HIV gp120 induces, NF-kappaB dependent, HIV replication that requires procaspase 8.
ABSTRACT HIV envelope glycoprotein gp120 causes cellular activation resulting in anergy, apoptosis, proinflammatory cytokine production, and through an unknown mechanism, enhanced HIV replication.
We describe that the signals which promote apoptosis are also responsible for the enhanced HIV replication. Specifically, we demonstrate that the caspase 8 cleavage fragment Caspase8p43, activates p50/p65 Nuclear Factor kappaB (NF-kappaB), in a manner which is inhibited by dominant negative IkappaBalpha. This caspase 8 dependent NF-kappaB activation occurs following stimulation with gp120, TNF, or CD3/CD28 crosslinking, but these treatments do not activate NF-kappaB in cells deficient in caspase 8. The Casp8p43 cleavage fragment also transactivates the HIV LTR through NF-kappaB, and the absence of caspase 8 following HIV infection greatly inhibits HIV replication.
Gp120 induced caspase 8 dependent NF-kappaB activation is a novel pathway of HIV replication which increases understanding of the biology of T-cell death, as well as having implications for understanding treatment and prevention of HIV infection.
- SourceAvailable from: Julià Blanco[show abstract] [hide abstract]
ABSTRACT: Syncytia arising from the fusion of cells expressing a lymphotropic human immunodeficiency virus (HIV)-1-encoded envelope glycoprotein complex (Env) gene with cells expressing the CD4/CXCR4 complex undergo apoptosis through a mitochondrion-controlled pathway initiated by the upregulation of Bax. In syncytial apoptosis, phosphorylation of p53 on serine 15 (p53S15) precedes Bax upregulation, the apoptosis-linked conformational change of Bax, the insertion of Bax in mitochondrial membranes, subsequent release of cytochrome c, caspase activation, and apoptosis. p53S15 phosphorylation also occurs in vivo, in HIV-1(+) donors, where it can be detected in preapoptotic and apoptotic syncytia in lymph nodes, as well as in peripheral blood mononuclear cells, correlating with viral load. Syncytium-induced p53S15 phosphorylation is mediated by the upregulation/activation of mammalian target of rapamycin (mTOR), also called FKBP12-rapamycin-associated protein (FRAP), which coimmunoprecipitates with p53. Inhibition of mTOR/FRAP by rapamycin reduces apoptosis in several paradigms of syncytium-dependent death, including in primary CD4(+) lymphoblasts infected by HIV-1. Concomitantly, rapamycin inhibits p53S15 phosphorylation, mitochondrial translocation of Bax, loss of the mitochondrial transmembrane potential, mitochondrial release of cytochrome c, and nuclear chromatin condensation. Transfection with dominant negative p53 has a similar antiapoptotic action as rapamycin, upstream of the Bax upregulation/translocation. In summary, we demonstrate that phosphorylation of p53S15 by mTOR/FRAP plays a critical role in syncytial apoptosis driven by HIV-1 Env.Journal of Experimental Medicine 11/2001; 194(8):1097-110. · 13.21 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Apoptosis of CD4(+) T lymphocytes, induced by contact between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and its receptors, could contribute to the cell depletion observed in HIV-infected individuals. CXCR4 appears to play an important role in gp120-induced cell death, but the mechanisms involved in this apoptotic process remain poorly understood. To get insight into the signal transduction pathways connecting CXCR4 to apoptosis following gp120 binding, we used different cell lines expressing wild-type CXCR4 and a truncated form of CD4 that binds gp120 but lacks the ability to transduce signals. The present study demonstrates that (i) the interaction of cell-associated gp120 with CXCR4-expressing target cells triggers a rapid dissipation of the mitochondrial transmembrane potential resulting in the cytosolic release of cytochrome c from the mitochondria to cytosol, concurrent with activation of caspase-9 and -3; (ii) this apoptotic process is independent of Fas signaling; and (iii) cooperation with a CD4 signal is not required. In addition, following coculture with cells expressing gp120, a Fas-independent apoptosis involving mitochondria and caspase activation is also observed in primary umbilical cord blood CD4(+) T lymphocytes expressing high levels of CXCR4. Thus, this gp120-mediated apoptotic pathway may contribute to CD4(+) T-cell depletion in AIDS.Journal of Virology 09/2001; 75(16):7637-50. