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J Virol, July 1998, p. 5897-5904, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Antiapoptotic Activity of the Herpesvirus
Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and
Inhibition of Caspase-3-Like Activity
Tobias
Derfuss,
Helmut
Fickenscher,
Michael S.
Kraft,
Golo
Henning,
Doris
Lengenfelder,
Bernhard
Fleckenstein, and
Edgar
Meinl*
Institut für Klinische und Molekulare
Virologie, University of Erlangen-Nürnberg, D-91054 Erlangen,
Germany
Received 29 December 1997/Accepted 1 April 1998
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ABSTRACT |
Viruses have evolved different strategies to interfere with host
cell apoptosis. Herpesvirus saimiri (HVS) and other lymphotropic herpesviruses code for proteins that are homologous to the cellular antiapoptotic Bcl-2. In this study HVS-Bcl-2 was stably expressed in
the human leukemia cell line Jurkat and in the murine T-cell hybridoma
DO to assess its antiapoptotic spectrum and to gain further insight
into its mode of action. HVS- Bcl-2 prevented apoptosis that occurs as
a result of a disturbance of intracellular homeostasis by, for example,
DNA damage or menadione, which gives rise to oxygen radicals. In Jurkat
cells, HVS-Bcl-2 also inhibited apoptosis mediated by the death
receptor CD95. In DO cells, HVS-Bcl-2 did not interfere with
CD95-mediated apoptosis but blocked dexamethasone-induced cell death.
Mitochondrial damage is a central coordinating event in apoptosis
induced by different stimuli. To assess the integrity of mitochondria,
we used rhodamine 123, which is released upon disturbance of the
mitochondrial membrane potential, and determined the release of
cytochrome c into the cytosol. Both signs of mitochondrial damage were prevented by HVS-Bcl-2. This viral protein also inhibited the generation of caspase-3-like DEVDase activity and blocked the
cleavage of poly(ADP-ribose) polymerase, a natural substrate of
caspase-3-like proteases. In conclusion, HVS-Bcl-2 protects against a
great variety of apoptotic stimuli, stabilizes mitochondria, and acts
upstream of the generation of caspase-3-like activity.
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INTRODUCTION |
Apoptosis is used by the host as a
defense mechanism to eliminate virus-infected cells. The programmed
cell death of infected cells limits viral replication and may prevent
virus-induced malignancies. Apoptosis is characterized by distinct
biochemical and morphological changes, such as activation of caspases
(formerly called ICE-like proteases), mitochondrial depolarization with
release of cytochrome c, and nucleosomal DNA fragmentation.
A central role in the execution of apoptosis is performed by caspases.
Caspases are present in the cytosol as inactive proenzymes. They become
activated upon intramolecular cleavage and are thought to execute
apoptosis induced by different stimuli (45). To date, more
than 10 caspases which differ in their substrate specificities and
their susceptibility to protease inhibitors have been identified.
Caspase-3 and related caspases are involved in nuclear apoptosis and in
extranuclear apoptotic events such as the formation of apoptotic bodies
and exposure of phosphatidylserine (24, 35). When caspase-3
is activated by apoptotic stimuli, it activates a DNase, named
caspase-activated DNase (CAD), that degrades DNA during apoptosis. This
endonuclease CAD is present in the cytosol complexed with its inhibitor
ICAD. Caspase-3 cleaves ICAD, and this allows CAD to translocate to the
nucleus and degrade DNA (13, 48).
During the apoptotic process, both cellular and viral proteins and
nucleic acids are destroyed. Different viruses have developed a variety
of strategies to interfere with host cell apoptosis (56,
58). Lymphotropic herpesviruses such as human herpesvirus 8 (HHV-8) (6, 49), Epstein-Barr virus (EBV) (21),
and herpesvirus saimiri (HVS) (44) code for a protein that
shows homology to cellular members of the Bcl-2 family. An important
function of these antiapoptotic herpesvirus Bcl-2 homologs in the life
cycle of these viruses is suggested by the finding that lymphotropic herpesviruses of distantly related species code for a Bcl-2 homolog (14, 61).
Bcl-2 family members are key regulators in the development of
apoptosis. Cellular Bcl-2 is a multifunctional protein, and different
mechanisms have been implicated in the protection of cells from
apoptotic stimuli: cellular Bcl-2 or Bcl-xL can prevent the
loss of mitochondrial membrane potential that is induced by a number of
apoptotic stimuli (36, 55). In some experimental systems,
Bcl-2 is also active when specifically targeted to the endoplasmic
reticulum (67). In addition, Bcl-2 and Bcl-xL
interact with several proteins participating in cell death regulation, such as the mammalian homolog of CED-4 (Apaf-1), Raf-1, BAG-1, calcineurin, p53BP-2, and other members of the Bcl-2 family (reviewed in references 32 and 46).
