JOURNAL OF VIROLOGY, Dec. 2001, p. 11437–11448
0022-538X/01/$04.00⫹0 DOI: 10.1128/JVI.75.23.11437–11448.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 23
Vaccinia Virus Infection Disarms the Mitochondrion-Mediated
Pathway of the Apoptotic Cascade by Modulating the
Permeability Transition Pore
SHAWN T. WASILENKO, ADRIENNE F. A. MEYERS, KATHLEEN VANDER HELM,
AND
MICHELE BARRY*
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
Received 25 June 2001/Accepted 29 August 2001
Many viruses have evolved strategies that target crucial components within the apoptotic cascade. One of the
best studied is the caspase 8 inhibitor, crmA/Spi-2, encoded by members of the poxvirus family. Since many
proapoptotic stimuli induce apoptosis through a mitochondrion-dependent, caspase 8-independent pathway,
we hypothesized that vaccinia virus would encode a mechanism to directly modulate the mitochondrial
apoptotic pathway. In support of this, we observed that Jurkat cells, which undergo Fas-mediated apoptosis
exclusively through the mitochondrial route, were resistant to Fas-induced death following infection with a
crmA/Spi-2-deficient strain of vaccinia virus. In addition, vaccinia virus-infected cells subjected to the proapoptotic stimulus staurosporine exhibited decreased levels of both cytochrome c released from the mitochondria and caspase 3 activation. In all cases we found that the loss of the mitochondrial membrane potential,
which occurs as a result of opening the multimeric permeability transition pore complex, was prevented in
vaccinia virus-infected cells. Moreover, vaccinia virus infection specifically inhibited opening of the permeability transition pore following treatment with the permeability transition pore ligand atractyloside and
t-butylhydroperoxide. These studies indicate that vaccinia virus infection directly impacts the mitochondrial
apoptotic cascade by influencing the permeability transition pore.
An important function of the cell-mediated immune response is the detection and elimination of virus-infected cells
as a means to arrest viral propagation. To do this, immune
effector cells rely on the production of cytokines and the recognition of virus infected cells by cytotoxic T lymphocytes
(CTL) to induce programmed cell death, or apoptosis (31).
Apoptosis results in a variety of cellular changes including cell
shrinkage, DNA fragmentation, chromatin condensation, and
finally the formation of apoptotic bodies. These changes are
mediated by biochemical events involving a family of cysteine
proteases termed caspases (45, 61). Following an apoptotic
stimulus, caspases become proteolytically activated and function to cleave cellular proteins, including other members of the
caspase family. Recently, it has also become clear that within
cells instructed to die, mitochondria play a central role in the
execution of apoptosis (13). The induction of apoptosis results
in both structural and physiological alterations to mitochondria, including disruption of electron transport and energy
metabolism, production of reactive oxygen species, loss of the
membrane potential, and release of proapoptotic proteins, including cytochrome c (37).
In order for a virus to replicate and disseminate within a
host, manipulation of the apoptotic process is essential (2, 48,
64). To ensure their survival, viruses have evolved strategies
that target crucial components within the apoptotic cascade.
For example, virus-encoded inhibitors of apoptosis have been
identified that either directly or indirectly modulate caspase
* Corresponding author. Mailing address: Department of Medical
Microbiology and Immunology, 671 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
Phone: (780) 492-0702. Fax: (780) 492-9828. E-mail: michele.barry
@ualberta.ca.
activation. One of the best-studied viral caspase inhibitors is
the cowpox virus-encoded cytokine response modifier A
(crmA), also known as Spi-2. crmA/Spi-2 inhibits both Fas- and
tumor necrosis factor (TNF)-induced apoptosis via interaction
with caspase 8 (33, 59, 71). In a similar manner, baculoviruses
encode P35, a broad-spectrum caspase inhibitor that protects
infected cells from apoptosis (8, 70). As well, baculoviruses and
African swine fever virus modulate the activation and activity
of caspases through the expression of inhibitors of apoptosis
(IAPs) (11, 14, 46). In addition to modulating caspase activity,
viruses have also developed strategies that interfere with other
components of the death pathway. For example, poxviruses
encode secreted TNF receptors that inhibit TNF-␣-induced
apoptosis by blocking ligand-receptor interactions (27, 52, 55).
Gammaherpesviruses and the poxvirus molluscum contagiosum encode viral Fas-associated death domain-like interleukin-1-converting enzyme inhibitory proteins (vFLIPs), which
interfere with recruitment of caspase 8 to the cytoplasmic domains of Fas and TNF receptor 1 (5, 28, 60). Additionally,
adenovirus has evolved an elaborate scheme to stimulate the
internalization of cell surface Fas (53, 62).
Since mitochondria play a central role in cell death, viruses
have also established mechanisms to modulate the mitochondrial component of the apoptotic pathway. Members of the
cellular Bcl-2 family influence the integrity of the mitochondria
(10, 21), and many viruses encoding Bcl-2-like proteins have
been identified. Viral Bcl-2 homologues with antiapoptotic
function have been found in adenovirus (65) and African swine
fever virus (1, 7) as well as in members of the gammaherpesvirus family including Epstein-Barr virus, equine herpesvirus 2,
herpesvirus saimiri, Kaposi’s sarcoma-associated herpesvirus,
bovine herpesvirus, herpesvirus ateles, alcelaphine herpesvirus
1, and murine gammaherpesvirus 68 (64). In addition, novel
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WASILENKO ET AL.
virus gene products that act at the mitochondrial checkpoint
but lack homology to Bcl-2 have also been identified. VMIA,
encoded by human cytomegalovirus (HCMV), inhibits apoptosis and the release of cytochrome c in HeLa cells through
interaction with the adenine nucleotide translocator (ANT)
subunit of the permeability transition (PT) pore (20). M11L,
encoded by the rabbit-specific poxvirus myxoma virus, localizes
to the mitochondria and inhibits staurosporine-induced loss of
mitochondrial membrane potential and apoptosis (17).