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) can induce apoptosis by a cornucopia of distinct mechanisms. A soluble Env derivative, gp120, can kill cells through signals that are transmitted by chemokine receptors such as CXCR4. Cell surface-bound Env (gp120/gp41), as present on the plasma membrane of HIV-1-infected cells, can kill uninfected bystander cells expressing CD4 and CXCR4 (or similar chemokine receptors, depending on the Env variant) by at least three different mechanisms. First, a transient interaction involving the exchange of lipids between the two interacting cells ('the kiss of death') may lead to the selective death of single CD4-expressing target cells. Second, fusion of the interacting cells may lead to the formation of syncytia which then succumb to apoptosis in a complex pathway involving the activation of several kinases (cyclin-dependent kinase-1, Cdk1; checkpoint kinase-2, Chk2; mammalian target of rapamycin, mTOR; p38 mitogen-activated protein kinase, p38 MAPK; inhibitor of NF-kappaB kinase, IKK), as well as the activation of several transcription factors (NF-kappaB, p53), finally resulting in the activation of the mitochondrial pathway of apoptosis. Third, if the Env-expressing cell is at an early stage of imminent apoptosis, its fusion with a CD4-expressing target cell can precipitate the death of both cells, through a process that may be considered as contagious apoptosis and which does not involve Cdk1, mTOR, p38 nor p53, yet does involve mitochondria. Activation of some of the above- mentioned lethal signal transducers have been detected in patients' tissues, suggesting that HIV-1 may indeed trigger apoptosis through molecules whose implication in Env-induced killing has initially been discovered in vitro.Cell Death and Differentiation 09/2005; 12 Suppl 1:916-23. · 8.37 Impact Factor
HIV gp120 Induces, NF-kB Dependent, HIV Replication
that Requires Procaspase 8
Gary D. Bren1, Sergey A. Trushin1,2, Joe Whitman1, Brett Shepard1, Andrew D. Badley1,2*
1Division of Infectious Diseases, Mayo Clinic, Rochester, Minnesota, United States of America, 2Program in Translational Immunovirology and Biodefense, Mayo Clinic,
Rochester, Minnesota, United States of America
Background: HIV envelope glycoprotein gp120 causes cellular activation resulting in anergy, apoptosis, proinflammatory
cytokine production, and through an unknown mechanism, enhanced HIV replication.
Methodology/Principal Findings: We describe that the signals which promote apoptosis are also responsible for the
enhanced HIV replication. Specifically, we demonstrate that the caspase 8 cleavage fragment Caspase8p43, activates p50/
p65 Nuclear Factor kB (NF-kB), in a manner which is inhibited by dominant negative IkBa. This caspase 8 dependent NF-kB
activation occurs following stimulation with gp120, TNF, or CD3/CD28 crosslinking, but these treatments do not activate NF-
kB in cells deficient in caspase 8. The Casp8p43 cleavage fragment also transactivates the HIV LTR through NF-kB, and the
absence of caspase 8 following HIV infection greatly inhibits HIV replication.
Conclusion/Significance: Gp120 induced caspase 8 dependent NF-kB activation is a novel pathway of HIV replication which
increases understanding of the biology of T-cell death, as well as having implications for understanding treatment and
prevention of HIV infection.
Citation: Bren GD, Trushin SA, Whitman J, Shepard B, Badley AD (2009) HIV gp120 Induces, NF-kB Dependent, HIV Replication that Requires Procaspase 8. PLoS
ONE 4(3): e4875. doi:10.1371/journal.pone.0004875
Editor: Derya Unutmaz, New York University School of Medicine, United States of America
Received September 25, 2008; Accepted February 18, 2009; Published March 16, 2009
Copyright: ? 2009 Bren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Grant Support: Dr. Andrew Badley is supported by the following grants: NIH R01 AI62261, R01 AI40384 and a Burroughs Wellcome Award ID #1005160.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The HIV env is a pleotrophic molecule which causes a range of
effects on human cells, by ligating either the CD4 or chemokine
receptors, env can cause activation, anergy, and/or apoptosis of
the receptor bearing cell . In addition, HIV env can
independently enhance HIV replication , potentially through
NFAT activation .
A prominent effectof gp120 on host cells is induction of apoptosis.
Depending upon cell type and activation status, gp120 induced
apoptosis can occur following CD4 crosslinking, or CXCR4
crosslinking,and despite early reports to the contrary, suchapoptotic
signaling cascades are caspase dependent [4–6]. The molecular
signals which initiate gp120 induced apoptosis include the Fas/Fas
ligand system and/or P38 MAPK . In either situation,
mitochondrial depolarization, release of cytochrome c, and
formation of the apoptosome ensue . This activates effector
caspases 9 and 3,which function to activate initiatorcaspases suchas
caspase 8 to amplify the apoptotic cascade , and they also cleave
host regulatory and structural proteins which promote the
phenotypic characteristics of apoptosis.
Recently, a non-apoptotic role for procaspase 8 has become
recognized: Nuclear factor kB (NF-kB) activation in response to
antigen receptor, Fc receptor, or TLR2, 3, 4 ligation requires the
presence of procaspase 8 [10,11]. In response to these stimuli,
procaspase 8 complexes with Ikkb, resulting in phosphorylation
and proteasomal degradation of Ikba, followed by phosphoryla-
tion and nuclear translocation of p65 [10,11]. More recently,
TRAF6 has been suggested to bind caspase 8, promoting the
movement of this complex into lipid rafts . The interaction of
TRAF6 with caspase 8 is enhanced by caspase 8 processing ,
suggesting that cleavage of the caspase 8 zymogen enhances the
ability of caspase 8 to activate NF-kB. Also, the structurally related
cFLIP can initiate NF-kB activation via TRAF2 , in a manner
that is enhanced by its prior cleavage by caspase 8 .
