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HIV-1 protease processes procaspase 8 to cause
mitochondrial release of cytochrome
c
, caspase cleavage
and nuclear fragmentation
Z Nie
1,2
, BN Phenix
1,2
, JJ Lum
1,2
, A Alam
3
, DH Lynch
4
,
B Beckett
2
, PH Krammer
5
, RP Sekaly
3
and AD Badley*
,1,2,6
1
Department of Biochemistry, Microbiology and Immunology, University of
Ottawa, Ottawa, Ontario, Canada
2
Ottawa Health Research Institute, Ottawa, Ontario, Canada
3
Institut de Recherches Cliniques de Montreal, Montreal, Quebec, Canada
4
Immunex Corporation, Seattle, Washington, USA
5
Tumor Immunology Program, German Cancer Research Center, Heidelberg,
Germany
6
Division of Infectious Diseases, Mayo Clinic and Foundation, Rochester,
Minnesota, USA
* Corresponding author: Andrew Badley, MD, FRCPC, Division of Infectious
Diseases, Mayo Clinic, 200 First Street NW, Rochester, Minnesota 55905,
USA. Tel: 507-255-7761; Fax: 507-255-7767.
Received 15.3.02; revised 17.6.02; accepted 17.6.02
Edited by RA Knight
Abstract
Infection of T cells with HIV-1 induces apoptosis and
modulates apoptosis regulatory molecules. Similar effects
occur following treatment of cells with individual HIV-1
encoded proteins. While HIV-1 protease is known to be
cytotoxic, little is known of its effect on apoptosis and
apoptosis regulatory molecules. The ability of HIV-1 protease
to kill cells, coupled with the degenerate substrate specificity
of HIV-1 protease, suggests that HIV-1 protease may activate
cellular factor(s) which, in turn, induce apoptosis. We
demonstrate that HIV-1 protease directly cleaves and
activates procaspase 8 in T cells which is associated with
cleavage of BID, mitochondrial release of cytochrome
c
,
activation of the downstream caspases 9 and 3, cleavage of
DFF and PARP and, eventually, to nuclear condensation and
DNA fragmentation that are characteristic of apoptosis. The
effect of HIV-1 protease is not se en in T cell extracts which ha ve
undetectable levels of procaspase 8, indicating a specificity
and requirement for procaspase 8.
Cell Death and Differentiation (2002) 9, 1172 ± 1184.
doi:10.1038/sj.cdd.4401094
Keywords: apoptosis; HIV; HIV-1 protease; caspase 8; mitochon-
drial permeability transition pore complex, cytochrome c
Abbreviations: AICD, activation induced cell death; ATCC,
American type cell culture; ATP, adenine trinucleotide phosphate;
BA, bongkrekic acid; BSA, bovine serum albumin; CHAPS,
cholamidopropyl dimethylammonio propane sulfonate; DFF, DNA
fragmentation factor; DMSO, dimethyl sulfoxide; DTT, dithiothreitol;
EDTA, ethylene diamine tetracetic acid; EGTA, ethylene glycol
tetracetic acid; FLIP, FLICE-like inhibitory peptide; HEPES,
hydroxyethyl piperazine ethane sulfonic acid; HIV, human
immunode®ciency virus; HIV-1 PI, HIV-1 protease inhibitor; HPLC,
high performance liquid chromatography; HRP, horseradish
peroxidase; PAGE, poly crylamide gel electropheresis; PARP, poly
(ADP Ribose) polymerase; PBL, peripheral blood lymphocyte;
PMSF, phenylmethylsulfonyl ¯uoride; SDS, sodium dodecyl
sulphate; TRAIL, TNF related apoptosis inducing ligand
Introduction
HIV-1 infection results in CD4 T cell apoptosis which
contributes to CD4 T cell depletion in infected individuals.
Multiple mechanisms have been proposed to explain
enhanced CD4 T cell apoptosis in HIV-1 infected persons.
HIV-1 infected accessory cells, including macrophages,
develop the ability to induce apoptosis of autologous
uninfected CD4 T cells by producing the apoptosis inducing
ligand, Fas (APO-1/CD95) Ligand (FasL).
1±10
AICD of T cells
is a physiologic response to activation
11 ± 14
which is greater in
HIV-1 infected individuals than in uninfected controls,
15 ± 19
and is potentially induced by tat and/or gp120 cross linking the
CD4 receptor
3,20 ± 22
resulting in increased expression of Fas
Ligand, TNF or TRAIL.
17,23
A third form of HIV-1 induced CD4
T cell death follows direct infection of a CD4 T cell with HIV-1,
and is independent of Fas receptor ligation.
8,15,24,25
While numerous HIV-1 proteins, including tat,
26 ± 28
gp120,
20±22
Nef,
29±32
vpr,
33 ± 35
and protease
36 ± 39
have been
implicated as direct mediators of infected CD4 T cell death, the
molecular mechanisms , whereby some of these HIV-1 specific
proteins induce apoptosis, including the mechanisms asso-
ciated with HIV-1 protease induced death, are unclear.
HIV-1 protease, a late regulatory protein in the HIV-1 life
cycle, functions as a homodimer
40
to cleave HIV-1
polyprotein. While ectopic expression of HIV-1 protease
induces apoptosis in a variety of cell types, including
human CD4 T cells,
36±39
coincubation of nuclei with HIV-1
protease does not induce the nuclear changes of
apoptosis,
40
suggesting that cytosolic factor(s) must be
activated by HIV-1 protease which in turn either directly or
indirectly causes nuclear fragmentation. The presence of
active HIV-1 protease within the cytosolic fraction of
infected cells
41,42
raises the possibility that cleavage of
non viral proteins by HIV-1 protease may contribute to the
cytotoxicity of HIV-1 infection. In support of this view, HIV-1
protease substrate specificity is not restricted to viral
proteins, since Bcl-2, actin, laminin B and pro-interleukin-
1 are cleaved by HIV-1 protease both in vitro and in
vivo.
