.

Analysis of Apoptosis and Cell Death—Changes in nuclear morphology were assessed by fixing the cells grown on poly-L-lysine-coated glass coverslips in 4% (v/v) formaldehyde at room temperature for 30 min. The fixed cells were stained with DAPI (10 µg/ml), and nuclear morphology was analyzed by fluorescence microscopy. Apoptosis was quantitated by counting a minimum of 200 cells. Analysis of phosphatidylserine exposure during apoptosis was performed by flow cytometric analysis of annexin V-fluorescein isothiocyanate/propidium iodide-stained cells (34) using a Beckman Coulter Epics XL flow cytometer (Beckman Coulter, Fullerton, CA) (argon laser, excitation wavelength, 488 nm). A minimum of 10,000 events was acquired in list mode while gating the forward and side scatters to exclude cell debris and analyzed in FL-1 and FL-3.

Analysis of Pro-caspase Processing and Caspase Activity—The processing of caspase-3 was followed by flow cytometric detection of HeLa cells immunostained for active caspase-3 using the C92–605 monoclonal antibody that specifically recognizes the active form of caspase-3 but not its pro-form as previously reported and validated against Western blot analysis and in situ caspase-3 activity (35, 36). In brief, the cells were harvested by trypsin-EDTA dissociation, washed twice in ice-cold phosphate-buffered saline, fixed, and then stained with a phycoerythrin conjugated anti-caspase-3 antibody (1:20) according to the manufacturer's instructions (BD Pharmingen). Cells were then analyzed using a Beckman Coulter Epics XL flow cytometer (Beckman Coulter). A minimum of 10,000 events was acquired in list mode while gating the forward and side scatters to exclude cell debris and analyzed in FL-2 for neo-epitope appearance. For the assessment of caspase-3/-7 (DEVDase) activity, the cells were seeded in 60-mm dishes and infected or treated with apoptosis-inducing compounds as described above. At the indicated time points, the cells were harvested, resuspended in caspase lysis buffer (40 mM sucrose, 50 mM NaCl, 5 mM EGTA, 2 mM MgCl2, 10 mM HEPES, pH 7.0), and freeze-thawed 3 times, and the supernatant was collected (15,000 x g, 30 min). Eighty µg of sample protein was assayed for DEVDase activity with 20 µM N-acetyl-Asp-Glu-Val-Asp-aminofluoromethylcoumarin ({lambda}ex = 355 nm; {lambda}em = 538 nm) in caspase assay buffer (100 mM HEPES, 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol, pH 7.25) (37). In some experiments, cell extracts were prepared from mock- or virus-infected or transfected cells and combined with recombinant active human caspase-3 (0.3 µg/ml) to investigate the effect of viral proteins on caspase-3 activity.

Immunoprecipitation Assays—HeLa cells grown in 35-mm dishes were infected with CVB4 at a multiplicity of infection of 15 in growth medium containing 2% (v/v) heat-inactivated fetal bovine serum. After 8 h, the medium was removed, the cells were washed twice with Met-free minimal essential medium (MEM), and the cells were further incubated for 30 min at 37 °C with Met-free MEM before supplementation and incubation with L-[35S]methionine (1.1 MBq/ml) for a further 90 min. Under these conditions, the shut-down of cellular protein synthesis by CVB4 infection (38) causes viral proteins to be the main proteins that are radiolabeled. After washing the cells twice in ice-cold radioimmune precipitation assay (RIPA) buffer (150 mM NaCl, 10 mM Tris-Cl, pH 7.4, containing complete protease inhibitor mixture (1 tablet/10 ml)), they were scraped into RIPA buffer supplemented with 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.1% SDS, the cells were vortexed, and supernatants were prepared by centrifugation (15,000 x g for 30 min at 4 °C). Immunoprecipitation was carried out by incubating supernatant with antibody (1 µl/100 µl of supernatant) for 45 min on ice followed by the addition of protein A-Sepharose and a further 1-h incubation on ice with gentle mixing. The Sepharose beads were removed by centrifugation, washed 3 times with radioimmune precipitation assay buffer supplemented with 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, and 0.1% SDS before boiling for 5 min in SDS-PAGE loading buffer, and the immunoprecipitated proteins were resolved using 12.5 and 15% gels. The viral proteins were visualized by autoradiography on Hyperfilm MP preflashed with a Sensitize Preflash Unit (Amersham Biosciences). Western blot analysis of the immunoprecipitated proteins using an anti-caspase-3 antibody confirmed that caspase-3 was correctly immunoprecipitated in our experiments.