The large number of viruses encoding proteins that function
to maintain the integrity of the mitochondria led us to hypothesize that vaccinia virus, a member of the poxvirus family,
would employ a mechanism to directly modulate the mitochondrial apoptotic pathway. To determine whether vaccinia virus
infection could inhibit the mitochondrion-mediated apoptotic
pathway, we monitored the ability of vaccinia virus strain
Copenhagen, which is naturally devoid of the caspase 8 inhibitor crmA/Spi2, to inhibit Fas- and staurosporine-mediated
apoptosis. We show here, for the first time, that vaccinia virus
modulates the apoptotic mitochondrial pathway by inhibiting
the PT pore, thereby preserving the mitochondrial membrane
potential and retaining cytochrome c.
MATERIALS AND METHODS
Cell and viruses. Jurkat cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (both from Gibco BRL Life Technologies,
Inc.), 100 M 2-mercaptoethanol, 50 U of penicillin/ml, and 50 g of streptomycin/ml (RHFM). Stably transfected Jurkat cells were generated as previously
described (3) and maintained in RHFM supplemented with 800 g of G418/ml.
Recombinant vaccinia virus strain Copenhagen expressing beta-galactosidase
(VV65) was a gift from G. McFadden (Robarts Research Institute, London,
Ontario, Canada) (26). VV65 was routinely propagated in baby green monkey
kidney (BGMK) cells, a gift from S. Dales, and grown in Dulbecco’s modified
Eagle medium supplemented with 10% newborn calf serum (both from Gibco
BRL Life Technologies, Inc.), 50 U of penicillin/ml, 50 g of streptomycin/ml,
and 2 mM glutamine. Viruses were isolated as previously described (57).
Virus infection. Jurkat cells were infected at a multiplicity of infection (MOI)
of 10 PFU per cell in 200 l of RHFM at 37°C. After 1 h, the cells were
supplemented with additional RHFM for 4 h and incubated at 37°C under 5%
CO2 before induction of apoptosis. The efficiency of virus infection was routinely
quantified by colorimetric analysis using the lacZ gene. In all experiments, the
efficiency of infection was found to be greater than 95%. When necessary, VV65
was UV inactivated for 60 min prior to infection and cytosine arabinoside (Sigma
Chemical Co.) was added to a final concentration of 40 g/ml.
Antibodies. The p20 fragment of caspase 3 (C3p20) was amplified by PCR
from pSKII:CPP32 using the forward oligonucleotide 5⬘-GGATCCTCTGGAA
TATCCCTGGAC-3⬘ containing a BamHI restriction site and the reverse oligonucleotide 5⬘-GTCGACGTCTGTCTCAATGCCACA-3⬘ containing a SalI restriction site. Amplified C3p20 was subcloned into pGex4T-3 (Pharmacia
Biotech) to construct pGex4T-3:C3p20. pGex4T-3-C3p20 was transformed into
BL21DE3, and protein expression was induced by the addition of 0.1 mM
isopropyl--D-thiogalactopyranoside (IPTG) (Rose Scientific Ltd.). Glutathione
S-transferase (GST)-C3p20 was purified utilizing glutathione Sepharose 4B according to the manufacturer’s instructions (Pharmacia Biotech). Pet15b-Bid, a
gift from X. Wang (University of Texas Southwestern Medical Center, Dallas,
Tex.) was used to express His-tagged Bid (40). Recombinant His-tagged Bid was
purified using a Ni2⫹ column according to the manufacturer’s instructions (Novagen). Rabbits were immunized by injection of 500 g of bacterially expressed
GST-caspase 3 or His-Bid in Freund’s complete adjuvant (Gibco BRL Life
Technologies). At monthly intervals the animals were boosted with 500 g of
antigen in Freund’s incomplete adjuvant (Gibco BRL Life Technologies), and
antiserum was collected 10 days after the fourth boost. The anti-cytochrome c
antibody (clone 7H8.2Cl2) was purchased from PharMingen. Anti-human Fas
immunoglobulin M (IgM) (clone CH11) was purchased from Upstate Biotechnology. Goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated
antibodies were purchased from Bio-Rad and used at dilutions of 1:3,000 and
1:10,000, respectively.
J. VIROL.
Apoptosis induction. Cells were induced to undergo apoptosis by addition of
either 250 ng of activating anti-Fas/ml or 1 to 5 M staurosporine (Sigma
Chemical Co.) as outlined in Results.
Chromium release assay. 51Cr release assays were performed as previously
described (4). Briefly, cells were labeled with 100 Ci of 51Cr at 37°C for 1 h.
Labeled target cells were incubated with 250 ng of anti-Fas clone CH11/ml, and
51
Cr release was quantitated after 8 h. 51Cr release was calculated by the following equation: percent lysis ⫽ 100 ⫻ (sample release ⫺ spontaneous release)/
(total release ⫺ spontaneous release). Standard deviations were generated from
three replicates.
Cytochrome c release assay. Cytochrome c release was monitored as previously described (25, 49). Following apoptosis treatment, 2 ⫻ 106 or 5 ⫻ 106
Jurkat cells were permeabilized by incubation in digitonin lysis buffer containing
75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose, and 190 g
of digitonin (Sigma Chemical Co.)/ml. Cells were incubated on ice for 10 min,
after which the mitochondria-containing pellet and the cytosolic supernatant
were separated by centrifugation at 10,000 ⫻ g for 5 min. Mitochondrial pellets
were resuspended in 0.1% Triton X-100–25 mM Tris (pH 8.0) prior to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
Measurement of mitochondrial membrane potential. Changes in mitochondrial membrane potential were quantified by staining cells with tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes) (16, 18, 43). Cells were loaded
with TMRE by a 30-min incubation (at 37°C, under 5% CO2) in RHFM containing 0.2 M TMRE. As a control, cells were also treated with a membrane
uncoupler, carbonyl cyanide m-chlorophenylhydrazone (ClCCP) (Sigma Chemical Co.), at a final concentration of 50 M, for 15 min at 37°C under 5% CO2.