36,43
Although some of the proteins cleaved by HIV-1
protease
36,43
are important in the regulation of apoptosis,
none alone is sufficient to induce apoptosis. We propose
Cell Death and Differentiation (2002) 9, 1172 ± 1184
ã
2002 Nature Publishing Group All rights reserved 1350-9047/02 $25.00
www.nature.com/cdd
that HIV-1 protease cleaves alternate apoptosis regulatory
molecules in such a manner that they develop the ability to
induce apoptosis.
Results
HIV-1 protease induces HeLa nuclear apoptosis
and DNA fragmentation in cell-free system
To determine if HIV-1 protease induces nuclear fragmenta-
tion, we modified a previously described cell-free system.
44,45
Cytoplasmic extracts from Jurkat T cells were treated with or
without HIV-1 protease and co-incubated with HeLa nuclei.
The nuclear membranes and chromatin of nuclei incubated
with untreated cytoplasmic extracts were intact (Figure 1A), in
contrast to nuclei coincubated with HIV-1 protease treated
cytoplasmic extracts which were marginated (Figure 1B) and/
or fragmented (Figure 1C). These nuclear effects of HIV-1
protease were inhibited by an HIV-1-PI (Figure 1D). Similarly,
nuclei incubated with HIV-1 protease treated cytoplasmic
extracts developed internucleosomal DNA cleavage (as
determined by DNA ladder analysis) which was also inhibited
by HIV-1-PI (Figure 2A). As a control, the human aspartyl
protease renin was used to treat cytoplasmic extracts, and, by
contrast, the renin treated cytoplasmic extracts did not induce
DNA laddering (Figure 2B), despite maintaining activity as
determined by cleavage of the fluorogenic renin substrate 1
(fluorescence of control cytosols=0 relative fluorescence
units, fluorescence of cytosols=22431 relative fluorescence
units). Since HIV-1 protease alone does not directly induce
the nuclear changes of apoptosis
40
(data not shown),
cytoplasmic signals must necessarily be activated by HIV-1
protease which, in turn, leads to the nuclear events of
apoptosis.
Caspase cascade is activated in cell extracts
treated with HIV-1 protease
We next assessed procaspase 8 and procaspase 3
processing after treatment of Jurkat cytoplasmic extracts with
HIV-1 protease. Both the 18 kd active fragment of caspase 8
and the 17 kd active fragment from caspase 3
46,47
were
detected following HIV-1 protease treatment but not in control
cytosols nor in renin treated cytoplasmic extracts (Figure 3A).
In HIV-1 protease treated, but not untreated cytoplasmic
extracts, cytochrome cwas released from mitochondria into
the cytoplasmic compartment (Figure 3B) in a comparable
manner to the release of cytochrome cseen with
recombinant active caspase 8 or Granzyme B, indicating
mitochondrial activation in treated cytoplasmic extracts.
Figure 1 HIV-1 protease induces the nuclear changes of apoptosis. Jurkat cytosols (1 mg) were treated with recombinant HIV-1 protease at 308C for 3 h and then
co-incubated with HeLa nuclei. Treated nuclei were imaged under microscopy by Hoechst 33342 staining. (A) Nuclei incubated with Jurkat cytosols without HIV-1
protease treatment. (B) Nuclei incubated with Jurkat cytosols treated with HIV-1 protease, resulting in fragmentation of the nuclear membrane and chromatin
condensation or (C) margination of chromatin. (D) The induction of apoptotic changes were completely inhibited by HIV-1-PI
HIV-1 protease activates caspase 8
ZNieet al
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Cell Death and Differentiation
Following the mitochondrial release of cytochrome c
48,49
into cytosols, cytosolic cytochrome ccomplexes with
APAF-1 in the presence of dATP to form the apoptosome
which allows the autoactivation of procaspase 9.
50,51
In
those samples where cytochrome crelease was seen,
procaspase 9 cleavage was also present, suggesting
formation of the apoptosome and downstream caspase
activation (Figure 3C).
We next determined whether HIV-1 protease mediated
cleavage of procaspase 8 is responsible for mitochondrial
activation. BID is a cytosolic member of the Bcl-2 family of
apoptosis regulatory proteins
52
that is cleaved by caspase
8 to create a truncated form of BID (tBID) which
translocates to mitochondria and causes the release of
cytochrome cinto the cytosol.
53
The p15 tBID form was
detected in the HIV-1 protease treated cytoplasmic extracts
but not in untreated cytosols (Figure 3D). Conversely, while
HIV-1 protease may cleave Bcl-2
43
we did not detect Bcl-2
cleavage in this assay (Figure 3D) although it was
observed after 4 h (data not shown). Following mitochon-
drial activation and downstream effector caspase activation,
cellular substrates, including PARP, are cleaved. Consis-
tent with our data indicating caspase activation in HIV-1
protease treated cytoplasmic extracts, but not untreated
cytoplasmic extracts, the 85 kd fragment of activated PARP
was seen only in HIV-1 protease treated cytoplasmic
extracts (Figure 3D). These data suggest that activated
caspase 8 cleaves BID to initiate the mitochondrial events
which lead to apoptosis. Kinetic analysis of cleavage of
procaspase 8 and cytochrome crelease was performed at
308C to slow the reaction, and analysed using Western
blot. In these experiments cytochrome cwas released after
the cleavage of procaspase 8 into its 18 kd active
fragments (Figure 3E).