Since HIV env initiates apoptosis and stimulates HIV replication,
we questioned whether these events were related, and if so, whether
procaspase 8 was involved in the enhanced HIV replication.
Jurkat and I9.2 T cells (ATCC) as well as primary human CD4
T cells were grown in RPMI 1640 supplemented with 10% fetal
bovine serum and 2 mM Glutamine. Primary human peripheral
blood lymphocytes and cells from HIV-infected patients were
obtained following informed consent. This protocol was reviewed
and approved by the Mayo Clinic institutional review board
(protocol #1039-03). 293T cells were cultured in DMEM plus
10% FBS and 2 mM Glutamine. HIV infections were performed
using HIV IIIb using a high MOI of 2.5 mg/ml p24 in the
infected supernatant. TNFa (R&D Systems, Minneapolis, MN)
was used at 10 mg/ml where indicated. Anti-CD3 (Ortho Biotech,
Rariton, NJ) and anti-CD28 (BD Pharmingen, San Jose, CA) were
PLoS ONE | www.plosone.org1March 2009 | Volume 4 | Issue 3 | e4875
used at 1 ug/ml to mimic T cell receptor activation. Where
indicated camptothecin (Sigma, St. Louis, MO) was used at
10 mm. Gp120 was purchased from Immunodiagnostics (Woburn,
MA), SDF was purchased from R&D systems (Minneapolis, MN).
CD4 T cells were isolated (98% CD4 T cells as determined by
flow cytometry) from the blood of healthy volunteer blood donors
by using RossetteSep CD4 enrichment cocktail in accordance with
the manufacture’s protocol (StemCell Technologies, Vancouver,
British Columbia, Canada). The expression of activation markers
as CD69 and HLA-DR on resting CD4 T cells were determined
by flow cytometry.
Resting CD4 T cells (CD4+/CD692/HLA-DR-) were incubated
with HIV-164 gp120IIIB (Immuno Diagnostics,Inc.Woburn,MA)
or gp120 IIIB pretreated with soluble CD4 (1:2 ratio) (Immuno
Diagnostics, Inc. Woburn, MA) at concentrations of 1 mg/ml/
26106cells for 30 minutesonice andthenincubated for 24 hours at
37uC. The following day, cell death was analyzed by staining with
AnnexinV-Cy-5 following the manufacturer’s instructions (BD
Biosciences). All experiments were performed at least three times.
PCR, Plasmids and Transfection
Nef was PCR amplified from DNA extracted from normal or
HIV IIIB infected Jurkats and I9.2 cells using primers and
protocol described by Zhu, et al . The procaspase 8 cDNA was
a gift from Dr. Marcus Peter. The genes encoding p50, p65, IkBa,
and IKKc were PCR amplified from a human cDNA library and
cloned into pcDNA3 (Invitrogen, Carlsbad, CA). The Casp8p43
construct was created by site directed mutagenesis of the serine at
amino acid 375 to a stop codon. A dominant negative IkBa was
created to block IKK activation of NF-kB by PCR generated
mutagenesis of serines 32 and 36 to Alanine . For mammalian
expression, the constructs were cloned into either pEGFP or
pcDNA3 (Invitrogen, Carlsbad, CA). All plasmids were confirmed
by DNA sequence analysis and tested for expression prior to
The luciferase reporter constructs HIV-1 LTR Luc and HIV-1
LTR DKB Luc have been previously described . The TK-
Renilla plasmid, purchased from Promega (Madison, WI), was
used as an internal control in all reporter plasmid transfections.
Transfection efficacies of 30–40% are routinely achieved in these
experiments as assessed by parallel transfections with expression
vectors encoding fluorescent proteins. Results are expressed as
luciferase per Renilla expression in order to normalize for
variability between transfection efficiency and cell viability,
between experimental groups, and between experiments.
Jurkat and I9.2 T cells were transfected with 1 ug plasmid/10^6
cells using an Electro Square Porator T820 (BTX, San Diego, CA)
at 300 volts for 10 msec. Transfection of primary CD4 T cells was
done using AMAXA. For the caspase 8 reconstitution experi-
ments, I9.2 Jurkat cells were electroporated with mammalian
expression vectors coding for GFP or GFP-caspase 8 along with
the HIV LTR-Luciferase and TK-Renilla reporter plasmids and
incubated 18 hours at 37uC. 10^6 cells were then incubated with
or without either 1 ug/ml HIV IIIB gp120 + 0.5 ug.ml soluble
CD4 or 20 nM SDF-1a for 24 hours at 37uC. The cells were then
harvested and HIV LTR activity was measured using the Dual
Light Assay Kit (Promega, Madison, WI) as per the manufactur-
Stable Cell Lines
The Jurkat T-derivative cell line, I9.2, deficient in procaspase 8
protein expression, was transfected with expression vectors encoding
for either green fluorescent protein (GFP), or procaspase 8 wild type
conjugated to GFP (GFP-casp 8 WT). After transfection, the cells
were placed in media containing 800 ug/ml Geneticin and cultured
for 14 days passing cells every three days with fresh media and
Geneticin. The cells were then checked for GFP expression by
fluorescent microscopy and for protein expression by western
blotting. The transfected cells were maintained in media containing
500 ug/ml Geneticin. For electroporation experiments the Genet-
icin was removed 24 hours in advance.