Figure 1
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  		FIGURE 1.
Coxsackievirus B4 infection suppresses staurosporine-induced apoptosis. HeLa cells were infected with CVB4 at a multiplicity of infection of 15 for 6 h or mock-infected for 6 h before exposure to staurosporine (STS, 0.5 µM) or carrier solvent (Me2SO) for a further 4 h. A, flow cytometric analysis of apoptosis using annexin V (AV) binding and propidium iodide (PI) uptake. The cells were isolated, labeled, and analyzed as described under "Experimental Procedures." B, statistical analysis of the non-apoptotic (AV–/PI–), early apoptotic (AV+/PI–), and late apoptotic (AV+/PI+) population of cells. All data are the mean ± S.D. of 3–4 independent experiments and were analyzed for difference from mock-infected cells (a, p < 0.001) or from mock-infected cells treated with staurosporine (b, p < 0.001). C, temporal development of the antiapoptotic response to CVB4 infection. HeLa cells were infected with CVB4 at a multiplicity of infection of 15 before exposure to staurosporine (STS, 0.5 µM) or carrier solvent (Me2SO) for a further 4 h at the indicated time points or mock-infected for 6 h followed by a 4-h exposure to staurosporine. Each bar shows the % non-apoptotic cells (AV–/PI–)(black) or the combined % of early and late apoptotic cells (AV+/PI–plus AV+/PI+). D, CVB4 infection prevents the development of apoptotic nuclear morphology. HeLa cells were either mock-infected (a and c) or infected with CVB4 (b and d) and treated with staurosporine (c and d) or carrier solvent (a and b) as above before fixing and staining with DAPI.

 
Cloning of CVB4 Proteins 2BC and 2B and Ectopic Expression in HeLa Cells—The coding sequences for 2BC and 2B were amplified by reverse transcription-PCR and cloned into pGEM-3Z. The sequence was confirmed to be CVB4 2BC and 2B by sequencing. For the transfection and ectopic expression of the viral proteins, pGEM-3Z was linearized with SalI, extracted with phenol/chloroform, and precipitated. Copy RNA was generated using T7 Cap Scribe and purified by phenol/chloroform extraction followed by chloroform extraction and precipitated. The RNA pellet was dried and resuspended in nuclease-free water, and its integrity was confirmed by electrophoresis on a 1.2% agarose gel. The transfection was carried out using TransMessenger transfection reagent and 4 µg of RNA as described by the manufacturer (Qiagen).

Statistical Analysis of the Data—All data are given as means ± S.D. of at least three independent experiments. Comparison of treatments against controls was made using a paired Student's t test or ANOVA using Bonferroni's post-hoc test for multiple comparisons. The significance level chosen for the statistical analysis was p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Coxsackievirus B4 Infection of HeLa Cells Confers Resistance to Staurosporine-induced Apoptosis—Infection of HeLa cells with CVB4 did not induce any detectable signs of apoptosis over the first 10 h as demonstrated by a lack of phosphatidylserine exposure, absence of chromatin condensation, and maintained viability (Fig. 1). By this time, only few cells had rounded up as a result of the infection, whereas by 24 h, ~25% of the cells had rounded up and displayed altered nuclear morphology, but only half of which was typical of apoptosis (round and highly condensed), whereas the others had features of CPE (deformed and elongated nuclei with partial chromatin condensation) (data not shown) (18, 19, 29, 39). Measurement of annexin V binding showed that the number of apoptotic cells (AV+/PI–) at 24 h was around 5%, which was slightly lower than after 10 h of infection (8 ± 1%) (Fig. 1B), whereas the (secondary) necrotic population (AV+/PI+) had significantly increased. This confirms that CVB4 infection induced a marginal and transient apoptotic response that later in the infection was halted with necrotic-like CPE becoming the predominant form of cell death. As expected, when HeLa cells were treated with the apoptosis-inducing agent staurosporine (0.5 µM), a third of the cell population was clearly apoptotic by 4 h, as evidenced by a significant increase in the number of cells binding fluorescein isothiocyanate-labeled annexin V (Fig. 1, A and B) and a comparable increase in the number of cells with typical apoptotic nuclei, i.e. displaying condensed and fragmented chromatin (Fig. 1D and Table 1). However, when HeLa cells were infected with CVB4 for 6 h before treatment, there was a significant suppression in the extent of apoptosis induction by staurosporine. The number of cells with phosphatidylserine exposed was reduced from 34.4% ± 3.7 in mock-infected cells treated with staurosporine to 11.3% ± 2.7 in CVB4-infected cells exposed to the inducer of apoptosis (p < 0.001) (Fig. 1, A and B). Likewise, the loss of cell viability (as measured by propidium iodide uptake) was significantly suppressed (Fig. 1, A and B). CVB4 infection prevented the appearance of fragmented chromatin and apoptotic body formation after staurosporine treatment, reducing the population of cells with apoptotic nuclear morphology from 40 ± 2 to 17 ± 1% (p < 0.001) (Table 1). However, many nuclei showed an elongated and somewhat condensed distribution of the chromatin (Fig. 1D). Taken together, these data show that the Enterovirus CVB4, like poliovirus (19, 28), can generate an antiapoptotic state in human cells that becomes manifest after 6 h of infection (Fig. 1C).