Activation of caspase 8 leads to the activation of
downstream caspases
We next determined the kinetics of caspase activation. HIV-1
protease induced the processing of procaspase 8 as early as
1 min after adding HIV-1 protease at 378C (Figure 4A), and
cleavage of procaspase 3 into its 17 kd active fragment was
seen within 5 m. The relationship between caspase 8
cleavage and the cleavage of caspase 3, 9 and DFF were
next evaluated in reaction mixtures incubated at 308C. In
these experiments HIV-1 cleavage of both procaspase 8 and
3 induced by HIV-1 protease was inhibited by HIV-1-PI pre-
treatment, but only procaspase 3 cleavage was inhibited by
the caspase 8 inhibitor (IETD-fmk) (Figure 4B). The lack of
procaspase 8 inhibition by z-IETD-fmk indicates that
procaspase 8 activation is a consequence of HIV-1 protease,
rather than a result of autocatalysis. Thus both the timing of
caspase 3 cleavage (Figure 4A) and its inhibition by z-IETD-
fmk (Figure 4B) indicate that the cleavage of procaspase 3
depends upon prior caspase 8 activation. Furthermore,
cleavage of procaspase 9 occurred after the cleavage of
procaspase 8 (Figure 4B) and was inhibited by saquinavir and
partially inhibited by z-IETD-fmk (Figure 4B). Therefore both
caspase 3 and 9 activation occur after caspase 8 activation.
We also determined that cleavage of DFF (a DNAse,
activated by caspase 3, that contributes to nuclear fragmenta-
tion) into its 10 kd form occurred after 4 h in the treated
cytosols, and its cleavage was inhibited by HIV-1-PI and by z-
IETD-fmk (Figure 4B). These results demonstrate that HIV-1
protease treatment of cytoplasmic extracts results in
procaspase 8 processing which precedes and contributes to
processing of caspases 9 and 3 as well as DFF.
HIV-1 protease cleaves caspase 8 but not
caspase 3
The ability of HIV-1 protease to cleave pro-interleukin 1 into its
active subunits
54
infers that it may function as a caspase, a
suggestion that is supported by our data in Jurkat cytoplasmic
A
B
Figure 2 HIV-1 protease induces internucleosomal DNA fragmentation. (A)
DNA gel from nuclei incubated with Jurkat cytosols in the presence or absence
of HIV-1 protease with or without HIV-1 PI. (B) DNA gel from nuclei incubated
with Jurkat cytosols and treated with or without HIV-1 protease or renin
(control)
Cell Death and Differ entiati on
HIV-1 protease activates caspase 8
ZNieet al
1174
extracts showing that HIV-1 protease cleaves and activates
procaspase 8. To investigate this possibility further, recombi-
nant GST-caspase 8 was directly incubated with HIV-1
protease (Figure 5A). Within 1 min of co-incubation of
recombinant GST-procaspase 8 with HIV-1 protease, cas-
pase 8 is cleaved specifically by HIV-1 protease, as
demonstrated by the lack of autocatalysis of GST-caspase 8
and the inhibition of HIV-1 protease cleavage by HIV-1-PI
(HIV-1-PI does not inhibit caspase 8 activity (data not
shown)). Importantly, coincubation of HIV-1 protease with
full-length recombinant GST-caspase 8 generates p18
fragments, which have previously been associated with
caspase 8 activity.
47,55,56
To confirm the activity of the p18
caspase 8 fragments, we tested the ability of GST-caspase 8
treated with HIV-1 protease to cleave caspase 3, yet such
experiments did not result in caspase 3 cleavage (data not
shown). However, when HIV-1 protease was added after
GST-caspase 8 was cleaved by HIV-1 protease (to inhibit
remaining HIV-1 protease activity) (Figure 5B, top), and then
cytoplasmic extracts added, caspase 3 was cleaved (Figure
5B, bottom), suggesting the requirement of a mitochondrial
amplification step to cleave caspase 3. In these experiments
the effects of GST-caspase 8 cleavage products on caspase
3 were inhibited by z-IETD-fmk (Figure 5B, bottom). In
contrast to our results with GST-caspase 8, incubation of
recombinant caspase 3 with HIV-1 protease did not result in
cleavage, yet co-incubation of caspase 3 with Granzyme B
did, as previously described
57
(Figure 5C).
HIV-1 protease cleavage of procaspase 8 occurs at
an atypical site
The pattern of procaspase 8 cleavage that follows HIV-1
protease cleavage appears distinct from that seen with active
caspase 8 treatment (compare Figures 5A and B with Figures
3A, E and 4A, B), suggesting that the HIV-1 protease
cleavage site is different than the usual caspase 8 cleavage
site. We instead propose that HIV-1 protease generates
active caspase 8 (cleaved at an atypical site, Figure 5A, B),
which then activates more procaspase 8 (cleaved at the
typical site) resulting in the generation of p43, p41 and p18
fragments (Figures 3A, E and 4A, B).
To assess this possibility further, two sets of experi-
ments were performed. First we mutated the typical
cleavage of caspase 8. The initial cleavage event of
procaspase 8 activation occurs at ASP374,
56
within the
domain VETDSEEQ. Using a sequence coupled predictive
method of Markov chain theory,
58
this sequence would be
predicted to be cleaved by HIV-1 protease with a high
degree of likelihood. We therefore mutated this domain to
VDPDSDKQ, using site directed mutagenesis, as this
sequence is extremely unlikely to be cleaved by HIV-1
protease.