Homology of cellular Bcl-2 family members is restricted to distinct
Bcl-2 homology regions, BH1, BH2, BH3, and BH4. Most of the cellular
Bcl-2 members have a membrane anchor at their C terminus and are
localized at the outer mitochondrial, outer nuclear, and endoplasmic
membranes. The functions of the different Bcl-2 homology domains could
be identified to some extent. BH1 and BH2 domains are involved in Bcl-2
homodimer formation. The BH3 domain of death agonists like Bax or Bak
is required for heterodimerization with Bcl-xL and Bcl-2
and to promote apoptosis. The BH4 domain of Bcl-2 and
Bcl-xL is involved in binding to death-regulatory proteins like Raf-1, Bag-1, calcineurin (32, 46), and CED-4
(27). The HVS-encoded Bcl-2 is shorter than cellular Bcl-2
or Bcl-xL and lacks a strong homology to the BH3 and BH4
domains but contains, like its cellular counterpart, a membrane anchor
at the C terminus (44, 53). Cellular Bcl-2 can be converted
to a death promoter upon cleavage by caspases (7), and this
death-promoting activity is dependent on BH3. It was suggested that the
lack of BH3 allows the viral Bcl-2 homologs to evade regulation by
caspases (7).
HVS is a lymphotropic and oncogenic herpesvirus. This virus persists
for life in its natural host, the squirrel monkey, without causing
apparent disease. It causes leukemia and lymphoma in other New World
primate species. HVS transforms T cells from New World monkeys
(50), rhesus monkeys (39), and humans
(3) to stable growth in cell culture (reviewed in references
17 and 40). HVS-Bcl-2 is
expressed predominantly during lytic replication of HVS (31)
and can inhibit apoptosis induced by Sindbis virus (44).
The purpose of this study was to analyze the repertoire of apoptotic
stimuli against which HVS-Bcl-2 is protective, to compare the
functional properties of HVS-Bcl-2 and cellular Bcl-xL, and to gain further insight into the mode of action of this viral antiapoptotic effector. We report that HVS-Bcl-2 protected against apoptosis induced by irradiation, dexamethasone, and menadione, which
gives rise to oxygen radicals. Inhibition of CD95-mediated cell death
was seen in Jurkat cells but not in DO cells. HVS-Bcl-2 protected
against this broad range of apoptotic stimuli by stabilizing mitochondria and by functioning upstream of the activation of caspase-3.
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MATERIALS AND METHODS |
Cell lines, plasmids, and transfections.
The complete
sequence of open reading frame 16 of HVS strain C488 (30),
which codes for HVS-Bcl-2, was amplified by PCR, cloned with the pCR2.1
vector (Invitrogen, De Schelp, The Netherlands), and confirmed by
sequencing by the Dye Dideoxy terminator method (ABI, Weiterstadt,
Germany). HVS-Bcl-2 was excised with Asp718 and
NotI from pCR2.1 and then inserted into the eukaryotic
expression vector pCEP4 (Invitrogen). The cDNA of Bcl-XL
(4) (kindly provided by C. Thompson and L. Boise) was
excised from pBluescript with PvuII and NotI and
subsequently also inserted into pCEP4.
The human T-cell leukemia line Jurkat was transfected by
electroporation. The conditions were as follows. A total of 5 × 106 cells were suspended in 250 µl of CG medium
(Vitromex, Selters, Germany) with 25 µg of DNA of pCEP4-HVS-Bcl-2,
pCEP4-Bcl-xL, or the unmodified pCEP4 and electroporated at
210 V and 960 µF. Additionally, the murine T-cell hybridoma line DO
was transfected by electroporation (230 V and 960 µF) with
pCEP4-HVS-Bcl-2 or unmodified control vector. Jurkat cells and DO cells
were kept in RPMI supplemented with 10% fetal bovine serum, 2 mM
glutamine, and 50 µg of gentamicin per ml.
Stable transfectants were isolated with hygromycin B (Boehringer
Mannheim) at a final concentration of 500 µg/ml for Jurkat
cells and
1,200 µg/ml for DO cells. Transfected clones were generated
by
seeding 1 to 100 cells in 96-well plates.
Transcript analysis.
The expression of HVS-Bcl-2 in stably
transfected cell lines was analyzed by RNase protection. To construct a
riboprobe template for RNase protection, a 561-bp fragment of
pCEP4-HVS-Bcl-2 was excised with SnaBI and PvuII
and cloned in pBluescript. This fragment consists of 250 bp of
HVS-bcl-2, 263 bp of pCEP4, and 48 bp of pCR2.1. As a
positive control for the expression of HVS-Bcl-2, we used the
transformed T-cell lines Ha-S-T and Hk-S-T, which were derived from
Callithrix jacchus and produce infectious virus (39). Total cellular RNA was prepared by the acidic phenol
extraction method (10). RNase protection was carried out as
described in standard protocols (54).