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  		TABLE 1
Coxsackievirus B4 infection suppresses apoptosis in HeLa cells

Cells were mock-infected or infected with CVB4 for 6 h before being treated with staurosporine (0.5 µM), actinomycin D (0.5 µg/ml), TRAIL (40 ng/ml), or carrier solvent (Me2SO) for a further 4 h. At the end of the incubation period the cells were fixed, and the nuclei were stained with DAPI as described under "Experimental Procedures." At least 200 nuclei were counted and scored for apoptotic morphology (condensed and fragmented chromatin). The data are the means ± S.E. from four independent experiments.

 
The Antiapoptotic Effect of Coxsackievirus B4 Is Not Restricted to Staurosporine but Occurs also with Actinomycin D and TRAIL—CVB4-infected HeLa cells were treated with apoptotic inducers actinomycin D and TRAIL 6 h post-infection. Comparison with mock-infected cells showed that the degree of apoptosis induction by these agents was clearly suppressed. The numbers of both annexin V-positive cells (Fig. 2A) and also those showing fragmented and condensed nuclei (Fig. 2B) were significantly reduced in the infected cells compared with the controls (Table 1). Taken together, our results imply that CVB4 infection opposes both the mitochondrial and death receptor pathways of apoptosis. We did, however, observe one noticeable difference when we examined the transcription inhibitor actinomycin D. Although virus infection was able to reduce the number of apoptotic cells induced by this treatment as well as those above, in this case we did not see the preservation of cell viability. Instead, the decrease in the number of apoptotic cells was balanced by an equivalent increase in the number of propidium iodide-positive cells. This strongly suggests that in the case of actinomycin D, CVB4 infection has either caused a shift in the mode of cell death away from apoptosis and toward primary necrosis or has accelerated the transition from apoptosis to secondary necrosis such that at any time point the overall number of purely apoptotic cells appeared reduced.


Figure 2
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  		FIGURE 2.
Coxsackievirus B4 infection suppresses actinomycin D and TRAIL-induced apoptosis. HeLa cells were infected with CVB4 at a multiplicity of infection of 15 for 6 h or mock-infected for 6 h before exposure to actinomycin D (Act D, 0.5 µg/ml) or TRAIL (40 ng/ml) or carrier solvent (Me2SO) for a further 4 h. A, flow cytometric analysis of apoptosis using AV binding and PI uptake. The cells were isolated, labeled, and analyzed as described under "Experimental Procedures." B, CVB4 infection prevents the development of apoptotic nuclear morphology. HeLa cells were either mock-infected (a and c) or infected with CVB4 (b and d) and treated with actinomycin D (a and b) or TRAIL (c and d) before fixing and staining with DAPI.