58
Both WT and mutant forms of GST-procaspase
8 were then reacted with HIV-1 protease. Analysis of
cleavage products by Western blot revealed identical
banding patterns, suggesting that HIV-1 protease cleavage
of procaspase 8 does not occur at this site.
To further address whether HIV-1 protease initiates
cleavage at this site, HIV-1 protease was incubated with
fluorogenic substrate z-IETD-AFC (Figure 6). Both active
caspase 8 and Granzyme B caused cleavage of z-IETD-
AFC, yet consistent with our mutational data, HIV-1
protease did not directly cleave z-IETD-AFC, supporting
the concept that HIV-1 protease activates procaspase 8 at
a site distinct from the typical activation site.
HIV-1 protease induced apoptotic signaling
requires procaspase 8
Our cumulative data thus demonstrate a direct effect of HIV-1
protease on procaspase 8, which is associated with the
downstream events of apoptosis including cleavage of BID,
release of cytochrome c, activation of caspases 9 and 3, as
well as cleavage of DFF and PARP. It remains possible that
HIV-1 protease initiated cleavage of other factors (e.g. other
initiator caspases) may also occur to initiate apoptotic
signaling. Thus, we assessed the ability of HIV-1 protease
to initiate apoptotic signaling in cells which are deficient in
procaspase 8. Cytosolic extracts of JB6 cells and I9.2 cells
which are a procaspase 8 deficient T cell derivatives were
treated with or without HIV-1 protease and cleavage of
procaspase 8, BID, procaspase 3 assessed. As expected,
while JB6 and I9.2 cells had undetectable levels of
procaspase 8, Jurkat T cell procaspase 8 was processed by
HIV-1 protease. Only in the Jurkat T cell extracts treated with
HIV-1 protease, was there any evidence of cleavage of BID or
of procaspase 3 (Figure 7), indicating that the presence of
procaspase 8 in Jurkat T cells is required for activation of the
downstream apoptotic signaling events, since the absence of
procaspase 8 in JB6 and I9.2 cells prevents downstream
apoptotic signaling.
Direct infection of HIV-1 causing cell death is
correlated with HIV protease expression and
requires active caspase 8
To determine whether HIV-1 protease expression is corre-
lated with the induction of apoptosis, we analyzed the
expression of protease in relation to the timing of apoptosis
in Jurkat T cells acutely infected with HIV-1. In this model of
acute HIV-1 infection, cell death by apoptosis occurs several
days following infection,
15
is inhibited by z-VAD-fmk, z-IETD-
fmk (Figure 8A) and by Saquinavir (data not shown), and is
associated with caspase 8 and PARP cleavage (Figure 8B).
Further cell death coincides with detectable expression of
HIV-1 protease (Figure 8C). Freshly isolated PBL were also
collected from six HIV-1 negative controls and from two
untreated patients infected with HIV-1 were analyzed for
expression of HIV-1 protease and for PARP cleavage into an
85 kd apoptosis characteristic fragments.
59,60
In control
patient 1 (who had an upper respiratory tract infection) and
HIV-1 patients 2 to 6 the 85 kd PARP fragment was present,
indicating that PBLs from these patients were undergoing
apoptosis (Figure 8D). Conversely the 85 kd PARP was not
present in control patient 2 and HIV patients 1. Expression of
HIV-1 protease was seen only in HIV-1 patients 2 ± 6 who had
high levels of viral replication (4500,000 copies/ml) and
importantly HIV-1 protease was not detected in HIV-1 patient
1 who had a low level of viral replication (1600 copies/ml).
Thus, given previous literature which demonstrate that HIV-1
Cell Death and Differentiation
HIV-1 protease activates caspase 8
ZNieet al
1175
A
B
C
Cell Death and Differ entiati on
HIV-1 protease activates caspase 8
ZNieet al
1176
protease cleaves actin into an HIV-1 protease specific pattern
in vivo, as well as in vitro,
36
our observations that apoptosis in
HIV-1 infection coincides with HIV-1 protease expression
support a possible role for HIV-1 protease apoptosis
associated with directly infected cells.
Discussion
The demonstration of caspase activation by HIV-1 protease is
significant for several reasons. First, the ability of HIV-1
protease to induce T cell apoptosis represents another
potential mechanism whereby HIV-1 may cause death of
HIV-1 infected T cells. This mechanism applies only to cells
directly infected by HIV-1, as addition of HIV-1 protease to cell
cultures does not influence cell viability (data not shown). The
relative importance of this mechanism, in comparison to other
proposed mechanisms of HIV-1 associated T cell depletion
(reviewed in
61
), including AICD, autologous cell mediated
killing, and direct virus induced killing associated with gp120,
Nef, Tat and/or Vr is however unclear. Secondly, the ability of
viruses to influence apoptosis has been well characterized,
62
and a number of virally encoded proteins have been shown to
interact with members of the caspase family to inhibit
apoptosis: these include baculovirus IAP and p35, Adeno-
virus E1B-19k, Cowpox Crm-A, Epstein Barr virus BHRF1,
62
and gamma herpes virus FLIP.
63
In contrast, HIV-1 protease
is an example of a virally encoded protein that activates
caspase 8 to promote apoptosis.