To trigger the permeability transition, cells were treated with either 1 M
staurosporine or 300 M t-butylhydroperoxide (both from Sigma Chemical Co.)
for 1 or 2 h, respectively. Prior to flow cytometric analysis, cells were washed with
phosphate-buffered saline (PBS) containing 1% fetal calf serum. TMRE fluorescence was acquired through the FL-2 channel equipped with a 585-nm filter
(band pass, 42 nm). Data were acquired on either 10,000 or 20,000 cells per
sample with fluorescence signals at logarithmic gain. Data were analyzed with
CellQuest software, and standard deviations were generated from three independent experiments.
Detection of DNA fragmentation. DNA fragmentation was assessed using the
terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)
kit (Roche Diagnostics Co.). Briefly, cells were harvested, washed in PBS containing 1% fetal calf serum, fixed in 2% paraformaldehyde, and permeabilized in
0.1% Triton X-100. Fixed and permeabilized cells were incubated for 1 h at 37°C
in a solution containing 25 mM Tris (pH 6.6), 200 mM cacodylate, 1 mM CoCl2,
0.6 nM fluorescein-12-dUTP, and 25 U of terminal deoxynucleotidyltransferase
(Roche Diagnostics Co.). Analysis was performed on a Becton Dickinson FACScan equipped with an argon laser at 15 mV with an excitation wavelength of 488
nm. Emission wavelengths were detected through the FL-1 channel equipped
with a 530-nm filter (band pass, 20 nm). Data was acquired on 10,000 cells per
sample with light scatter signals at linear gain and fluorescence signals at logarithmic gain.
In vitro reconstitution assay. Mitochondria were purified as previously described (24, 49). For each test sample, 4 ⫻ 107 cells were washed in buffer A
containing 20 mM morpholinepropanesulfonic acid (MOPS; pH 7.4), 100 mM
sucrose, and 1 mM EGTA. Cells were resuspended in buffer B containing 20 mM
MOPS (pH 7.4), 100 mM sucrose, 1 mM EGTA, 5% Percoll (Sigma Chemical
Co.), and 190 g of digitonin/ml. Following a 15-min incubation on ice with
intermittent inversion, nuclei were pelleted at 2,500 ⫻ g for 10 min at 4°C. The
pellet was discarded, and the supernatant was centrifuged at 15,000 ⫻ g for 15
min at 4°C. The mitochondrial fraction was collected and washed three times in
buffer A and resuspended in buffer C containing 20 mM MOPS (pH 7.4), 300
mM sucrose, and 1.0 mM EGTA. The protein concentration of the mitochondrial fraction was determined using the bicinchoninic acid (BCA) kit from Pierce
Chemical Company. For the in vitro assay, 6 g of purified mitochondria was
either incubated with 2, 5, or 10 ng of recombinant Bid in the presence or
absence of 0.25 g of purified granzyme B or treated with 5, 10, or 15 mM
atractyloside (Sigma Chemical Co.) for 40 min at 37°C. Granzyme B was purified
from YT-Indy cells was as previously described (9, 23). Following the addition of
granzyme B and His-tagged Bid, the samples were incubated at 37°C for 60 min.
Samples were then centrifuged at 15,000 ⫻ g to separate the mitochondrial pellet
from the supernatant prior to SDS-PAGE analysis.
Immunoblotting. Cellular lysates were analyzed by electrophoresis on an SDS–
15% polyacrylamide gel. Proteins were transferred to nitrocellulose membranes
(Osmonics Inc.) using a semidry transfer apparatus (Tyler Research Instruments) for 2.5 h at 500 mA. Membranes were blocked for at least 3 h in PBS
containing 0.1% Tween and 5% skim milk. Caspase 3 and Bid were detected
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FIG. 1. Vaccinia virus strain Copenhagen protects cells from anti-Fas-mediated death. Jurkat cells were either mock infected or infected with
either vaccinia virus strain Copenhagen or vaccinia virus strain Western Reserve at an MOI of 10. Following 5 h of infection, cells were treated
with 250 ng of anti-Fas antibody/ml to induce apoptosis, and cell death was monitored 8 h later by 51Cr release. As controls, Jurkat cells that
overexpress SPI-2 and Bcl-2 were also treated with anti-Fas, and Jurkat cells were pretreated for 30 min with 100 M zVAD.fmk prior to addition
of anti-Fas. Standard deviations were generated from three replicates.
using polyclonal rabbit anti-caspase 3 and anti-Bid at a dilution of 1:10,000.
Cytochrome c was detected using a monoclonal antibody at a 1:1,000 dilution. All
primary antibodies were incubated with the membranes overnight at 4°C. Membranes were probed with either a goat anti-mouse (1:3,000) or a goat anti-rabbit
(1:10,000) horseradish peroxidase-conjugated antibody. Proteins were visualized
with a chemiluminescence detection system according to the manufacturer’s
directions (Amersham Pharmacia Biotech).
RESULTS
Vaccinia virus strain Copenhagen-infected cells are resistant to anti-Fas-mediated apoptosis. To determine whether
vaccinia virus infection could inhibit the mitochondrion-mediated apoptotic pathway, we monitored the ability of vaccinia
virus strain Copenhagen to inhibit anti-Fas-induced apoptosis
of Jurkat cells. We utilized Jurkat cells, since Fas-induced
apoptosis occurs exclusively via the mitochondrial pathway in
this cell line (51). In addition, we utilized a strain of vaccinia
virus, strain Copenhagen, which is naturally devoid of the
caspase 8 inhibitor crmA/Spi-2 (19). Jurkat cells were either
mock infected or infected with vaccinia virus strain Copenhagen. At 5 h postinfection, apoptosis was triggered by the addition of anti-Fas and cell death was measured by 51Cr release.
As shown in Fig. 1, mock-infected cells treated with anti-Fas
displayed approximately 30% 51Cr release. This release was
completely inhibited by pretreating the cells with the broadspectrum caspase inhibitor zVAD.fmk, indicating that cytolysis
was directly dependent on caspase activation. Infection of
Jurkat cells with vaccinia virus strain Western Reserve, which
encodes a functional Spi-2, drastically reduced the levels of
51
Cr released. As expected, Jurkat cells stably transfected with
either Spi-2 or Bcl-2 were protected from anti-Fas-triggered
death. Most significantly, Jurkat cells infected with vaccinia
virus strain Copenhagen, lacking a functional crmA/Spi-2, also
inhibited death mediated via the Fas pathway, clearly suggesting that vaccinia virus strain Copenhagen employs an additional antiapoptotic mechanism.