Induction of apoptosis.
Dexamethasone (Sigma, Deisenhofen,
Germany), the anti-CD95 monoclonal antibody (MAb) CH-11 (Immunotech,
Marseille, France), FLAG-CD95 ligand (57), irradiation, and
menadione (Sigma) were applied to induce cell death. Menadione was
dissolved in phosphate-buffered saline (PBS) at 100 mM and stored in
aliquots at 
20°C. MAb CH-11 was used for human Jurkat cells, while
the FLAG-CD95 ligand was chosen for murine DO cells, since MAb CH-11,
directed to human CD95, does not cross-react with murine CD95. The
FLAG-CD95 ligand was cross-linked with the anti-FLAG MAb M2 (Integra,
Fernwald, Germany).
Isolation of the cytosolic fraction.
The cytosolic fraction
was obtained essentially as described previously (29). Cells
were washed twice in ice-cold PBS and counted. Subsequently,
106 cells were suspended in 50 µl of ice-cold buffer A
(20 mM HEPES KOH [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 10 µg each of aprotinin and leupeptin [Sigma] per ml).
After incubation for 15 min on ice, the cells were disrupted by four
strokes in a 2-ml Kontes Dounce homogenizer with the B pestle (Kontes
Glass Co., Vineland, N.J.). First, the material was centrifuged at
750 × g for 10 min at 4°C. The resulting supernatant
was further centrifuged at 14,000 × g for 15 min at
4°C. The supernatant from this final centrifugation represents the
cytosolic fraction, of which 20 µg of protein was used for Western
blotting to detect cytochrome c.
Quantification of caspase activity.
The cells were treated
with the anti-CD95 MAb CH-11 at the indicated concentrations (see Fig.
7) for 4 h at 37°C. After treatment, 5 × 105
cells were washed and resuspended in chilled lysis buffer (1% Triton
X-100, 130 mM NaCl, 10 mM Tris [pH 7.4]). DEVD-aminomethylcoumarin (Alexis, Gruenberg, Germany) was used as a substrate to determine the
DEVDase activity. The amount of free aminomethylcoumarin was determined
by using a fluorometer (Victor; Wallac, Freiburg, Germany) with a
390-nm excitation filter and a 460-nm emission filter. The specificity
of the enzymatic reaction was assessed by using DEVD-CHO as an
inhibitor for the in vitro reaction.
Western blots.
Bcl-xL expression was analyzed
with a polyclonal rabbit antibody (Dianova, Hamburg, Germany).
Poly(ADP-ribose) polymerase (PARP) was detected with MAb C2-10
(Pharmingen, Hamburg, Germany), cytochrome c was stained
with MAb 7H8.2C12 (Pharmingen), and caspase-3 was analyzed with a MAb
(C31720) from Transduction Laboratories (Lexington, Ky.). A lysis
buffer consisting of 50 mM Tris-HCl (pH 8.0), 1% Triton X-100, 150 mM
NaCl, 2 mM EDTA, 5 mM NaF, and 10 µg each of aprotinin and leupeptin
(Sigma) per ml was used to analyze the expression of
Bcl-xL. PARP and caspase-3 were detected in cell lysates
prepared with 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate,
0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris (pH 8.0). The
cytoplasmic fraction, which was prepared as described above, was used
to detect released cytochrome c. Proteins were separated
under reducing conditions by SDS-polyacrylamide gel electrophoresis and
electroblotted on Immobilon P membrane (Millipore, Eschborn, Germany).
The blots were blocked with PBS containing 5% low-fat milk and 0.05%
Tween 20. They were incubated with the primary Abs and then with a
1:1,000 dilution of peroxidase-conjugated goat anti-mouse
F(ab)2 or a peroxidase-conjugated donkey anti-rabbit F(ab)2 fragment (Amersham, Braunschweig, Germany). Blots
were developed by using the enhanced chemiluminescence Western blot detection system (Amersham).
Cell death detection and flow cytometry.
DNA fragmentation
was detected essentially as described previously (23).
Briefly, the cells were washed in PBS and incubated in a lysis buffer
(1% Nonidet P-40, 100 mM EDTA, 50 mM Tris-HCl [pH 7.5]) for 10 s. The supernatant was obtained after centrifugation at 260 × g, and the lysis step was repeated with the pellet. These conditions result in an enrichment of DNA from cells undergoing apoptosis, because their nuclei are less resistant to detergent lysis.