D
E
Figure 3 Caspase activation and mitochondrial release of cytochrome coccurs in Jurkat cytosols treated with HIV-1 protease. Jurkat cytosols were treated with
HIV-1 protease and cleavage of procaspases 8, 3, 9 and the caspase substrates, BID, Bcl-2 and PARP were assessed along with mitochondrial release of
cytochrome c.(A) Cleavage profiles of procaspase 8 and 3 indicating active p18 and p17 fragments respectively in the cytosols treated with HIV-1 protease, but not
those treated with renin. (B) Jurkat cytosols were incubated with HIV-1 protease, recombinant active caspase 8 or Granzyme B, fractionated and analysed for
cytochrome ccontent in the total reaction mixture, mitochondrial fractions or mitochondria free cytosolic fraction. (C) Cleavage profile of procaspase 9 indicating
p35 fragment in cytosols treated with HIV-1 protease, as well as cytochrome crelease from mitochondria. HSP 70 is analysed as a control mitochondria specific
protein. (D) The cleavage of BID, Bcl-2 and PARP induced by HIV-1 protease treatment of cytosols. PCNA is included as an internal control. (E) Jurkat cytosols
(1 mg) were treated with HIV-1 protease at 308C, and assayed at the indicated times for analysis of caspase 8 cleavage and cytochrome c release. PCNA was used
as an internal control
Cell Death and Differentiation
HIV-1 protease activates caspase 8
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1177
The HIV-1 genome is translated as polyprotein fusions
that require processing by HIV-1 protease. These poly-
proteins are processed by HIV-1 protease in two cellular
compartments: first, as membrane-associated polyproteins
that are cleaved for viral assembly and maturation, and
second, as free polyproteins within the cytosols
41,42
of
infected cells. Previous studies have shown that HIV-1
protease can induce apoptosis in both transfected and
microinjected cells.
43,64,65
Furthermore, a variety of cellular
proteins, including the antiapoptotic regulating protein Bcl-2
and cell structure proteins such as laminin B and
cytoskeleton proteins, are substrates of HIV-1 protease in
vitro and in vivo.
39,43,54,65,66
These observations suggest
that the degenerate substrate specificity of HIV-1 protease
allows protease to activate proteins which initiate apoptosis
cascades.
In the present study, we have developed a cell-free
system to characterize the mechanisms by which HIV-1
B
A
Figure 4 Activation of procaspase 8 by HIV-1 protease leads to cleavage of
downstream caspases. (A) At the indicated times, 100 mg cytosol proteins
were probed with anti-caspase 8 and 3. (B) In parallel cleavage of caspase 9
and DFF were assessed. As indicated either the caspase 8 inhibitor z-IETD-
fmk or HIV-1-PI were used
A
B
C
Figure 5 HIV-1 protease directly cleaves GST-procaspase 8 but not
procaspase 3. Purified recombinant GST-procaspase 8 (A) was incubated
for the indicated times with HIV-1 protease, with or without HIV-1-PI and
analyzed for cleavage. Reactions were stopped at the indicated times by
addition of gel loading buffer. (B) GST-caspase 8 was incubated with HIV-1
protease for 30 min and analyzed for caspase 8 cleavage (top). Thereafter
reactions were stopped by the addition of HIV-1 PI, and Jurkat cytosols added
and analyzed for caspase 3 cleavage (bottom). (C) Treatment of procaspase 3
with HIV-1 protease does not alter procaspase 3, whereas Granzyme B results
in cleavage of procaspase 3
Cell Death and Differ entiati on
HIV-1 protease activates caspase 8
ZNieet al
1178
protease induces apoptosis. Cell-free systems have been
successfully used to identify the apoptotic molecules and
their signal pathways.
44,45,67
In our system, treatment of
cytosols with HIV-1 protease initiates a pathway that
involves activation of both caspases and mitochondrial
events involved in apoptosis (Figure 9). Further, we
demonstrate that the apical and requisite event in this
pathway is the cleavage of procaspase 8 by HIV-1
protease, which in turn activates BID, causes mitochondrial
release of cytochrome c, activation of caspases 9 and 3 as
well as cleavage of DFF and PARP. The requirement for
mitochondria in this apoptosis cascade is demonstrated by
observations that GST caspase 8 activated by HIV-1
protease does not cleave caspase 3. Only when GST
caspase 8 was incubated with HIV-1-PR, PI added (to
inhibit protease) and the entire reaction added to
cytoplasmic extract was caspase 3 activated (Figure 5B).
However, as it is now recognized that activated caspase 8
can initiate apoptosis directly via caspase 3 (type 1
pathway) or indirectly via mitochondrial activation, cyto-
chrome-crelease and caspase 9 processing (type 2
pathway),
68,69
we cannot exclude the possibility that HIV-
1 protease mediated apoptosis may involve both type 1 as
well as type 2 signaling pathways. Indeed, when T cell
extracts treated with HIV-1 protease in the presence or
absence of the mitochondrial PTPC inhibitor BA were
analysed for caspase 3 and caspase 9 activation, BA
resulted in partial, but incomplete inhibition of caspase 3
and 9 activation, thereby indicating that both type I and type
II pathways are likely involved (data not shown). The
results are consistent with previous work which demon-
strate that activation of procaspase 8 is sufficient to induce
changes in a cell-free system that are similar to those seen
during apoptosis in vivo.
47
In the present study we demonstrate that procaspase 8
is required for HIV-1 protease induced apoptosis, as both
JB6 and I9.2 cells which are deficient in procaspase 8 do
not develop the molecular changes of apoptosis following
HIV-1 protease treatment. However, since our evidence
that HIV protease activates caspase 8 physiologically is
indirect, it remains possible that it may also act on different
substrates to initiate death pathways. Additional studies are
underway to address these possibilities.