Nuclear Protein Extraction and Electromobility Shift
293T cells were transfected with empty vector or plasmids
coding for full length procaspase 8 or p43 caspase 8. After six
hours at 37uC, nuclear proteins were harvested as previously
described , 5 ug of nuclear protein was allowed to bind to a
32-P labeled, double stranded, oligonucleotide encompassing the
GACTTTCCGCTGGGGACTTTCCAGGG-39) at room tem-
perature in the presence or absence of antibodies specific for the
NF-kB transcription factors p50 or p65 (Rel A) (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). The DNA-nuclear protein
complexes were run on a 6% non-denaturing polyacrylamide gel
at a constant voltage of 170 volts. The gel was then dried onto
filter paper and exposed to autoradiography film.
HIV-1 p24 Antigen Assay
Culture supernatants were harvested and assayed with the
RETROtek HIV-1 p24 ELISA kit (ZeptoMetrix Corp., Buffalo,
NY) for the presence of the HIV-1 antigen p24.
Standard error was calculated by standard deviation divided by
‘n’. Comparison was made using T tests with significance being
defined as p,0.05.
Human CD4 T-cells (10^6) were incubated with or without
1 ug/ml HIV IIIB gp120 + 0.5 ug/ml soluble CD4 (Immuno-
diagnotics Inc., Woburn, MA), or 0.1 ug/ml anti-CD95 (CH11
clone from Upstate Biotechnologies, Billerica, MA) at 37uC for
48 hours. The cells were harvested and lysed in 20 mM Tris/HCl
pH 7.5, 150 mM NaCl, 0.1% Triton X-100 with protease
inhibitors (2 ug/ml Aprotinin, 10 ug/ml Leupeptin, 2 ug/ml
Pepstatin, and 1 mM PMSF) for ten minutes on ice. The lysates
were centrifuged at 12,0006g and the supernatants run on 10%
PAGE, transferred to PVDF membrane and western blotted with
antibodies specific for human caspase 8 (c-14 clone form Dr.
Marcus Peter) and B-actin.
HIV gp120 potently induces HIV replication
Gp120 has been proposed to mediate death of uninfected CD4
T cells; however whether gp120 kills resting CD4 T cells in the
absence of activation is controversial. Therefore, we first analyzed
the effect of gp120 on resting CD4 T cells from HIV negative
donors (Figure 1A). Primary T cells from HIV-negative donors
were first infected with HIV, and then stimulated with stimuli
known to induce apoptosis: TNF, an agonistic anti-Fas antibody
(CH11), viral gp120, and camptothecin (CPT). Doses were chosen
that induced similar degrees of apoptosis (Figure 1B). Strikingly,
each of these proapoptotic signals induced HIV replication. In
addition, those stimuli that induce procaspase 8 activation (TNF,
CH11, and gp120) induced more viral replication than does
Caspase 8 and HIV replication
PLoS ONE | www.plosone.org2 March 2009 | Volume 4 | Issue 3 | e4875
camptothecin, which acts to induce mitochondrial apoptosis
independent of caspase 8 activation . Equally striking is the
observation that gp120 induced the greatest p24 production of all
the caspase 8 dependent stimuli. We next assessed the impact of
gp120 and other apoptosis inducers on HIV replication in primary
CD4 T cells from HIV-infected patients (Figure 1C). CD4 T cells
from four HIV-infected donors were isolated by negative selection,
cultured overnight, and then stimulated with gp120 (IIIb), and
other apoptosis inducing agents as indicated, and analyzed for p24
production 18 hours later (Figure 1C). No correlations were
observed between in vitro p24 production, and CD4 count or viral
load. HIV gp120 induced maximal HIV replication, yet in this
context, HIV replication reflected virus produced from cells
infected with HIV in vivo. Clearly, HIV gp120 is a potent inducer
of HIV replication in primary HIV-infected CD4 T cells, although
the mechanisms underlying this enhanced replication are
HIV Replication that Occurs During Apoptosis is NF-kB
NF-kB is a transcription factor that is a key regulator of
inflammation, apoptosis, immune activation, cell proliferation, and
viral replication. Multiple stimuli are capable of activating NF-kB
including, but not limited to, proinflammatory cytokines (e.g.,
TNF-a), activating cellular receptors (e.g., TCR), viral proteins
(e.g., EBV-LMP-1), DNA cleavage, chemotherapeutics (doxoru-
bicin), and oxidative stress. We have previously assessed the
involvement of NF-kB family members in HIV replication that
occurs during the peak of virus-induced death by demonstrating
that a super repressor form of IKBa blocks HIV replication,
thereby implicating NF-kB family members as the dominant
transcriptional mechanism that drives such HIV replication .