To begin to determine the point at which vaccinia virus
strain Copenhagen-infected Jurkat cells were resistant to cell
death, we monitored anti-Fas-mediated caspase 3 activation.
Caspase 3 activation was assessed by Western blot analysis
using an antibody raised against the large subunit of the active
caspase. Over 8 h, uninfected cells treated with anti-Fas
showed processing of caspase 3 from the full-length 32-kDa
procaspase to the mature 19- and 17-kDa forms (Fig. 2A). In
contrast, cells infected with vaccinia virus strain Copenhagen
and treated with the anti-Fas antibody exhibited only minor
amounts of active caspase 3 (Fig. 2B), indicating that infected
cells were protected from apoptosis.
Since infection of Jurkat cells with vaccinia virus strain
Copenhagen inhibited the activation of caspase 3, and since
mitochondrial release of cytochrome c is necessary for caspase
3 activation, we assessed the ability of vaccinia virus strain
Copenhagen to inhibit the release of cytochrome c following
treatment with anti-Fas (13, 37, 39). To detect the release of
cytochrome c, cells were fractionated into mitochondrial and
cytosolic fractions and the release of cytochrome c was detected by Western blot analysis. Using this approach, apoptotic
extracts demonstrated the translocation of cytochrome c from
the mitochondria to the cytosolic fraction (Fig. 3A). Translocation of cytochrome c was first detected as early as 4 h following the addition of anti-Fas and was found to increase over
time (Fig. 3A). As a control, we also monitored cytochrome c
release in Jurkat cells that overexpress the antiapoptotic protein Bcl-2. As previously documented, Bcl-2 expression inhibited the translocation of cytochrome c to the cytosol (Fig. 3B)
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FIG. 2. Vaccinia virus strain Copenhagen infection inhibits activation of caspase 3. Jurkat cells were either mock infected or infected with
vaccinia virus strain Copenhagen at an MOI of 10 and treated with 250 ng of anti-Fas antibody/ml for 2, 4, 6, or 8 h. At the times indicated, cells
were permeabilized with digitonin, and caspase 3 processing was monitored by Western blot analysis. (A) Mock-infected Jurkat cells; (B) Jurkat
cells infected with vaccinia virus strain Copenhagen.
(35, 66). Fas-mediated release of cytochrome c was also monitored in Jurkat cells infected with vaccinia virus strain Copenhagen. As shown in Fig. 3C, infection with vaccinia virus strain
Copenhagen significantly interfered with the release of cytochrome c, indicating that vaccinia virus strain Copenhagen
inhibits the Fas-mediated apoptotic pathway upstream of cytochrome c release.
Vaccinia virus strain Copenhagen infection inhibits cytochrome c release in isolated mitochondria but not cleavage of
Bid. The ability of vaccinia virus strain Copenhagen to inhibit
Fas-induced cytochrome c release suggested to us that the
virus could potentially hinder apoptosis by interfering with
activation of the proapoptotic Bcl-2 family member Bid. During Fas-mediated apoptosis, Bid is cleaved by caspase 8,
prompting the translocation of truncated Bid to the mitochondria, resulting in the release of cytochrome c (38, 40). Additionally, the serine proteinase granzyme B, which is released
from activated CTL, is also able to induce apoptosis through
the cleavage of Bid (3, 24). To investigate the potential inhibition of Bid activation by vaccinia virus strain Copenhagen,
we performed an in vitro apoptotic reconstitution assay involving isolated mitochondria, recombinant Bid, and purified granzyme B. Mitochondria were purified from both mock-infected
and virus-infected Jurkat cells as well as from Jurkat cells
stably expressing Bcl-2. To ensure mitochondrial purity, virusinfected samples were subjected to Western blot analysis to
detect Spi-1, a known vaccinia virus cytoplasmic protein (unpublished data) (34). Isolated mitochondria were incubated
with increasing amounts of recombinant Bid, either in the
presence or in the absence of purified granzyme B, and proteolytic cleavage of Bid was monitored by Western blot analysis. Figure 4A demonstrates that in the presence of purified
mitochondria and granzyme B, recombinant Bid underwent
proteolytic cleavage and activation in this assay. In agreement
with previously published results, Bid was also processed in the
presence of isolated mitochondria from cells overexpressing
Bcl-2 (22, 44) (Fig. 4B). The processing of Bid was found to be
unaltered when mitochondria from vaccinia virus-infected cells
were used in the same assay, indicating that vaccinia virus
infection does not inhibit the proteolytic processing of Bid
(Fig. 4C).
Since vaccinia virus infection did not suppress granzyme
B-induced Bid cleavage, we next asked if mitochondria isolated
from vaccinia virus-infected cells could inhibit granzyme Binduced cytochrome c release in the presence of Bid. Mitochondria isolated from mock-infected Jurkat cells were incubated with increasing amounts of recombinant Bid in the
presence of granzyme B, resulting in the translocation of cytochrome c from the mitochondria to the supernatant (Fig.
4D). As shown in Fig. 4D, an increase in the amount of cytochrome c translocation was detected following the addition of
granzyme B and increasing amounts of recombinant Bid. At 5
FIG. 3. Vaccinia virus strain Copenhagen inhibits cytochrome c translocation. Jurkat cells were either mock infected or infected with virus.
Following infection, cells were treated with 250 ng of anti-Fas/ml for 2, 4, 6, or 8 h to induce cytochrome c translocation. At the times indicated,
cells were permeabilized with digitonin and fractionated into the mitochondrion-containing membranous fraction and the soluble cytoplasmic
fraction, and cytochrome c was assessed by Western blot analysis. (A) Mock-infected Jurkat cells; (B) mock infected Jurkat cells that overexpress
Bcl-2; (C) Jurkat cells infected with vaccinia virus strain Copenhagen.