Treatment of HIV-1 infected patients with inhibitors of
HIV-1 protease has dramatically reduced both morbidity
and mortality associated with this infection. Thus far, two
reasons for the improved outcomes are apparent: first,
protease inhibitors are potent inhibitors of viral replication
70
and second, this class of drugs possesses intrinsic
immunomodulatory properties including antiapoptotic ef-
fects.
61,71
We suggest that direct inhibition of HIV-1
protease also reduces protease induced apoptosis of
infected cells to further reduce HIV-1 associated T cell
death. Further research is therefore required to determine
the contribution of this form of cell death on the
pathogenesis of HIV-1 disease, and the effect of HIV-1
protease mutations on the pathogenesis of HIV-1 induced
immunodeficiency.
Materials and Methods
Preparation of cell-free extracts
Cell-free extracts were freshly prepared from human Jurkat T
lymphoblastoid cells (ATCC, Rockville, MD, USA) as described
previously
44,45
with some modifications. Briefly, cells (0.5610
6
cells/ml) were harvested by centrifugation at 16006gfor 5 min at
48C. The cell pellet was washed twice with ice-cold PBS (pH 7.4),
followed by a single wash with ice-cold caspase buffer (20 mM PIPES,
100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS, 250 mM
sucrose, pH 7.2).
72
After centrifugation, the cells were resuspended
with two volumes of ice-cold complete caspase buffer which was
supplemented with protease inhibitors (100 mmPMSF,10mg/ml
leupeptin, 2 mg/ml aprotinin) and then transferred to a 2-ml dounce
homogenizer. After sitting on ice for 15 min, the cells were disrupted
with 50 strokes of B-type pestle (Fisher Scientific Ltd, Nepean, ON,
Canada). Cell disruption (495%) was confirmed by examination of
5ml aliquot of suspension under a light microscope after staining with
Figure 6 Effect of active caspase 8, Granzyme B and HIV protease on z-
IETD-AFC. The caspase 8 autoactivation cleavage site fluorogenic substrate
z-IETD-AFC was incubated with recombinant active caspase 8, Granzyme B,
or with either 0.1 or 1 mg of HIV protease as indicated, and fluorescence
measured every 2 min for 30 min Figure 7 Extracts from Jurkat T cells, JB6 or I9.2 cells were treated with HIV-
1 protease, and analysed for caspase 8, caspase 3 and BID cleavage. PCNA
is used as an internal control
Cell Death and Differentiation
HIV-1 protease activates caspase 8
ZNieet al
1179
Trypan blue. The nuclei were removed by the centrifugation at
10006gfor 10 min at 48C. Protein concentrations were determined
with BCA protein assay kit (Pierce Chemical Co, Rockford, IL, USA).
JB6 cells and I9.2 cells which are procaspase 8 deficient T cell
derivatives, were a kind gift of Dr. S Nagata
73
and Dr. J Blenis
74
respectively. JB6 and I9.2 cells were handled in an identical manner to
the method described for Jurkat T cells above.
Preparation of HeLa nuclei
HeLa cell (ATCC, Rockville, MD, USA) nuclei isolation was performed
as described.
75
Nuclei were freshly prepared for each experiment from
the 80% confluent cultures of HeLa cells. Cells were washed three
times with ice-cold PBS (pH 7.4), followed by a single wash with ice-
cold nuclear buffer (10 mM PIPES, 80 mM KCL, 20 mM NaCl, 250 mM
sucrose, 5 mM EGTA, 1 mM DTT, 0.5 mM spermidine, 0.2 mM
spermine, 1 mg ml protease inhibitors, pH 7.4). The cell pellet was
resuspended with two volumes of ice-cold nuclear buffer. The cells
were disrupted with 50 strokes of B-type pestle and 495% lysis
confirmed by Trypan blue exclusion. Nuclei were pelleted (10006gfor
10 min at 48C) and washed twice with nuclear washing buffer (10 mM
PIPES, 10 mM KCl, 2 mM MgCl
2
,1mMDTT,10mM cytochalasin B,
1mgml
71
protease inhibitors, pH 7.4).
HIV-1 protease treatment of cytosolic extracts
Cell extracts treated with HIV-1 protease were carried out in 100 ml
cell-free reaction buffer (complete caspase buffer supplemented with
10 mM phosphocreatine, 2 mM ATP and 150 mg/ml creatine
phosphokinase). The concentration ratio of cytosol proteins and
HIV-1 protease was 1000 : 1. The final concentration of HIV-1 protease
was between 0. 5 ± 1 mg per reaction mixture. HIV-1 protease was
purchased (Bachem Bioscience Inc - King of Prussia, PA, USA) with a
specific activity of 1.81610
4
mmole/min/mg at 378C, with a purity of
496% by SDS ± PAGE and a single peak by RP ± HPLC. Where
indicated, the HIV-1 protease inhibitors (HIV-1-PI) Saquinavir 10 mM
(Roche Laboratories, Mississauga, Ontario, used for data described in
Figures 3 and 4) or Nelfinavir 7 mM (Agouron Laboratories,
Mississauga, Ontario, Canada), used for data described in all Figures
except 3 and 4) were used. Where indicated the human aspartyl
protease renin (Sigma Aldrich Canada Ltd, Oakville, ON, Canada) was
used as a control. Renin substrate 1 (Molecular Probe Inc., Eugene,
OR, USA) was used to measure renin activity in cytosol mixtures
according to the supplied protocol. z-IETD-fmk (Enzyme Systems
Products, Livermore, CA, USA) was used in some experiments as
indicated, at 100 mM dissolved in DMSO (Sigma, Irvine, UK).