We next asked whether gp120-induced HIV replication is
mediated by transcriptional upregulation.
First, we treated cells from HIV-infected patients with gp120, in
the presence or absence of a dominant negative form of the NF-kB
inhibitor IkBa. Cells were then cultured overnight and p24
production monitored. Cells treated with gp120 and transfected
with vector control had a significant induction of p24 compared to
those not treated with gp120. The cells transfected with DN IkBa
had significantly impaired p24 production, indicating that gp120
induction of HIV replication is NF-kB dependent (Figure 2A).
Next, we transfected Jurkat T cells with a luciferase reporter
construct under control of HIV LTR. In parallel, TK-Renilla was
co-transfected as a control for transfection efficiency. Cells were
then stimulated with gp120/sCD4 in order to isolate the effect of
gp120 signaling to signaling only through the CXCR4 receptor;
and luciferase expression was measured and normalized to TK-
Renilla (Figure 2B). Indeed, gp120 treatment increased luciferase
expression demonstrating a transcriptional mechanism underlying
the effects of HIV gp120 on HIV replication. This effect required
caspase 8 because the Jurkat-derived caspase 8 deficient cell line,
I9.2, which expresses CXCR4 (MCF in isotype Jurkat 31.8;
CXCR4 in Jurkat = 73.6; MCF isotype in I9.2 = 31.3 CXCR4
in I9.2 = 69.4,) failed to upregulate HIV LTR activity after
treatment with gp120 (Figure 2B). Of interest, I9.2 cells also failed
to upregulate LTR activity in response to TNF, which also induces
caspase 8 activation. Finally, we observed that only gp120, but not
SDF-1a, which is the natural ligand for CXCR4, caused
upregulation of HIV LTR activity in a caspase 8 dependent
manner (Figure 2C). The inability of caspase 8 deficient cells to
respond to gp120 by increasing HIV LTR activation is specifically
due to the lack of caspase 8 since I9.2 cells reconstituted with
procaspase 8 have a restored response to gp120 stimulation
(Figure 2D). We therefore focused attention on gp120 induced
caspase 8 dependent HIV LTR activation.
Casp8p43 Cleavage Intermediate Activates NF-kB Driven
Activation of the HIV LTR
Recently, caspase 8 and certain of its homologs (caspase 10, c-
FLIP, FADD) have been identified as modulators of the NF-kB
response . Because caspases are critical to apoptosis induced
by HIV, we questioned whether they or their cleavage products
also might initiate HIV transcription. First, we confirmed that
gp120 treatment results in procaspase 8 cleavage (Figure 3A).
Consequently, we generated plasmids expressing full length
Figure 1. HIV gp120 induces HIV replication in resting CD4 T
cells. (A) Primary CD4 T cells were harvested, sorted into the resting
subset, and stimulated with gp120, and analyzed for death. (B) Primary
CD4 T cells harvested from uninfected donors were infected or not with
HIV IIIb and cultured for seven days. The infected cells were then
divided and cultured at 10^6/ml with media, 10 ng/ml TNF, 0.5 ug/ml
CH11 anti-Fas receptor antibody, 1.0 ug/ml gp120 IIIb, or 10 uM
camptothecin. A p24 ELISA was performed on culture supernatants as
per the manufacturer’s instructions. (C) PBLs were harvested from HIV-
positive donors. The cells were washed three times and cultured over
night in RPMI 1640 plus 10% FBS. The cells were then washed three
additional times and cultured at 1610^6 without stimulation, or with
10 ng/ml TNF, 0.5 ug/ml CH11 anti-Fas antibody, 1.0 ug/ml HIV IIIb
gp120, or 10 uM camptothecin for 18 hours at 37uC. Supernatants were
tested for the presence of the HIV protein p24 by ELISA.