After centrifugation, the supernatants were pooled and SDS was added to
a final concentration of 1%. Subsequently the material was digested
with RNase A and subsequently with proteinase K (Boehringer Mannheim).
The DNA was precipitated, washed with 70% ethanol, separated on an
agarose gel, and stained with ethidium bromide.
Cell death was quantified by flow cytometry as follows. The cells were
collected, washed once, incubated for at least 10 min
in PBS containing
20 µg of propidium iodide (PI) per ml, and analyzed
with a flow
cytometer (FACStrak; Becton Dickinson, Heidelberg,
Germany). Viable and
dead cells were distinguished by both forward-scatter
analysis and
fluorescence caused by PI uptake. The specific cell
death was
calculated as 100 × (percent experimental cell death
percent spontaneous cell death in medium)/(100%
percent spontaneous
cell death). Alternatively, cell death was quantified by a histone
release enzyme-linked immunosorbent assay (cell death detection
kit;
Boehringer Mannheim). The histone released after treatment
is
delineated as the enrichment factor, which was calculated as
described
by the manufacturer, i.e., optical density after treatment/optical
density of control cells.
Changes in mitochondrial membrane potential were evaluated with
rhodamine 123 (Molecular Probes, Leiden, The Netherlands).
Rhodamine
123 is taken up by intact mitochondria and released
upon permeability
transition of the mitochondria (
5,
20).
Cells that had been
treated to undergo apoptosis were incubated
with rhodamine 123 for 30 min at 37°C, washed, incubated with
PI, and then analyzed with the
FACStrak.
Annexin-V binding to phosphatidylserine exposed on the outer part of
the cell membrane was detected by incubating the cells
with fluorescein
isothiocyanate-conjugated annexin-V (Pharmingen)
for 30 min at room
temperature with a calcium-containing buffer
as specified by the
manufacturer. Then the solution was diluted
1:10 and measured by flow
cytometry within the next 30 min.
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RESULTS |
Expression of HVS-Bcl-2 and Bcl-xL in stably
transfected cells.
The expression of HVS-Bcl-2 in stably
transfected cell lines was analyzed by RNase protection assays.
Differences in the expression levels of different clones from Jurkat
cells were observed. Strong expression of HVS-Bcl-2 was detected in
clones Ju-H3, Ju-H10, and Ju-H2, with the highest level being found in
clone Ju-H3 (Fig. 1A). The clones in Fig.
1 were used for the following experiments and compared with four clones
that were transfected with the empty vector. Four HVS-transfected
clones derived from the murine T-cell hybridoma line DO expressed
HVS-Bcl-2 to a similar but lower degree (Fig. 1A). Western blot
analysis confirmed the overexpression of Bcl-xL in
transfected Jurkat cells (Fig. 1B). A low level of constitutively
expressed Bcl-xL in wild-type Jurkat cells could also be
detected under appropriate conditions (data not shown).
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FIG. 1.
Expression of HVS-Bcl-2 and Bcl-xL in
transfected cell lines. (A) Transcripts of HVS-Bcl-2 were detected by
RNase protection. Different HVS- bcl-2-transfected clones
derived from Jurkat cells (Ju) or DO cells were analyzed. Control cells
contained the expression vector pCEP4 without insert. In cell lines
transfected with HVS-bcl-2, the riboprobe of 700 nucleotides
(nt) specifically protected a fragment of 321 nucleotides. This
protected fragment of 321 nucleotides consists of 250 nucleotides of
HVS-bcl-2, 48 nucleotides of pCR2.1, and 23 nucleotides of
pCEP4. As a positive control, we used T-cell cultures from
Callithrix jacchus that release infectious virus and
transcribe HVS-bcl-2 (31). When RNA from these
cells is used, the applied riboprobe protects a fragment of about 250 nucleotides, representing the calculated length of the viral transcript
that is mirrored in the riboprobe. (B) The expression of
Bcl-xL in transfected Jurkat cells was detected by Western
blotting.
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Protection from apoptosis induced by CD95 ligation, oxygen
radicals, irradiation, and dexamethasone.
HVS-Bcl-2 and
Bcl-xL protected Jurkat cells from CD95-mediated apoptosis.
The results of one representative experiment of three with similar
results are shown in Fig. 2A. This figure
shows the mean percentages of cell death development after CD95
treatment of four different clones per transfected gene. When analyzing individual clones, we noted that there was a correlation between the
expressed amount of HVS-Bcl-2 (Fig. 1) and the degree of protection from apoptosis. Upon treatment with 1 ng of anti-CD95 per ml, all
tested HVS-Bcl-2-expressing clones were completely protected from cell
death while 35% of the cells from four control clones died at this
concentration of anti-CD95. Upon treatment with 10 ng of anti-CD95 per
ml, clone Ju-H3, with the highest expression, showed only 2% cell
death, clone H10 showed 17%, clone H2 showed 17%, clone H8 showed
31%, and control cells showed 74% specific cell death after 24 h.