Nuclei incubation with HIV-1 protease treated
cytoplasmic extracts
First, a mixture of cytoplasmic extracts and HIV-1 protease were
incubated at 308C for 4 h in cell-free reaction buffer. Then, aliquots of
20 ml HIV-1 protease treated cytoplasmic were incubated with 80 mlof
HeLa cell nuclei (5610
6
nuclei) at 378C in nuclear apoptosis buffer
(nuclear washing buffer supplemented with 2 mM ATP and 5 mM
EGTA). Apoptotic nuclei were determined by Hoechst staining and
DNA fragmentation assay.
Hoechst staining
HeLa nuclei were stained with Hoechst 33342 (Molecular Probes,
Eugene, OR, USA) as previously described
67
in fixing buffer (10%
formaldehyde, 50% glycerol, 100 mM NaCl, 2 mM KCl, 1 mM MgCl2,
0.1 mM EDTA, 1 mg/ml Hoechst 33342, 5 mM HEPES, pH 7.8). The
stained nuclei were imaged under fluorescence microscopy (Zeiss
AxioCAM, Jena, Germany).
DNA fragmentation assay
The DNA fragmentation assay was performed as described.
76
Briefly
2±5610
6
nuclei were pelleted for 20 min at 48C, and dispersed in
Figure 8 Jurkat T cells were infected or mock infected (HIV-) with HIV
IIIb
,in
the presence or absence of z-IETD-fmk, z-VAD-fmk or z-DEVD-fmk, and
assessed for viability (A). Infected cells harvested on day 8 were then
analyzed for caspase 8 and PARP cleavage (B), or for HIV protease
expression (C). HIV protease expression and PARP cleavage were also
assessed in bulk PBL from HIV positive or negative patients, as indicated (D)
Cell Death and Differ entiati on
HIV-1 protease activates caspase 8
ZNieet al
1180
30 ml of lysis buffer (10 mM Tris, 100 mM NaCl, 25 mM EDTA, 0.5%
Sarkosyl) by gentle vortexing. Forty micrograms protease K (Qiagen
Inc., Mississauga, ON, Canada) was added and incubated at 528C
overnight. Then, 40 mg RNase (Sigma, Irvine, UK) was added and
incubated for 2 h at room temperature. The fragmented DNA in the
lysates was detected by 2% agarose gel electrophoresis.
SDS ± PAGE and Western blot
For Western blot analysis, 50 ± 200 mg of cytosolic proteins were
fractionated on 4 ± 15% gradient polyacrylamide gels (Biorad
Laboratories Canada Inc., Hercules, CA, USA), then transferred
onto PVDF membranes (Millipore, Bedford, MA, USA) for 1 h at
100 V using transfer buffer (25 mM Tris, 192 mM glycine, 20%
methanol). The membranes were blocked by incubation in TBS
buffer (20 mM Tris, 500 mM NaCl, 0.05% Tween, pH 7.5)
containing 5% milk for overnight at 48Cor2hatroom
temperature and washed five times with TBS buffer. Then, the
membranes were blotted for 1 h at room temperature with the
various dilutions of primary antibodies, specifically, monoclonal
anti-caspase 8 (Biosource International, Camarillo, CA, USA), anti-
caspase 9 (Medical & Biological Laboratories Co., Watertown, MA,
USA), anti-cytochrome c(BD Pharmingen, Mississauga, ON,
Canada), anti-PARP (Oncogene, Darmstadt, Germany) and anti-
Bcl-2 (Calbiochem, La Jolla, CA, USA), anti-PCNA (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), rabbit anti-caspase 3
77
and
rabbit anti-cFLIP (Alexis Biochemicals, San Diego, CA, USA), goat
anti-BID, anti-actin and anti-DFF45 (Santa Cruz Biotechnology).
The blots were washed five times with TBS and developed with
HRP linked secondary antibodies, sheep anti-mouse Ig, donkey
anti-rabbit Ig (Amersham Pharmacia Biotech, Oakville, ON,
Canada) and anti-goat IgG (Santa Cruz Biotechnology). All the
blots were developed by SuperSignal (Pierce, Rockford, IL, USA),
an enhanced chemiluminescence method, following the manufac-
turer's protocol.
Generation of recombinant caspase 3 and
caspase 8
GST-caspase 8 was made by subcloning full-length cDNA caspase 8
into pGEX-4T-1 (Amersham Pharmacia Biotech) and expression of
GST ±Caspase 8 performed by IPTG stimulation at 308C, according to
the manufacturers instructions, in the presence of 100 uM EGTA and
EDTA. The human caspase 3 cDNA was amplified by RT ± PCR with
the following primers: 5-GATGGAGAACACTGAAAAACTC-3 and 5-
ATCCAACCAACCATTTCTTTAGTG-3 from Jurkat total RNA and
subcloned into BamHI and EcoRI sites of pBSKS+(Stratagene, Cedar
Creek, TX, USA) and sequenced. To produce recombinant caspase-3,
the cysteine 163 of the active site was mutated to serine in order to
avoid autocatalysis. The mutagenesis was performed by overlapping
PCR using PBSKS+caspase-3 as the template, and the mutation was
then confirmed after cloning and sequencing of the PCR product. The
caspase-3-C163S was then subcloned into pGEX2TK (Amersham
Pharmacia Biotech) and transformed into DH5 alpha. Purified
Caspase 3 was made as previously described, followed by removal
of the GST tag by thrombin digestion.