 


Figure 3
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  		FIGURE 3.
Coxsackievirus B4 infection does not prevent caspase-3 proteolytic activation. HeLa cells were infected with CVB4 at a multiplicity of infection of 15 for 6 h or mock-infected for 6 h before exposure to inducers of apoptosis or carrier solvent (Me2SO) for a further 2 or 4 h. This was followed by flow cytometric analysis of caspase-3 using an antibody that specifically only recognizes the cleaved active p17/p20 fragments as described under "Experimental Procedures." A, histogram showing staurosporine (STS, 0.5 µM)-induced caspase-3 activation. The dashed lines represent mock-infected cells, whereas the continuous lines represent CVB4-infected cells. Top histogram, control 4 h; middle histogram, staurosporine 2 h; bottom histogram, staurosporine 4 h. B, time course of caspase-3 activation in mock-infected (black bars) and CVB4-infected cells (gray bars) exposed to staurosporine. All data are the mean ± S.D. of four independent experiments. C, histogram showing actinomycin D (0.5 µg/ml)-induced caspase-3 activation. The thin lines show solvent control-treated cells for 4 h (dashed lines, mock-infected cells; continuous lines, CVB4-infected cells); the thick lines show cells treated with actinomycin D for 4h (dashed lines, mock-infected cells; continuous lines, CVB4-infected cells). D, same as C but using TRAIL (40 ng/ml) as the apoptosis inducer.

 
Coxsackievirus B4 Does Not Prevent the Activation of the Apoptotic Machinery—To investigate the mechanism of this apparent CVB4-induced protection from apoptosis, we examined the processing of procaspase-3 to its active fragments. For this we used the C92–605 monoclonal antibody that specifically recognizes the active form of caspase-3 but not the pro form and allows the quantitative analysis of caspase-3 proteolytic activation (36). Given that activation of this execution caspase-3 occurs toward the end of the apoptotic cascade, a decrease in the activation of this caspase would reflect an upstream interference with the apoptotic machinery. However, Fig. 3 clearly shows that CVB4 infection did not suppress the formation of active caspase-3 after induction of apoptosis by staurosporine. Likewise, there was no significant decrease in the levels of active caspase-3 after actinomycin D treatment (36.0% ± 5.6 gated in mock-infected cells versus 32.2% ± 3.0 in CVB4-infected cells) or after TRAIL receptor activation (31.7% ± 3.0 gated in mock-infected cells versus 27.3% ± 3.4 in CVB4-infected cells). Therefore, our results demonstrate that the execution of apoptosis was prevented by CVB4 despite the apparently normal activation of the apoptotic machinery.

Detection of a Caspase-3 Inhibitory Activity in Coxsackievirus B4-infected Cells—A possible explanation for our findings would be that although caspase-3 is proteolytically cleaved, it is not enzymatically active. This hypothesis was tested by preparing cell extracts from the mock-infected and CVB4-infected HeLa cells exposed to staurosporine as above. As shown in Table 2, staurosporine produced a large increase in DEVDase activity in mock-infected cells. However, in cells infected with CVB4 for 10 h and treated with staurosporine, there was a 32% decrease in DEVDase activity as compared with the activity measured in the corresponding mock-infected cells. Thus, although caspase-3 was proteolytically cleaved to the same extent in both mock and virus-infected cells (Fig. 3), the caspase was not fully active (Table 2), suggesting that CVB4 induces a caspase-inhibitory activity in infected cells. To further confirm that the inhibition was due to a direct interference of caspase-3 activity and not to inhibition of its correct proteolytic activation or inhibition of a related caspase that can metabolize DEVD, such as caspase-7, we tested the capacity of virus-infected cell extracts to inhibit exogenously added recombinant caspase-3. Cell extracts prepared from mock- and CVB4-infected cells had very little intrinsic DEVDase activity (Table 2). Table 3 presents the activity detected after the addition of 0.3 µg/ml of recombinant caspase-3 and shows that the activity measured in the presence of extracts from CVB4-infected cells was substantially suppressed despite the addition of equal amounts of recombinant caspase-3. These findings clearly demonstrate that CVB4 generates a caspase-3 inhibitory activity that was at least partially responsible for the antiapoptotic effect reported here.


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  		TABLE 2
Coxsackievirus B4 infection suppresses caspase-3 activity in HeLa cells

Cells were mock-infected or infected with CVB4 for 6 h before being treated with staurosporine (0.5 µM) for 4 h. At the end of the incubation period cell lysates were prepared and tested for DEVDase activity as described under "Experimental Procedures." The data are the means ± S.E. from four independent experiments.

 


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  		TABLE 3
Coxsackievirus B4 infection leads to the expression of a protein that directly inhibits caspase-3 activity

Cells were mock-infected or infected with CVB4 for 10 h before lysis and preparation of post-mitochondrial supernatant. Recombinant human caspase-3 was subsequently added to equal amounts of cell extract protein, and its DEVDase activity was assayed at 37 °C as described under "Experimental Procedures." The data are the means ± S.D. from four independent experiments.