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FIG. 4. Vaccinia virus infection protects against granzyme B-mediated cytochrome c release from isolated mitochondria by a mechanism
downstream of Bid activation. Mitochondria were isolated from mock-infected Jurkat cells, Jurkat cells overexpressing Bcl-2, and Jurkat cells
infected with vaccinia virus strain Copenhagen. Purified mitochondria were incubated for 60 min at 37°C with 2, 5, or 10 ng of recombinant Bid
either in the presence or in the absence of granzyme B. Following treatment, samples were fractionated into mitochondria-containing and soluble
fractions and the proteins were resolved by SDS-PAGE. (A through C)Western blot analysis of Bid cleavage in mitochondria isolated either from
mock-infected cells (A), from cells overexpressing Bcl-2 (B), or from vaccinia virus strain Copenhagen-infected cells (C). (D through F) Western
blot analysis of cytochrome c translocation from purified mitochondria to supernatant fractions in mitochondria isolated either from mock-infected
cells (D), from mock-infected cells overexpressing Bcl-2 (E), or from vaccinia virus strain Copenhagen-infected cells (F).
and 10 ng of Bid, we found that some cytochrome c translocation was independent of Bid cleavage, as previously reported
(40, 67) (Fig. 4D). The release of cytochrome c was completely
abolished in mitochondria isolated from Jurkat cells overexpressing the antiapoptotic protein Bcl-2 (Fig. 4E). Inhibition of
cytochrome c translocation was also seen in mitochondria isolated from vaccinia virus-infected cells (Fig. 4F). Taken together, these results indicated that cytochrome c release was
inhibited in mitochondria isolated from infected cells and suggested that vaccinia virus infection directly modulated the mitochondrial arm of the apoptotic pathway.
Vaccinia virus strain Copenhagen-infected cells are resistant to staurosporine-mediated apoptosis. In view of our findings demonstrating that vaccinia virus strain Copenhagen
could inhibit Fas-mediated cytochrome c release and cytochrome c release from isolated mitochondria, we assessed the
ability of vaccinia virus strain Copenhagen to directly inhibit
the mitochondrial route to apoptotic death. To determine if
vaccinia virus strain Copenhagen could directly inhibit the mitochondrial cascade, we treated cells with the proapoptotic
reagent staurosporine, which triggers the mitochondrion-mediated apoptotic pathway (6, 58). Mock-infected or virus-infected Jurkat cells were treated with staurosporine, and the
levels of DNA fragmentation were measured using the
TUNEL assay and flow cytometry. As shown in Fig. 5, untreated cells demonstrated low levels of DNA fragmentation
(Fig. 5a, d, and f). Upon staurosporine treatment, 42% of the
mock-infected Jurkat cell population showed DNA fragmentation (Fig. 5b). Preincubation with the broad-spectrum
caspase inhibitor zVAD.fmk completely inhibited staurosporine-induced DNA fragmentation, clearly demonstrating that
staurosporine-induced DNA fragmentation occurred via
caspase activation, as expected (Fig. 5c). In addition, stably
transfected Jurkat cells that overexpress Bcl-2 were also found
to be resistant to apoptosis, indicating that staurosporine-me-
diated cell death occurred via the mitochondrial pathway (Fig.
5d). Most importantly, vaccinia virus strain Copenhagen-infected Jurkat cells treated with staurosporine displayed clear
protection from apoptosis, with only 7% of the cells showing
DNA fragmentation (Fig. 5g). To determine if the block was
upstream of caspase 3 activation, cells were treated with staurosporine for 2, 4, or 6 h and caspase 3 processing was monitored by Western blot analysis. As shown in Fig. 6A, mockinfected Jurkat cells treated with staurosporine displayed rapid
conversion of the 32-kDa procaspase 3 to the active fragments.
This conversion was significantly inhibited both in Bcl-2-overexpressing cells and in cells infected with vaccinia virus strain
Copenhagen (Fig. 6B and C). Compared to mock-infected
cells, vaccinia virus strain Copenhagen-infected Jurkat cells
treated with staurosporine displayed drastic reductions in levels of the 19- and 17-kDa caspase 3 fragments and maintenance of the full-length 32-kDa proform, indicating that apoptosis inhibition occurred upstream of caspase 3 activation
(Fig. 6A and B).
Since Jurkat cells infected with vaccinia virus strain Copenhagen and induced to undergo apoptosis with anti-Fas inhibited cytochrome c translocation from mitochondria to the
cytosol, we investigated the possibility that staurosporine-induced cytochrome c translocation would be inhibited by virus
infection as well. Mock-infected cells treated with staurosporine exhibited a dramatic loss of cytochrome c from the mitochondrial fraction and subsequent accumulation in the cytoplasmic fraction (Fig. 7A). In contrast to cells treated with
anti-Fas antibody, cells treated with staurosporine displayed
cytochrome c translocation as early as 2 h posttreatment, and
we routinely detected complete translocation of cytochrome c
to the cytoplasmic fraction after 6 h of treatment. As anticipated, staurosporine-induced translocation of cytochrome c
was completely inhibited in Jurkat cells engineered to overexpress Bcl-2 (35, 66) (Fig. 7B). Most importantly, in cells in-
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FIG. 5. DNA fragmentation is blocked by vaccinia virus strain Copenhagen infection. Jurkat cells were either mock infected or infected with
vaccinia virus strain Copenhagen. Following infection, cells were treated with 2.5 M staurosporine for 2 h, and DNA fragmentation was assessed
by TUNEL as outlined in Materials and Methods. (a) Untreated Jurkat cells; (b) Jurkat cells treated with staurosporine; (c) Jurkat cells treated
with staurosporine in the presence of 100 M zVAD.fmk; (d) Jurkat cells overexpressing Bcl-2; (e) Jurkat cells overexpressing Bcl-2 treated with
staurosporine; (f) Jurkat cells infected with vaccinia virus strain Copenhagen; (g) Jurkat cells infected with vaccinia virus and treated with
staurosporine.
fected with vaccinia virus strain Copenhagen, cytochrome c
release was drastically reduced (Fig. 7C). In contrast to the
situation in mock-infected cells, complete translocation of cytochrome c from the mitochondrial to the cytosolic fraction
was inhibited by vaccinia virus infection, and cytosolic cytochrome c was only partially evident at 4 and 6 h post-staurosporine treatment.