Caspase 8 and HIV replication
PLoS ONE | www.plosone.org3 March 2009 | Volume 4 | Issue 3 | e4875
caspase 8, or the Casp8p43 cleavage product, and assessed their
ability to cause transactivation of a HIV LTR luciferase reporter
construct. In such experiments, we observed that the Casp8p43
cleavage product was a more potent inducer of HIV LTR activity
than full length caspase 8 (Figure 3B). Moreover, this effect is
inhibited by coexpression of the regulatory subunit (IKKc) of the
IKK signaling complex or a super repressor form of IKBa
(Figure 3C). Casp8p43 failed to transactivate the HIV LTR with
deletions of the NF-kB binding domains (DkB), which indicates
that Casp8p43-mediated transactivation of the wild-type HIV
LTR is mediated specifically by NF-kB. Finally, EMSA analysis
confirmed Casp8p43 driven NF-kB activation (Figure 3D) induced
by the p50/p65 heterodimer, as indicated by inhibition by p50 or
p65 antibodies. Such data link apoptosis signaling to NF-kB
activation, in a manner that requires caspase cleavage (as
Casp8p43 is only present after caspase 8 activation), potentially
as a homeostatic attempt of a dying cell to block its own death by
initiating NF-kB driven activation of antiapoptotic regulatory
proteins. Moreover, our hypothesis provides and explanation for
prior observations : Jurkat T cells stably transfected with HIV
LTR-Luc were stimulated to die by ultraviolet (UV) irradiation
and LTR driven luciferase expression measured. HIV transcrip-
tion was induced by UV, and also was inhibited by a pan-caspase
inhibitor (Z-VAD-Fmk), indicating that HIV LTR activation by
UV requires caspase activation . Because UV irradiation
causes apoptosis by activating a caspase 8 dependent death
pathway , these data are consistent with our hypothesis that
caspase 8 cleavage intermediates drive HIV replication. In
contrast, apoptosis induced by staurosporin (a direct mitochon-
driotoxin, which does not activate caspase 8), induced high levels
of death, but did not activate the HIV LTR .
HIV Infection of Caspase 8 Deficient T Cells Results in
Reduced Apoptosis and Impaired HIV Replication
Given our experimental data suggesting that the caspase 8
cleavage product p43 drives HIV transcription, we opted to assess
the relevance of this observation during experimental HIV
infection. Since caspase inhibitors do not prevent procaspase 8
autoprocessing which generates p43 , we used I9.2 cell lines
deficient in caspase 8. We confirmed expression of both CD4 and
Figure 2. Enhanced HIV replication by gp120 requires NF-kB and caspase 8. (A) Primary CD4 T cells from HIV-infected patients were
transfected with vector control or dominant negative IkB and treated with gp120 as indicated. P24 was measured the following day. (B) Jurkat or I9.2
cells were transfected with a luciferase reporter under the control of the HIV LTR promoter and a Renilla expressing plasmid as an internal control and
cultured 18 hours with or without HIV gp120 (5 ug/ml) plus sCD4 (2.5 ug/ml) where indicated TNF was used as a positive control. The cells were
harvested and the luciferase activity was measured and expressed in terms of fold increase over the control. (C) Jurkat or I9.2 cells transfected as
above and treated with SDF as indicated and HIV LTR Luc measured, normalized to TK-Renilla, and expressed as fold increase over control. (D) I9.2
cells were transfected with empty vector (left) or procaspase 8 (right), stimulated with gp120 or SDF, and HIV LTR activity measured.
Caspase 8 and HIV replication
PLoS ONE | www.plosone.org4March 2009 | Volume 4 | Issue 3 | e4875
CXCR4 in I9.2 cells (data not shown). Consistent with previous
observations, infection of Jurkat T cells results in significant
apoptosis that peaks on day 9, and high levels of viral replication,
as measured by HIV p24 production. By contrast, and as would be
predicted if caspase 8 were essential for both HIV-induced
apoptosis and HIV replication, infection of I9.2 cells resulted in
less death and less viral replication (Figure 4A). Successful infection
of these cells was confirmed by DNA PCR for HIV Nef, indicating
that the initial events of attachment, insertion and reverse
transcription occur in both Jurkat and I9.2 cells (Figure 4B), albeit
to a lesser degree in the I9.2 cells. We therefore directly assessed
whether caspase 8 is required for HIV replication by using a
system that does not depend on HIV infection. Jurkat T cells or
caspase 8-deficient I9.2 cells were transfected with a luciferase
reporter construct under control of the HIV LTR, along with TK-
Renilla as a control for transfection efficacy. These cells were then
stimulated with agents known to induce HIV replication: TNF or
CD3/CD28 crosslinking and luciferase measured. Whereas,
Jurkat T cells responded to such treatment with robust luciferase
expression, I9.2 cells did not produce significant luciferase in
response to TNF or CD3/CD28. However, when I9.2 cells were
first reconstituted with procaspase 8, I9.2 cells became responsive
to both TNF and CD3/CD28 stimulation of the HIV LTR
(Figure 4C). These data demonstrate that caspase 8 can restore
HIV LTR-driven transcription in caspase 8 deficient I9.2 cells.