 
Coxsackievirus 2BC Is Responsible for the Inhibition of Caspase-3—The inhibition of caspase-3 activity could have resulted from a direct molecular interaction between the caspase and one or more viral proteins. To test this hypothesis, we performed immunoprecipitation experiments using cell extracts prepared under the following experimental conditions. HeLa cells were infected with CVB4 for 8 h and then incubated with methionine-free medium for 30 min before replacing this medium with methionine-free medium containing [35S]methionine for 90 min. Under these conditions, the synthesis of the host cell's own proteins is shut down, and virus proteins form the major product and consequently are labeled preferentially (38). Mock-infected cells were labeled and processed for lysate preparation similarly (Fig. 4A). Both lysates were then supplemented with unlabeled recombinant human caspase-3 (2.25 µg/100 µl) and incubated at 4 °C for 1 h. Finally, the caspase-3 was precipitated by the addition of an anti-caspase-3 antibody. Precipitated proteins were analyzed by SDS-PAGE and autoradiography. Under these circumstances the caspase-3 itself is not radiolabeled and will not be detected in the gel; however, any virus proteins that may co-precipitate would be detected. We observed a radiolabeled protein band with an apparent molecular mass of 48 kDa was co-precipitated from the infected cell lysates. This band was not precipitated from control cell lysates mixed with recombinant caspase nor from infected cells in the absence of caspase-3 antiserum. We tentatively identified this band as CVB4 protein 2BC (computed Mr 48,228) (33). Our findings, therefore, suggest that 2BC binds to caspase-3 and that this might be responsible for the inhibition of its activity. The ability of 2BC to bind to caspase-3 was confirmed in immunoprecipitation experiments where CVB4 proteins 2BC and 2B were synthesized and radiolabeled in vitro by transcription/translation using the cloned gene. Recombinant caspase-3 was then added, incubated as above, and immunoprecipitated with recombinant human active caspase-3 using an anti-caspase-3 antibody (Fig. 4B). The co-immunoprecipitation experiments also showed that the binding to caspase-3 was specific to 2BC protein and did not occur with 2B protein (data not shown). However, protein-protein interaction between caspase-3 and 2BC is not proof that 2BC inhibits the activity of the caspase. To address this issue we expressed 2BC and 2B in HeLa cells by transfection using mRNAs encoding for these proteins. We then assessed the ability of lysates prepared from these transfected cells to inhibit the activity of recombinant caspase-3 activity in the same manner as we had earlier analyzed infected cell lysates. As shown in Fig. 5A, lysates prepared from cells transfected with mRNA for 2BC caused a 50% inhibition of caspase-3 activity, whereas those transfected with mRNA for 2B had no effect.


Figure 4
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  		FIGURE 4.
Coxsackievirus B4 protein 2BC co-immunoprecipitates with caspase-3. A, HeLa cells were radiolabeled with [35S]methionine during mock- or CVB4 infection before cell lysate and immunoprecipitation of caspase-3 using a polyclonal anti-active caspase-3 antibody as described under "Experimental Procedures." The immunoprecipitated proteins were resolved by SDS-PAGE and visualized by autoradiography of the co-immunoprecipitated (viral) proteins. A band corresponding to 48 kDa is indicated with arrows. B, co-immunoprecipitation of 48 kDa 2BC protein with recombinant human active caspase-3. 35S-Labeled 2BC protein was produced in a TNT reaction and mixed with recombinant active human caspase-3 before immunoprecipitation. Co-immunoprecipitated proteins were visualized by autoradiography. The left lane shows the radiolabeled proteins produced in the TNT reaction that include 48-kDa 2BC and 15–16-kDa globin plus three additional byproducts of the TNT reaction. Ab, antibody.

 
Inhibition of Caspase-3 by 2BC Confers Protection against Staurosporine-induced Apoptosis—To test the hypothesis that the inhibition of caspase-3 by 2BC mediates the anti-apoptotic effects of CVB4 infection, we ectopically expressed 2BC in HeLa cells and exposed them to staurosporine. As shown in Fig. 5B, the induction of apoptosis by staurosporine was suppressed (average 47% (n = 4)) in cells transfected with mRNA for 2BC compared with mock-transfected cells exposed to the apoptosis inducer or cells transfected with mRNA for luciferase and exposed to staurosporine (data not shown).