Vaccinia virus infection inhibits disruption of the mitochondrial inner membrane potential and opening of the PT pore.
During apoptosis the release of cytochrome c coincides with
loss of the inner mitochondrial membrane potential (41, 69).
Disruption of the inner mitochondrial membrane potential is
thought to occur due to the opening of the PT pore (12, 36, 37).
Since vaccinia virus strain Copenhagen infection inhibited cytochrome c translocation and apoptosis, we asked if vaccinia
virus infection was able to inhibit apoptosis by maintaining
mitochondrial integrity and the inner mitochondrial mem-
brane potential. Changes in the membrane potential were
monitored by assaying the uptake of TMRE, a fluorescent
mitochondrion-specific dye (18, 50). Disruption of the membrane potential in mock-infected and infected cells following
staurosporine treatment was monitored by TMRE fluorescence. In untreated Jurkat cells, 94% of the cells demonstrated
TMRE fluorescence, indicating an intact mitochondrial membrane potential (Fig. 8a). Upon staurosporine treatment, 48%
of the cells exhibited a reduction in TMRE fluorescence (Fig.
8b). As a control, Jurkat cells were treated with a membrane
uncoupler, ClCCP, resulting in the reduction of TMRE fluorescence in all cells (Fig. 8c). Jurkat cells treated with staurosporine in the presence of the caspase inhibitor zVAD.fmk still
demonstrated a loss in membrane potential (Fig. 8d), indicating that staurosporine-induced loss of the mitochondrial membrane potential occurred in a caspase-independent manner.
This is in contrast to what was observed with DNA fragmen-
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FIG. 6. Staurosporine-induced caspase 3 activation is inhibited by vaccinia virus strain Copenhagen infection. Jurkat cells were either mock
infected or infected with virus; following infection, they were treated with 5 M staurosporine for 0, 2, 4, or 6 h. At the times indicated, cells were
permeabilized with digitonin and the proteins were resolved by SDS-PAGE. The activation of caspase 3 was monitored by Western blot analysis.
(A) Mock-infected Jurkat cells; (B) mock-infected Jurkat cells that overexpress Bcl-2; (C) Jurkat cells infected with vaccinia virus strain
Copenhagen.
tation (Fig. 5c), because staurosporine directly induces the loss
of the PT in a caspase-independent manner whereas DNA
fragmentation requires caspase activation (58). As expected,
Jurkat cells overexpressing Bcl-2 were completely resistant to
the staurosporine-induced collapse of the inner membrane potential (Fig. 8e and f). Similarly, upon treatment with staurosporine, 87% of Jurkat cells infected with vaccinia virus maintained a TMRE-positive state (Fig. 8g and h), indicating that
vaccinia virus infection inhibited staurosporine-induced loss of
the inner mitochondrial membrane potential. The addition of
cytosine arabinoside (araC), an inhibitor of virus replication
and late gene expression, had no effect on the ability of vaccinia virus to inhibit staurosporine-induced loss of the inner
mitochondrial membrane potential, indicating that virus replication and late gene expression were not necessary (Fig. 8i and
j). In contrast, UV inactivation of the virus resulted in reversal
of this observation, clearly showing that a productive vaccinia
virus infection was necessary for the inhibition (Fig. 8k and l).
Controlled permeabilization of the inner and outer mitochondrial membrane is known to occur as a result of opening
a mitochondrial multiprotein complex known as the PT pore
(12, 36, 37). The PT pore consists of the outer-mitochondrialmembrane-localized voltage-dependent anion carrier (VDAC),
the inner-membrane-localized ANT, and the matrix protein
cyclophilin D (12, 36, 37). Two accessory proteins, hexokinase
and the peripheral benzodiazepine receptor, are also found
associated with the PT pore. Members of the Bcl-2 family
associate with components of the pore and modulate pore
activity, thereby inhibiting apoptosis (12, 37, 42).
Since infection of cells with vaccinia virus renders them
resistant to apoptosis and inhibits disruption of the mitochondrial inner-membrane potential, we asked if vaccinia virus
strain Copenhagen infection regulated apoptosis by modulating the activity of the PT pore. Pore-specific ligands can act
directly on components of the pore, resulting in dissipation of
the inner-membrane potential and the release of cytochrome c
(32, 68, 72). To ascertain if vaccinia virus infection could inhibit induction of the permeability transition and subsequent
apoptosis, we treated purified mitochondria with the ANT
ligand atractyloside and monitored cytochrome c release. Mitochondria were isolated from mock-infected, vaccinia virusinfected, or Bcl-2-overexpressing cells and induced to undergo
permeability transition with various concentrations of atractyloside, and the translocation of cytochrome c was monitored
by Western blot analysis. As shown in Fig. 9A, cytochrome c
translocation from mock-infected mitochondria was detected
following treatment with 5 mM atractyloside, and increasing
levels of cytochrome c release were detected with higher concentrations of atractyloside. Atractyloside-induced cytochrome
c translocation was completely inhibited in mitochondria iso-
FIG. 7. Staurosporine-induced cytochrome c release is inhibited by vaccinia virus strain Copenhagen infection. Jurkat cells were either mock
infected or infected with virus and were treated with 5 M staurosporine for 0, 2, 4, or 6 h. At the times indicated, cells were fractionated into
mitochondria-containing membrane fractions and cytoplasmic fractions by the addition of digitonin. The translocation of cytochrome c was
monitored by Western blot analysis. (A) Mock-infected Jurkat cells; (B) mock-infected Jurkat cells that overexpress Bcl-2; (C) Jurkat cells infected
with vaccinia virus strain Copenhagen.
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WASILENKO ET AL.