Our observations that caspase 8 is required for optimum NF-kB
dependent gp120 mediated HIV LTR activation, and that the
Casp8p43 cleavage product is a more potent activator of HIV
LTR than full length caspase 8 offers insights into the biology of
apoptosis. That Casp8p43 more efficiently drives HIV LTR
activity than does full length caspase 8 is implicitly intuitive since
all cells contain full length caspase 8, and it would be potentially
harmful for all cells to have NF-kB activation. However, upon
Figure 3. Casp8p43 induces NF-kB dependent HIV LTR transcription. (A) Jurkat T cells were treated with gp120 or CH11 anti-Fas antibody
and analyzed for caspase 8 cleavage by western blot. (B) Jurkat T cells transfected with kB luciferase reporter plasmid, TK-Renilla as a control for
transfection efficiency, and either control vector or vector expressing full length caspase 8, or p43 subunit of caspase 8. As a positive control, NF-kB
elements p65/p50 were used as indicated. Results are representative of three independent experiments. (C) Jurkat T cells were transfected with HIV
LTR or HIV LTR missing the KB motifs (HIV LTR D KB), along with TK-Renilla as a transfection control, and the Casp8p43 with or without IKKc, dominant
negative (DN) IkBa as indicated. Data are expressed as Luciferase units, normalized to TK-Renilla and representative of three independent
experiments. (D) 293T cells were transfected with the indicated constructs. The following morning nuclear extracts were prepared and incubated with
a P32 labeled HIV LTR NF-kB probe in the presence or absence of antibodies to NF-kB proteins p50 and p65. Then complexes were run on a 6%
nondenaturing gel, and analyzed using autoradiography.
Caspase 8 and HIV replication
PLoS ONE | www.plosone.org5March 2009 | Volume 4 | Issue 3 | e4875
receipt of a death stimulus, NF-kB activation likely represent a
compensatory reaction of that dying cell in an attempt to
upregulate KB dependent antiapoptotic gene transcription. In
an HIV-infected cell, the compensatory NF-kB response has the
additional effect of stimulating HIV LTR transcription, and HIV
replication. Furthermore, it is tempting to speculate that this death
initiated NF-kB response is a reason why HIV evolved to be
regulated transcriptionally by NF-kB.
When applied to HIV pathogenesis, such a model of apoptosis
resulting in NF-kB activation allows the prediction that inhibiting
caspase 8, and consequently inhibiting apoptosis, might result in
two independent beneficial outcomes for HIV-infected patients.
First, since causes of CD4 T cell loss include exaggerated rates of
apoptosis, induced by a variety of viral and host stimuli, inhibiting
caspase 8 activation would likely reduce the rate of CD4 T cell
loss. In addition, and perhaps unexpectedly, our current data
suggest that caspase 8 inhibition might have the additional benefit
of reducing viral replication. Attempts to therapeutically modify
apoptosis in vivo have principally focused on promoting apoptosis
for cancer therapy. Our observations suggest an additional reason
to explore inhibition of apoptosis in cells from HIV-infected
We have recently described a novel pathway of apoptosis
initiated by HIV, wherein HIV Pr, which is present and active
within the cystosolic fraction, interacts and cleaves procaspase 8,
creating a novel, HIV specific caspase 8 fragment, we call
Casp8p41 . Casp8p41 has two independent functions:
induction of mitochondrial dependent apoptosis [21,26], and
activation of NF-kB . Therefore, both gp120 and HIV
protease each contribute to enhanced HIV replication indirectly
through NF-kB activation, both in a caspase 8 dependent manner
(albeit different mechanisms of caspase 8 activation). It will be of
interest to determine whether other HIV specific proteins (eg., Tat
and Nef) which have also been ascribed the two seemingly
independent activities of apoptosis induction and enhanced viral
replication, might also be linked via caspase 8.
Knowledge that gp120 independently drives viral replication
offers insights into the means by which HIV achieves viral burdens
which sometimes exceed 106/ml: new viral particles initiate a
positive feedback loop, by acting upon infected cells to activate
NF-kB, enhance HIV transcription and produce even more
progeny viruses. In situations where this positive feedback loop is
unopposed (e.g., in the absence of antiviral immunity or antiviral
therapy), it becomes clear why viral burdens are highest
immediately post infection, and then reduce slightly to individuals
own ‘viral setpoint’. This positive feedback loop likely also occurs
in settings of drug interruptions; it takes several days to make the
first round of new virus, thereafter the activating effects of gp120
(and likely other proteins) ensure that the slope of viral rebound is
steep. Ultimately the viral setpoint in that setting reflects the
opposing forces of apoptosis driven HIV replication versus
antiviral immunity and any impairment in replicative fitness
caused by drug resistance. These data therefore have clear
implications for the therapeutic strategies aimed at reactivating
HIV from latency; consideration must be given to agents (perhaps
gp120) which activate procaspase 8. Finally, data in the current
report suggest that extreme caution be used for proposed
therapeutic vaccine strategies which plan to use env, as enhanced
replication might promote disease progression and drug resistance.
Conceived and designed the experiments: GDB ADB. Performed the
experiments: SAT JW BDS. Analyzed the data: GDB ADB. Wrote the
paper: GDB ADB.