77
Figure 9 Putative role of HIV protease in HIV pathogenesis
Cell Death and Differentiation
HIV-1 protease activates caspase 8
ZNieet al
1181
Cleavage reactions of recombinant caspases
Reactions to assess the ability of HIV-1 protease to cleave
recombinant caspases were performed under the following
conditions: 3 ml of purified recombinant GST-caspase 8 or caspase
3 were mixed with 10 ml of HIV-1 protease buffer (100 mM Na
acetate, 1 mM EDTA, 1 M NaCl, 1 mM DTT, 1 mg/ml BSA pH 4.7)
in the absence or presence of 0.5 mg HIV-1 protease (2 ml)
preincubated for 15 min at room temperature with either 2 mlof
methanol, or 2 ml of 10 mM Saquinavir in methanol. In the case of
caspase 3, Granzyme B (Enzyme Systems, Livermore, CA, USA)
was used as a positive control for cleavage, at the indicated
concentrations. The final reaction mixtures were incubated for the
indicated times at 378C. Cleavage products were then analyzed by
Western blot analysis.
Cytochrome
c
release assay
Cytochrome crelease assay was modified according to a previous
publication.
78
Crude cell extracts were supplemented with an ATP
regenerating system (10 mM phosphocreatine, 2 mM ATP and
150 mg/ml creatine phosphokinase). At various time points, HIV-1
protease treated cytosols were harvested and centrifuged twice at
15 000 g(48C) for 15 min to fractionate the cytosolic (supernatant)
fraction from the mitochondrial pellet. Aliquots of 20 ml cytosolic
protein (200 mg) were separated by 4 ± 15% gradient SDS ± PAGE and
probed with monoclonal antibody against cytochrome c. As indicated,
recombinant active caspase 8 (Biomol, Plymouth Meeting, PA, USA)
or Granzyme B (Enzyme Systems Products, Livermore, CA, USA)
were used as positive controls.
Caspase inhibitors
The caspase consensus site inhibitors z-DEVD-fmk, z-IETD-fmk and
z-VAD-fmk were purchased from Enzyme Systems. Independent
experiments were performed to validate the inhibitory effects of z-
DEVD-fmk, z-IETD-fmk or z-VAD-fmk on caspase activation. Jurkat T
cells were stimulated with recombinant leucine zipper Fas Ligand
(10 g/ml, Immunex) for 6 h at 378C in the absence or presence of z-
DEVD-fmk, z-IETD-fmk or z-VAD-fmk (Enzyme Systems), at con-
centrations ranging from 3 to 300 mM. Each inhibitor blocked
recombinant Fas Ligand (Immunex Corp, Seattle, WA, USA) induced
cell death at all concentrations, in a dose dependant manner (data not
shown).
Cells and HIV infection
Jurkat T cells were purchased from ATCC and maintained in RPMI
medium supplemented with 10% fetal calf serum (FCS, GIBCO).
For experiments using patient peripheral blood lymphocytes (PBL),
consenting patients or controls donated 20 mls of blood into
heparinized tubes, and PBLs extracted using ficol hypaque density
gradient centrifugation, and plastic adherence.
56
Resultant PBL
were cultured in RPMI 1640-10% human AB serum, supplemented
with penicillin/streptamycin and glutamine (Gibco). HIV infection
using HIV IIIb (NIH AIDS Reference Reagent Program) was
performed as previously described;
79
briefly virus containing
supernatants (or mock infected supernatants) were propogated in
PBMC from HIV uninfected donors. Cells are infected by overnight
culture with virus containing (or mock) supernatant (
45
373 pg of
p24/ml). Cell viability following infection was assessed by Trypan
blue exclusion.
Fluorogenic release assays
To assess the activity of different enzymes against z-IETD-AFC
(Enzyme Systems), caspase 8 (Enzyme Systems), 180 mgof
Granzyme B, 0.1 or 1.0_g of HIV protease were added to either
caspase 8 buffer (100 mM HEPES, pH 7.5, 10% v/v sucrose, 10 mM
DTT, 0.5 mM EDTA),
80
Granzyme B buffer (50 mM HEPES, pH 7.4,
0.1% CHAPS, 0.1 M NaCl, 10% v/v sucrose, 10 mM DTT)
81
or to HIV
protease buffer (100 mM Na Acetate, 4 mM EDTA, 300 mM NaCl,
pH 4.7)
82
to achieve a final volume of 500 ml. After 30 min, with the
reaction mixture in a fluorimeter (CytoFluor 2300, Millipore) adjusted to
400 nm excitation, 505 nm emission, 20 mlofz-IETD-AFC(20mM
stock) was added, and fluorescence release measured every 2 min
until 30 min.
82
Data presented representative of results obtained using
all three buffers. Independent experiments using the HIV protease
fluorogenic substrate. DABCYL-_-Abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-
Gln-EDAN (Bachem, King of Prussia PA, USA) were performed with
each buffer to confirm the activity of HIV protease under these
conditions (data not shown).
Acknowledgements
Informed consent was obtained from all subjects prior to blood collection,
and the study was reviewed and approved by the ethics committee of the
Ottawa Hospital/University of Ottawa. The authors gratefully acknowl-
edge the helpful discussions and advice of Dr. BWD Badley, as well as
the administrative expertise of A Carisse. BN Phenix and JJ Lum are
supported by a Studentship award from the Ontario HIV Treatment
Network (OHTN). AD Badley is supported by an OHTN Career Scientist
Award. This work is supported by grants from the Doris Duke Foundation
(#T98026), the Canadian Institute of Health Research (#HOP-36047),
the Canadian Foundation for AIDS Research and a Premier's Research
Excellence Award.
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