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FIG. 2.
HVS-Bcl-2 and Bcl-xL protect Jurkat cells
(Ju) from apoptosis mediated by CD95 (A), irradiation (B), and
menadione (C). Four different clones transfected with HVS-Bcl-2, four
clones transfected with Bcl-xL, and four control clones
were treated with anti-CD95 (A) or irradiated (B) at the indicated
dosage. The specific cell death (mean ± standard deviation
[SD]) was determined after 24 h (A) or 72 h (B) as
described in Materials and Methods. The data for the individual cell
lines expressing HVS-Bcl-2 or Bcl-xL and for the control
cells were pooled for presentation in panels A and B. To determine the
sensitivity to menadione-induced cell death, two different clones of
each group were studied and the data were pooled (C). The development
of apoptosis was quantified by a histone release enzyme-linked
immunosorbent assay and the enrichment factor (mean ± SD) was
calculated as described in Materials and Methods.
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Irradiation and menadione-induced oxygen radicals triggered cell death
in Jurkat cells. The kinetics of cell death development
after these two
treatments were different. The menadione-induced
cell death was evident
after 4 h, whereas the irradiation-induced
cell death was observed
after 48 to 72 h. HVS-Bcl-2 effectively
protected Jurkat cells
from cell death induced by irradiation
as detected by measuring PI
uptake (Fig.
2B). Protection from
menadione-induced apoptosis was
detected by measuring PI uptake
(data not shown) and in a histone
release assay (Fig.
2C). A similar
degree of protection from this type
of cell death was observed
with cellular Bcl-x
L (Fig.
2B
and C).
To gain further insight into the antiapoptotic properties of HVS-Bcl-2,
a second lymphoid cell line, the murine T-cell hybridoma
line DO, was
transfected with HVS-Bcl-2. This cell line readily
undergoes apoptosis
upon treatment with dexamethasone. HVS-Bcl-2
partially protected these
cells from dexamethasone-induced apoptosis
as seen by reduced DNA
fragmentation and by measurement of PI
uptake (Fig.
3A and C). In contrast, the same clones
were not
protected from CD95-mediated cell death (Fig.
3B and D).
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FIG. 3.
HVS-Bcl-2 blocks apoptosis mediated by dexamethasone but
not by CD95 in DO cells. (A and C) DO cells transfected with HVS-Bcl-2
or control vector were treated with 10 nM dexamethasone and assessed
for DNA fragmentation after 48 h (A). The development of cell
death after dexamethasone treatment was quantified by measuring PI
uptake after 48 h (C). Four clones transfected with HVS-Bcl-2 and
five control clones were analyzed. The specific cell death (mean ± SD) is indicated (C). (B and D) To assess the sensitivity to
CD95-mediated apoptosis, the same transfectants were treated with
FLAG-CD95 ligand and anti-FLAG MAb. These cells were analyzed for DNA
fragmentation 24 h after treatment with 10 ng of CD95 ligand per
ml (B). Development of cell death (mean ± SD) was quantified by
measuring PI uptake after 24 h, and the specific cell death
(mean ± SD) is indicated (D).
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Prevention of phosphatidylserine exposure.
As an early event
during apoptosis, cells expose phosphatidylserine on their outer cell
membrane. When fluorescein isothiocyanate-conjugated annexin-V was
used, the exposure of phosphatidylserine was detectable 2 to 4 h
after CD95 ligation in control cells. In HVS-Bcl-2- and Bcl-xL-transfected cells, however, binding of annexin-V
after CD95 ligation was considerably reduced (Fig.
4).
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FIG. 4.
HVS-Bcl-2 and Bcl-xL block CD95-induced
exposure of phosphatidylserine. The indicated Jurkat transfectants were
treated with the CD95-specific MAb CH-11 at 100 ng/ml, and binding of
annexin-V was determined at the indicated times after addition of the
MAb.
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Stabilization of mitochondria.
Two methods were applied to
assess the integrity of mitochondria, namely, the retention of the
fluorescent dye rhodamine 123 and the release of cytochrome
c into the cytoplasm. Rhodamine 123 is released when the
mitochondrial membrane potential is disturbed (5, 20).
Jurkat cells transfected with HVS-Bcl-2, Bcl-xL, or control
vector were treated with anti-CD95 and the maintenance of their
mitochondrial membrane potential was assessed. Protection from
apoptosis by HVS-Bcl-2 or Bcl-xL was associated with
stabilization of mitochondria. Double staining with rhodamine 123 and
PI showed that cells that exclude PI retain rhodamine 123 (Fig.