J. VIROL.
FIG. 8. Vaccinia virus strain Copenhagen infection inhibits staurosporine-induced disruption of the mitochondrial membrane potential. Jurkat
cells were either mock infected or infected with vaccinia virus and treated with 1 M staurosporine for 1 h, and the mitochondrial membrane
potential was determined using TMRE fluorescence. (a) Untreated Jurkat cells; (b) Jurkat cells treated with staurosporine; (c) Jurkat cells treated
with the membrane uncoupler ClCCP; (d) Jurkat cells treated with staurosporine in the presence of 100 M zVAD.fmk; (e) untreated Jurkat cells
overexpressing Bcl-2; (f) Jurkat cells overexpressing Bcl-2 treated with staurosporine; (g) untreated Jurkat cells infected with vaccina virus strain
Copenhagen; (h) vaccinia virus-infected cells treated with staurosporine; (i) Jurkat cells infected with vaccinia virus strain Copenhagen in the
presence of 40 g of araC/ml; (j) Jurkat cells infected with vaccinia virus strain Copenhagen in the presence of araC and staurosporine; (k)
untreated cells infected with UV-inactivated vaccinia virus strain Copenhagen; (l) Jurkat cells infected with UV-inactivated vaccinia virus and
treated with staurosporine.
lated from cells overexpressing Bcl-2, a known PT pore-modulating protein (42) (Fig. 9B). As shown in Fig. 9C, atractyloside-induced cytochrome c release from mitochondria isolated
from vaccinia virus-infected cells was also completely inhibited,
indicating that vaccinia virus strain Copenhagen infection inhibited the onset of atractyloside-induced mitochondrial permeability transition and the subsequent translocation of cyto-
chrome c. To further confirm that vaccinia virus infection
inhibited the opening of the PT pore, we treated Jurkat cells
with another permeability transition inducer, t-butylhydroperoxide, and monitored mitochondrial membrane potential using
TMRE fluorescence (68, 72). As shown in Fig. 9D, treatment
of mock-infected cells with t-butylhydroperoxide resulted in
disruption of the mitochondrial membrane potential, as shown
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VACCINIA VIRUS INHIBITS APOPTOSIS
11445
FIG. 9. Vaccinia virus strain Copenhagen inhibits opening of the PT pore. (A through C) Atractyloside-induced cytochrome c release is
inhibited by vaccinia virus strain Copenhagen infection. Mitochondria were purified and incubated at 37°C with 5, 10, or 15 mM atractyloside
(Atrac.) for 40 min. Following treatment, samples were fractionated into mitochondria-containing and soluble fractions, and translocation of
cytochrome c was analyzed by Western blot analysis. (A) Mitochondria isolated from mock-infected Jurkat cells; (B) mitochondria isolated from
mock-infected Jurkat cells overexpressing Bcl-2; (C) mitochondria isolated from vaccinia virus strain Copenhagen-infected Jurkat cells. (D)
Vaccinia virus inhibits t-butylhydroperoxide-induced disruption of the mitochondrial membrane potential. Jurkat cells were either mock infected
or infected with vaccinia virus and treated with 300 M t-butylhydroperoxide for 2 h. The mitochondrial membrane potential was determined using
TMRE fluorescence in the presence and absence of 100 M zVAD.fmk. Standard deviations were generated from three independent experiments.
by a decrease in TMRE fluorescence. Disruption of the membrane potential following t-butylhydroperoxide treatment was
not inhibited by zVAD.fmk, confirming previous findings that
indicate that t-butylhydroperoxide does not disrupt membrane
potential through caspase activation (68) (Fig. 9D). Infection
with vaccinia virus strain Copenhagen, however, prevented loss
of TMRE fluorescence, indicating that vaccinia virus infection
regulates the PT pore (Fig. 9D).
DISCUSSION
In order to survive and replicate within a host, viruses possess specific strategies to circumvent the multifaceted immune
response, including a variety of strategies to inhibit apoptosis
(63). The detection and apoptotic elimination of virus-infected
cells is mediated by specialized classes of lymphocytic cells
referred to as CTL and natural killer cells. The Poxviridae, of
which vaccinia virus is the prototypic member, are large double-stranded DNA viruses that encode many proteins essential
for evading the antiviral immune response (56). Until now, the
modulation of apoptosis by vaccinia virus has been attributed
primarily to expression of the serine proteinase inhibitor Spi-2
(33, 59, 71). Previous reports, however, have suggested that
vaccinia virus may foster an additional mechanism to interfere
with apoptosis (15, 33, 54). In this study we report for the first
time that vaccinia virus strain Copenhagen regulates the mitochondrial apoptotic pathway by inhibiting the PT pore.
Since recent advances in apoptosis have implicated mitochondria as a central control point in cell death, we specifically
asked if vaccinia virus employed a mechanism to interfere with
the mitochondrial component of apoptotic death (13, 37). Considering that numerous viruses encode mediators of this cascade, we predicted that vaccinia virus infection would also
result in maintenance of mitochondrial integrity following the
addition of a proapoptotic stimulus. To perform our studies we
utilized Jurkat cells, which, due to lower levels of caspase 8
activation, undergo Fas-mediated apoptosis exclusively through
the mitochondrial route (51). In addition, we chose to utilize
vaccinia virus strain Copenhagen, because it is naturally devoid
of the caspase 8-inhibitor crmA/Spi-2 (19). Using this approach we found that vaccinia virus strain Copenhagen infection inhibited cell death mediated through the Fas surface
receptor. Since this strain of vaccinia virus does not contain a
functional Spi-2 protein, this result clearly indicated that vaccinia virus strain Copenhagen employed a novel, Spi-2-independent antiapoptotic mechanism. Prior to this study, other
11446
WASILENKO ET AL.
researchers have suggested the existence of a Spi-2-independent antiapoptotic effect conferred by vaccinia virus infection.
Dobbelstein and Shenk found that some HeLa cells infected
with a vaccinia virus lacking Spi-2 were still protected from
apoptosis (15). In addition, Kettle and coworkers reported the
existence of a Spi-2-independent mechanism that was capable
of inhibiting cycloheximide-induced apoptosis (33). More recently, Shisler and Moss found that infection with a Spi-2deficient virus inhibited cleavage of the caspase 3 substrate
PARP (54).