Figure 5
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  		FIGURE 5.
Ectopically expressed protein 2BC but not 2B inhibits caspase-3 activity and suppresses staurosporine-induced apoptosis. A, HeLa cells were transfected with 2BC or 2B mRNA, and after 10 h a cell extract was prepared to which recombinant active caspase-3 was added and assayed for DEVDase activity as described under "Experimental Procedures." Controls included transfection with no mRNA and mRNA encoding for luciferase. B, HeLa cells were transfected with 2BC mRNA or mock-transfected, and after 8 h the cells were exposed to staurosporine (STS, 0.5 µM) or carrier solvent (Me2SO) for a further 4 h before flow cytometric analysis of apoptosis using AV binding and PI uptake as described under "Experimental Procedures." Control experiments using luciferase mRNA transfection showed no protection suppression from staurosporine-induced apoptosis. All data are the mean ± S.D. of four independent experiments. **, p < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Evidence has shown that the switch from apoptosis to necrotic-like cell death in poliovirus infection involves an unknown mechanism for inhibition of apoptosis (19, 28). Here, we show that the human Enterovirus CVB4 has a similar antiapoptotic activity in human cells. We also demonstrate that the ability of CVB4 to block cell death by apoptosis was not due to a failure of activation of the apoptosis pathway but through a direct inhibition of the activity of the main executioner protease, caspase-3, by CVB4 protein 2BC. The fact that the suppression of apoptosis occurred toward the end of the apoptosis cascade explains why both mitochondrially mediated and death receptor-mediated apoptosis were affected equally. The appearance of the anti-apoptotic phenotype is not immediate, requires 6 h to develop, and is preceded by a transient albeit minor pro-apoptotic response to the infection. Such a biphasic effect has been observed with other viruses including herpes simplex virus type 1 (40).

While our work was in progress, van Kuppeveld and co-workers (39) reported that Coxsackievirus B3 prevented apoptosis in response to actinomycin D and cycloheximide, and in an elegant series of experiments, they demonstrated that the antiapoptotic effect was mediated by the 2B protein. The mechanism of 2B action was identified to involve a decrease in Golgi and endoplasmic reticulum Ca2+ content. Endoplasmic reticulum Ca2+ is known to play a very important role not only in apoptosis but in cell death in general (41, 42). Indeed, the pro- and antiapoptotic properties of Bax and Bcl-2 have in part been attributed to their ability to modulate the ER Ca2+ content (43, 44). Taken together, these data show that coxsackieviruses may use at least two mechanisms to suppress apoptosis upon infection. A first mechanism operates at the level of ER Ca2+ content through 2B-mediated pore formation (45), and a second mechanism involves a direct inhibition of executioner caspase-3 by 2BC (this study). Both mechanisms may not necessarily operate together as one noticeable difference between the two studies is that in our experimental system the proteolytic activation of caspase-3 by the inducers of apoptosis was not significantly impaired. It is possible that a critical intracellular level of 2B is required for pore formation within the endoplasmic reticulum (45) and, hence, decreases its content of Ca2+. Here, we used a lower multiplicity of infection (15 versus 50 plaque-forming unit/cell), which is known to alter the cellular response to infection (23). This may alter the balance between the mechanisms causing the caspase-3 inhibition pathway to become the predominant antiapoptotic mechanism observed under our experimental conditions. Finally, we cannot rule out differences in apoptotic modulation between CVB4 and CVB3.

Virus infection suppressed the development of apoptotic nuclear morphology by all three of the inducers used here. However, CPE-like partial chromatin condensation and distortion was not eliminated. A similar observation was made in cells that were protected from actinomycin D- and cycloheximide-induced apoptosis by poliovirus infection (19) and Coxsackievirus B3 infection (39). However, the last two studies did not investigate the relationship between apoptosis, CPE, and the loss of cell viability. The down-regulation of apoptosis in CVB4-infected cells correlated with a delay in cell death and enhanced cell survival after staurosporine and TRAIL. The reason why actinomycin D-induced apoptosis but not cell death was prevented by CVB4 is presently unclear. However, the fact that the inhibition of mRNA synthesis by itself will be lethal may explain the observed rapid death irrespective of apoptosis suppression by the virus.