5). Likewise, HVS-Bcl-2- or
Bcl-xL-induced protection from irradiation-induced apoptosis was associated with intact mitochondrial membrane potential, as seen by retention of rhodamine 123 in HVS-Bcl-2- and
Bcl-xL-transfected cells (data not shown). Engagement of
CD95 on Jurkat cells triggered a dose-dependent release of cytochrome
c from mitochondria into the cytosol. The release of
cytochrome c into the cytoplasm was inhibited by HVS-Bcl-2
and by Bcl-xL (Fig. 6).
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FIG. 5.
HVS-Bcl-2 and Bcl-xL prevent mitochondrial
damage. The indicated transfectants were cultured with anti-CD95 (10 ng/ml) or left untreated. After 16 h, the cells were stained with
rhodamine 123 for 30 min at 37°C, washed, suspended in PBS containing
PI, and analyzed by flow cytometry.
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FIG. 6.
HVS-Bcl-2 and Bcl-xL block the release of
cytochrome c into the cytosol. The indicated transfectants
were treated for 4 h with the indicated concentrations of
anti-CD95. Subsequently, the cytosolic fraction was prepared and
analyzed by Western blotting for the presence of cytochrome
c.
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Inhibition of caspase-3-like activity.
Treatment of Jurkat
cells with anti-CD95 induced the activity of caspases recognizing the
sequence DEVD. The CD95-mediated induction of this enzymatic activity
was blocked by HVS-Bcl-2 and by Bcl-xL (Fig.
7). As an additional method for the
detection of activation of caspases, we analyzed the cleavage of PARP,
a natural substrate of caspase-3 and related caspases. CD95 ligation triggered the cleavage of PARP. This molecule has a molecular mass of
116 kDa, and its cleavage by caspase-3-related caspases leads to a
85-kDa fragment. The cleavage of PARP as a consequence of CD95 ligation
was inhibited by HVS-Bcl-2 and by Bcl-xL (Fig. 8). To clarify whether the activity of
caspase-3 was inhibited by HVS-Bcl-2, the level of the inactive 32-kDa
proform of caspase-3 was determined by Western blotting. It is known
that CD95 engagement triggers the cleavage of the inactive proform of
caspase-3 to generate its active subunits (12). In control
cells, CD95 engagement led to a dose-dependent disappearance of the
32-kDa form (Fig. 9). Upon the same
treatments, HVS-Bcl-2- and Bcl-xL-transfected cells
continued to show the uncleaved form at a similar level (Fig. 9). We
noted that the applied MAb to caspase-3 gave only a very weak staining
of small subunits of the active enzyme. Therefore, the disappearance of
the 32-kDa proform was a more reliable marker of caspase-3 activation
than was the generation of a small subunit.
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FIG. 7.
HVS-Bcl-2 and Bcl-xL block the appearance of
caspase-3-like DEVDase activity. The different transfectants were
treated with anti-CD95 at the indicated concentrations for 4 h or
left untreated. The DEVDase activity in the cell lysates was
measured by using DEVD-aminomethylcoumarin as a substrate. The
specific DEVDase activity was calculated as the difference of
DEVDase activity in the absence and presence of the inhibitor
DEVD-CHO. The increase in the DEVDase specific activity after CD95
engagement was determined. The mean values for three individual cell
clones expressing HVS-Bcl-2, of three clones expressing
Bcl-xL, and of a control cell line are shown.
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FIG. 8.
HVS-Bcl-2 and Bcl-xL block the CD95-induced
cleavage of PARP. The indicated transfectants had been treated with the
indicated concentrations of anti-CD95 for 4 h. The cleavage of
PARP was analyzed by Western blotting.
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FIG. 9.
HVS-Bcl-2 and Bcl-xL prevent the
CD95-induced cleavage of caspase-3. The indicated transfectants had
been treated with different concentrations of anti-CD95 for 4 h.
The content of the 32-kDa uncleaved proform of caspase-3 was analyzed
by Western blotting.
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DISCUSSION |
Antiapoptotic spectrum of HVS-Bcl-2.
We found that HVS- Bcl-2
inhibits apoptosis triggered by radiation-induced DNA damage, by
menadione (which gives rise to oxygen radicals), and by dexamethasone.
HVS-Bcl-2 can also interfere with apoptosis mediated by the death
receptor CD95. In the human T-cell leukemia line Jurkat, HVS-Bcl-2
effectively blocked CD95-mediated cell death. In contrast, in the
murine T-cell hybridoma DO, HVS-Bcl-2 did not inhibit CD95-mediated
apoptosis, although it conferred resistance to glucocorticoid-induced
apoptosis in these cells.