To define more clearly the mechanism of vaccinia virus apoptosis inhibition, we monitored both the activation of caspase
3 and the translocation of cytochrome c in response to treatment with anti-Fas. Jurkat cells infected with vaccinia virus
strain Copenhagen and treated with anti-Fas displayed reductions in both the activation of caspase 3 and the translocation
of cytochrome c compared to mock-infected cells, clearly indicating that the block in apoptosis was upstream of cytochrome c release. Similarly, apoptosis induced by staurosporine was also dramatically inhibited in cells infected with
vaccinia virus strain Copenhagen. Once again, both caspase 3
activation and cytochrome c release were inhibited, demonstrating that the novel mechanism employed by vaccinia virus
occurred upstream of cytochrome c release. Analysis of Bid
activation in an in vitro reconstitution assay indicated that
vaccinia virus did not inhibit cleavage of Bid, allowing us to
rule out the possiblity of interference with Bid activation.
Western blot analysis of cytochrome c in the same in vitro
reconstitution assay using mitochondria purified from infected
and uninfected cells once again revealed that vaccinia virus
interferes with the translocation of cytochrome c. Taken together, the data clearly suggest that vaccinia virus infection
inhibited apoptosis by functioning directly at the mitochondria.
Recently, M11L, encoded by the poxvirus myxoma virus, has
been shown to localize to the mitochondria and inhibit apoptosis (17). Numerous virus-encoded Bcl-2 homologues and a
novel protein from HCMV, vMIA, also inhibit apoptosis by
functioning at the mitochondria (20, 64). Interestingly, no open
reading frame exists in vaccinia virus strain Copenhagen that
displays homology with known virus-encoded or cellular apoptotic inhibitors, suggesting that vaccinia virus has evolved a
novel mechanism to inhibit cytochrome c release and apoptosis
(19).
Although the exact mechanism of cytochrome c release during apoptosis is still controversial, the release of cytochrome c
has been linked to disruption of the inner-mitochondrial-membrane potential (12, 36, 37, 41, 69). Dissipation of the innermitochondrial-membrane potential is a common and irreversible feature of apoptosis. We found that following treatment
with staurosporine, cells infected with vaccinia virus strain
Copenhagen retained the inner-mitochondrial-membrane potential, as monitored by TMRE fluorescence. This result supported our previous observations demonstrating that within
infected cells the integrity of the mitochondria was maintained
and cytochrome c translocation was inhibited. Loss of the
membrane potential occurs by triggering opening of the PT
pore, a phenomenon known as the “permeability transition”
(12, 36, 37). Since disruption of the inner-mitochondrial-membrane potential and the induction of apoptosis occurs as a
result of opening of the PT pore, our data suggested that
J. VIROL.
FIG. 10. Model of vaccinia virus-mediated apoptosis inhibition.
Fas initiated apoptosis occurs via activation of caspase 8 and by the
subsequent proteolytic cleavage of Bid. Once cleaved, Bid translocates
to the mitochondria, resulting in a loss of the inner-mitochondrialmembrane permeability transition and the release of cytochrome c.
The release of cytochrome c (Cyto. c) results in activation of caspase
9 via interaction with the adapter molecule Apaf1 and the subsequent
activation of caspase 3. Additionally, loss of the inner-membrane permeability transition can be triggered by staurosoporine and atractyloside. Vaccinia virus infection manipulates this pathway at two separate
points. First, the vaccinia virus-encoded serine protease inhibitor SPI2/crmA inhibits the activity of caspase 8. Second, vaccinia virus infection also inhibits apoptosis by modulating the PT pore, thereby preventing the loss of cytochrome c and activation of caspase 9.
perhaps vaccinia virus could maintain mitochondrial integrity
by regulating the opening of the PT pore. In support of this we
found that vaccinia virus infection inhibited cytochrome c release from mitochondria treated with increasing amounts of
atractyloside, a pore agonist known to induce the permeability
transition and the release of cytochrome c (32, 68, 72). In
addition, we found that vaccinia virus-infected cells treated
with the pro-oxidant t-butylhydroperoxide, which also causes
disruption of the mitochondrial transmembrane potential,
demonstrated preservation of the mitochondrial inner-membrane transmembrane potential (68). Thus, vaccinia virus infection inhibited the effects mediated by both atractyloside and
t-butylhydroperoxide, two reagents that trigger the permeability transition. In addition, both Bid and staurosporine can
induce apoptosis via a PT pore-dependent mechanism, and we
found that vaccinia virus infection also inhibited apoptosis
induced by these reagents (58, 67). Our data are therefore
compatible with the idea that vaccinia virus has evolved a
mechanism to directly modulate the permeability transition
and thereby inhibit apoptosis (Fig. 10).
Due to the multimeric nature of the PT pore, multiple targets for viral modulation are possible. Both pro- and antiapo-
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VACCINIA VIRUS INHIBITS APOPTOSIS
ptotic members of the Bcl-2 family associate with components
of the PT pore and modulate apoptosis (12, 37, 42). At least
one component of the PT pore has now been directly associated with virus-mediated apoptotic inhibition. The vMIA protein from HCMV associates with the ANT and inhibits apoptosis (20). Alternatively, proapoptotic viral proteins such as
Vpr encoded by human immunodeficiency virus and HBx encoded by hepatitis B virus induce apoptosis by interaction with
ANT and VDAC, respectively (29, 30, 47). Collectively, Vpr,
HBx, and vMIA modulate apoptosis by interacting with components of the PT pore and regulating the pore complex. Thus,
a similar scenario to account for vaccinia virus interference
with the mitochondrial apoptotic pathway is likely, and we are
currently looking for vaccinia virus proteins that interact with
known components of the pore. At present, however, the exact
composition of the PT pore is still controversial, and the identification of a novel vaccinia virus-encoded protein and its
exact mechanism of action will lead to a clearer understanding
of the PT pore and its regulation in the future. In addition,
further investigation into vaccinia virus modulation of the PT
pore and apoptosis will result in valuable information regarding virus-host interactions and the mechanisms of cell death.
ACKNOWLEDGMENTS
This work was supported by grants from the Canadian Institutes for
Health Research and the Alberta Heritage Foundation for Medical
Research (to M.B.). M.B. is the recipient of an Alberta Heritage
Foundation for Medical Research Scholar Award, and A.F.A.M. is the
recipient of a studentship from the Canadian Institutes for Health
Research.
We thank D. Burshtyn and S. Slemko for critical review of the
manuscript and H. Everett for helpful discussions.
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