The mechanism by which 2BC inhibits caspase-3 activity is not known, presumably some sections of the protein are able to combine with caspase-3, whereas others (perhaps the same ones) bring about an interference in enzymic function. However, the identity of these putative functional domains is not known. Our immunoprecipitation studies suggest that a complex is formed between caspase-3 and 2BC but not with 2B alone. However, the conclusion that the 2B portion of 2BC does not participate in the inhibition process rests on the assumption that 2B behaves in the same manner whether on its own or attached to 2C. There is evidence from subcellular distribution and functional studies that this may not be the case (46–50). Nevertheless, it is likely that the antiapoptotic property of 2BC resides in the 2C portion of the protein. Many functions have been attributed to 2C, although its exact role remains to be determined. Analysis of the sequence of CVB4 2BC using DiAlign (51) and ClustalW (52) multiple alignment tools identified regions in the protein with similarity to X-linked inhibitor of apoptosis protein (XIAP) and to serpins (Table 4). All identified regions of similarity reside in the 2C portion of the protein. Interestingly, the region of similarity identified in XIAP falls within the linker-BIR2 domains that have been demonstrated to mediate the inhibition of caspases-3 and -7 by XIAP (53–55). In addition, the software program predicted a serpin domain that may play a role in the inhibition of caspase-3. A recent report (56) proposed that poliovirus 2C contained several serpin similarity regions. The related poxvirus protein CrmA could provide a precedent since in addition to its well established serpin function, this protein inhibits several caspases that include not only caspases-1 and -8 (57) but also (albeit at high concentration) caspase-3 (58). However, under our experimental conditions, co-immunoprecipitation experiments with anti-caspase-3 antibody identified a major protein band corresponding to 48 kDa, whereas no band corresponding to 37 kDa (the molecular mass of protein 2C) was detected (Fig. 4A). This suggests that protein 2C alone did not bind to caspase-3 but, instead, that the 2BC precursor protein was responsible for the antiapoptotic properties of CVB4 reported here.


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  		TABLE 4
Alignment of Coxsackievirus 2BC (extracted from accession number P08292) with putative serpin and BIR domains

 
At least under in vitro conditions host cell death by apoptosis appears to be detrimental to the virus because it precedes virus-induced necrotic-like CPE and, thus, presumably decreases its replication (59). In many cases cell death by apoptosis occurs as a consequence of impaired or interrupted virus replication (18–22), but the mechanism responsible for the initiation of apoptosis is far from clear. A recent report (60) suggested that an early transient activation of the serine/threonine protein kinase, glycogen synthase kinase 3, through the downstream regulation of beta-catenin contributes to the induction of apoptosis after HeLa cell infection by CVB3. Both forms of cell death, i.e. necrotic-like CPE and apoptosis, have been documented in cardiomyocyte, neuron, and pancreatic beta cell death (17, 24, 25). However, how much either form of cell death individually contributes in vivo to viral replication and spreading and also subsequent viral-induced autoimmunity and chronic disease such as seen in dilated cardiomyopathy (61) remains to be investigated. Another facet of the interaction between virus-induced apoptosis and the host cell is its possible importance in persistent infection. Coxsackievirus RNA is known to persist in human tissues for many years (62), and there is clear evidence from work on poliovirus (21) and sindbis virus (63) that suppression of apoptosis may lead to viral persistence.

In conclusion, we report here that CVB4 infection protects HeLa cells from drug- and death receptor-induced apoptosis. The mechanism underlying the suppression of apoptosis did not involve a failure to activate the apoptotic pathways. Instead, it was found that CVB4 protein 2BC binds to and inhibits the apoptosis executioner caspase-3.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a studentship from the Medical Research Council. Present address: Cancer Research UK, Barts and The London, Queen Mary's School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom. Back

2 To whom correspondence should be addressed. Tel.: 44-1483-686449; Fax: 44-1483-300374; E-mail: g.kass{at}surrey.ac.uk.

3 The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IAP, inhibitors of apoptosis protein; CPE, cytopathic effects; CVB4, Coxsackievirus B4; PI, propidium iodide; AV, annexin V; XIAP, X-linked inhibitor of apoptosis protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; DEVDase, Asp-Glu-Val-Asp protease. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Don Nicholson (Merck Frosst) for generously providing recombinant human active caspase-3 and Dr. Andrzej Kierzek (University of Surrey) for help with the bioinformatic analysis of the sequences. We thank Dr. Margaret M. Willcocks for valuable technical assistance.



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