Previous studies on the anti-apoptotic properties of cellular Bcl-2
family members have shown that Bcl-2 and Bcl-x
L inhibited
apoptosis induced by oxygen radicals, DNA damage, or growth factor
or
serum withdrawal in virtually all systems (
32,
46). On
the
other hand, protection from apoptosis mediated by the death
receptor
CD95 has been observed in some cell types (
5,
9)
but not in
others (
26,
41). The observation that cellular
and viral
Bcl-2 family members protect against CD95-mediated cell
death only in
some cell types might be explained by different
signaling cascades that
are involved in CD95-mediated apoptosis.
A well-understood signaling
pathway used upon engagement of CD95
involves the recruitment of the
adapter molecule FADD and subsequent
activation of FLICE (caspase-8)
(
43). This pathway is inhibited
by FLICE inhibitory proteins
(FLIPs) that are encoded by HVS and
other rhadinoviruses such as HHV-8,
bovine herpesvirus 4, and
equine herpesvirus 2 (
2,
25,
57,
63). Recently, cellular
FLIPs inhibiting CD95-mediated cell death
have also been discovered
(reviewed in reference
62). A second apoptotic pathway used
upon CD95
engagement, which is mediated by Daxx, has been described
(
65). Daxx-mediated apoptosis could be blocked by Bcl-2,
while
the pathway that uses FLICE is not modulated by Bcl-2
(
65).
Taken together, this suggests that HVS-Bcl-2 and
HVS-FLIP might
be complementary in their ability to inhibit
CD95-mediated apoptosis.
In our analysis, HVS-Bcl-2 and cellular Bcl-x
L inhibited a
similar spectrum of apoptotic pathways. Previous studies comparing
different cellular Bcl-2 family members with each other found
that
Bcl-2 and Bcl-x
L are functionally similar (
26)
but may
block cell death differentially in some systems (
18,
52).
Possible biological role of HVS-Bcl-2.
Two kinds of apoptotic
stimuli that eventually lead to apoptosis of virus-infected cells can
be distinguished. First, the infected cell may undergo a
cell-autonomous apoptosis occurring without attack by immune cells. In
particular, replication of herpesviruses and alphaviruses
(19) results in death of the infected cell. HVS-Bcl-2
interfered with apoptosis that occurs due to disturbances of the
intracellular homeostasis. This indicates that a cell-autonomous
apoptosis can be blocked by HVS-Bcl-2. This antiapoptotic activity may
result in prolongation of the life span of the infected cell and
consequently in higher viral replication. This is in line with the
observation that HVS-Bcl-2 protects against apoptosis induced by
heterologous infection (44). Second, in the host,
virus-infected cells are attacked by cytotoxic T cells, natural killer
cells, and macrophages. Cytotoxic T cells use two effector mechanisms
to destroy their targets, the secretion of lytic granules and the CD95
ligand (1). HVS-Bcl-2 is able to block CD95-mediated cell
death. This might attenuate the effect of cytotoxic T cells. Exposure
of phosphatidylserine is an early cellular change during the
development of apoptosis (38). HVS-Bcl-2 prevented the
exposure of phosphatidylserine. Also, this might be beneficial for the
virus, because exposure of phosphatidylserine on the surface of
apoptotic cells triggers specific recognition and removal by phagocytes
(15, 60). Whether protection from apoptosis is associated
with prevention of phosphatidylserine exposure depends on the
particular antiapoptotic strategy. Bcl-xL also interfered
with phosphatidylserine exposure. Another antiapoptotic principle,
blocking the activity of the p21-activated kinase 2, prevents cell
death but not phosphatidylserine exposure (47).
Since lymphotropic herpesviruses like EBV, HHV-8, and HVS that code for
a Bcl-2 homolog are potentially oncogenic, a role
of the viral Bcl-2 in
tumor formation might be expected. Prevention
of apoptosis is
frequently a component of tumor formation. Overexpression
of cellular
Bcl-2 due to a t(14;18) translocation leads to a follicular
B-cell
lymphoma (
59). On the other hand, the expression pattern
of
HVS-Bcl-2 does not support a role of this protein in oncogenesis,
since
its expression is restricted to cultures with lytic replication:
in
human T cells transformed to stable growth by HVS, this virus
persists
episomally and only a limited number of genes are expressed
(
16,
30). HVS-
bcl-2 is not transcribed in
growth-transformed
human T cells (
31). These findings
suggest that the main function
of HVS-Bcl-2 is to prolong the life span
of lytically infected
cells, thereby increasing viral replication.
Similar to HVS, the
Bcl-2 homolog of HHV-8 is predominantly a
lytic-cycle gene (
6,
49). The EBV-encoded Bcl-2 is also
expressed mainly during lytic
replication, is dispensable for
B-lymphocyte transformation in
vitro (
37), and is not
expressed in posttransplantation lymphoproliferative
disorders
(
42). Nevertheless, a role of herpesvirus Bcl-2 homologs
in
the initiation and development of certain tumors seems conceivable,
since Bcl-2 may cooperate with cellular oncoproteins like c-myc
and
also viral oncoproteins like E1A to induce transformation
(
11).
Mode of action of HVS-Bcl-2.
We have analyzed the effects of
HVS-Bcl-2 on the stability of mitochondria and on the activation of
caspases. To assess the mitochondrial integrity, rhodamine 123, an
indicator of mitochondrial membrane potential, was applied and the
release of cytochrome c in the cytoplasm was determined.
Both methods showed that the mitochondrial damage during the apoptotic
process is blocked by HVS-Bcl-2. A disruption of mitochondrial
integrity has been recognized as part of an apoptotic pathway induced
by different cell death-inducing stimuli (36). Therefore,
the stabilization of mitochondria by HVS-Bcl-2 is in line with the
broad spectrum of antiapoptotic activity of HVS-Bcl-2.
Multiple connections exist between disruption of mitochondria and
activation of caspases (
5,
55). Loss of mitochondrial
membrane potential has been implicated in the release of cytochrome
c, which then participates in triggering apoptosis (
34,
66).
In cell extracts, an apoptotic program could be induced by
the
addition of dATP (
34). Three protein factors, designated
apoptotic
protease-activating factors (Apaf-1, Apaf-2, and Apaf-3),
were
found to be necessary and sufficient to reconstitute
dATP-dependent
caspase-3 activation in such cell extracts
(
68). Apaf-1 is the
mammalian homolog of CED-4, Apaf-2 is
cytochrome
c, and Apaf-3
has been identified as caspase-9
(
33). According to these findings,
caspase-9 binds to Apaf-1
in a cytochrome
c- and dATP-dependent
fashion and becomes
activated under these conditions (
33). The
active caspase-9
then cleaves and activates caspase-3. Caspase-3
and related caspases
play major roles in the execution of nuclear
apoptosis (
13,
48) and also in extranuclear changes such as
exposure of
phosphatidylserine (
24). Caspase-3 and related caspases
cleave the DNA repair enzyme PARP (
45). Caspase-3 recognizes
the sequence DEVD (
22). To determine whether HVS-Bcl-2
modulates
the activation of caspase-3 and related caspases, three
different
methods were used. First, DEVDase activity induced upon
engagement
of CD95 was quantified in cell extracts. The induction of
this
enzymatic activity was reduced by HVS-Bcl-2. Second, cleavage
of
PARP upon CD95 engagement was determined and found to be blocked
by
HVS-Bcl-2. Third, the activation of caspase-3 was monitored
by
determining the level of the inactive 32-kDa precursor form
of
caspase-3. In control cells, CD95 engagement induced the cleavage
and
disappearance of the 32-kDa form of caspase-3. After the same
treatments, in HVS-Bcl-2-transfected cells, no loss of the uncleaved
caspase-3 was detected. Together, these experiments indicate that
HVS-Bcl-2 acts upstream of caspase-3. This is consistent with
our
observation that HVS-Bcl-2 blocks phosphatidylserine exposure
(see
above), since caspase-3 is involved in phosphatidylserine
externalization (
24). In our assays, HVS-Bcl-2 and
Bcl-x
L were
similar with respect to the stabilization of
mitochondria and
inhibition of caspase-3-like activity. This is in line
with recent
studies indicating that Bcl-2 and Bcl-x
L
interfere with the activation
of caspases (
8,
51), regulate
the membrane potential of mitochondria,
and inhibit cytosolic
cytochrome
c accumulation (
20,
28,
29,
64).
Bcl-x
L has also been reported to maintain cell viability
after the activation of caspases (
5).
In conclusion, HVS-Bcl-2 protects against diverse apoptotic stimuli,
stabilizes mitochondria, and acts upstream of the generation
of
caspase-3 like activity.
| |
ACKNOWLEDGMENTS |
We thank L. Boise and C. Thompson for the Bcl-xL
cDNA.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB
466), the Bayerische Forschungsstiftung, the Bundesministerium für Bildung und Forschung, and the EU (Shared Cost Action Project on Immunoregulatory Aspects of T Cell Autoimmunity in Multiple Sclerosis).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Klinische und Molekulare Virologie, Schlossgarten 4, D-91054
Erlangen, Germany. Phone: 49-9131-853786. Fax: 49-9131-856493. E-mail:
ermeinl{at}viro.med.uni-erlangen.de.
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J Virol, July 1998, p. 5897-5904, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