1. Badley AD (2006) Cell Death During HIV Infection. Boca Raton: CRC Press.
2. Cicala C, Arthos J, Selig SM, Dennis G Jr, Hosack DA, et al. (2002) HIV
envelope induces a cascade of cell signals in non-proliferating target cells that
favor virus replication. Proc Natl Acad Sci U S A 99: 9380–9385.
3. Cicala C, Arthos J, Censoplano N, Cruz C, Chung E, et al. (2006) HIV-1 gp120
induces NFAT nuclear translocation in resting CD4+ T-cells. Virology 345:
4. Castedo M, Ferri KF, Blanco J, Roumier T, Larochette N, et al. (2001) Human
immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis
involves mammalian target of rapamycin/FKBP12-rapamycin-associated pro-
tein-mediated p53 phosphorylation. J Exp Med 194: 1097–1110.
5. Roggero R, Robert-Hebmann V, Harrington S, Roland J, Vergne L, et al.
(2001) Binding of human immunodeficiency virus type 1 gp120 to CXCR4
induces mitochondrial transmembrane depolarization and cytochrome c-
mediated apoptosis independently of Fas signaling. J Virol 75: 7637–7650.
6. Westendorp MO, Frank R, Ochsenbauer C, Stricker K, Dhein J, et al. (1995)
Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.
Nature 375: 497–500.
Figure 4. Optimum HIV replication requires caspase 8. (A) Jurkat
T cells (squares) or I9.2 cells (diamonds) were infected with HIV IIIb, and
analyzed daily for viability (red) and for P24 production (blue). (B) Two
days following infection, cells were harvested, and DNA extracted. The
DNA was assayed for HIV Nef by PCR. (C) Non-transfected Jurkat T cells
or I9.2 cells transfected with control vector or procaspase 8, and
stimulated with TNF or CD3/CD28 as indicated were analyzed for HIV
LTR driven expression normalized to Renilla.
Caspase 8 and HIV replication
PLoS ONE | www.plosone.org6March 2009 | Volume 4 | Issue 3 | e4875
7. Trushin SA, Algeciras-Schimnich A, Vlahakis SR, Bren GD, Warren S, et al.
(2007) Glycoprotein 120 binding to CXCR4 causes p38-dependent primary T
cell death that is facilitated by, but does not require cell-associated CD4.
J Immunol 178: 4846–4853.
8. Perfettini JL, Castedo M, Roumier T, Andreau K, Nardacci R, et al. (2005)
Mechanisms of apoptosis induction by the HIV-1 envelope. Cell Death Differ 12
Suppl 1: 916–923.
9. Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, et al. (1999) Ordering
the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2,
-3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144:
10. Lemmers B, Salmena L, Bidere N, Su H, Matysiak-Zablocki E, et al. (2007)
Essential role for caspase-8 in Toll-like receptors and NFkappaB signaling. J Biol
Chem 282: 7416–7423.
11. Su H, Bidere N, Zheng L, Cubre A, Sakai K, et al. (2005) Requirement for
caspase-8 in NF-kappaB activation by antigen receptor. Science 307:
12. Bidere N, Snow AL, Sakai K, Zheng L, Lenardo MJ (2006) Caspase-8 regulation
by direct interaction with TRAF6 in T cell receptor-induced NF-kappaB
activation. Curr Biol 16: 1666–1671.
13. Kataoka T, Tschopp J (2004) N-terminal fragment of c-FLIP(L) processed by
caspase 8 specifically interacts with TRAF2 and induces activation of the NF-
kappaB signaling pathway. Mol Cell Biol 24: 2627–2636.
14. Golks A, Brenner D, Krammer PH, Lavrik IN (2006) The c-FLIP-NH2
terminus (p22-FLIP) induces NF-kappaB activation. J Exp Med 203: 1295–1305.
15. Zhu T, Mo H, Wang N, Nam DS, Cao Y, et al. (1993) Genotypic and
phenotypic characterization of HIV-1 patients with primary infection. Science
16. Cohen L, Henzel WJ, Baeuerle PA (1998) IKAP is a scaffold protein of the
IkappaB kinase complex. Nature 395: 292–296.
17. Duh EJ, Maury WJ, Folks TM, Fauci AS, Rabson AB (1989) Tumor necrosis
factor alpha activates human immunodeficiency virus type 1 through induction
of nuclear factor binding to the NF-kappa B sites in the long terminal repeat.
Proc Natl Acad Sci U S A 86: 5974–5978.
18. McElhinny JA, MacMorran WS, Bren GD, Ten RM, Israel A, et al. (1995)
Regulation of I kappa B alpha and p105 in monocytes and macrophages
persistently infected with human immunodeficiency virus. J Virol 69:
19. Lee S, Lee HS, Baek M, Lee DY, Bang YJ, et al. (2002) MAPK signaling is
involved in camptothecin-induced cell death. Mol Cells 14: 348–354.
20. Asin S, Taylor JA, Trushin S, Bren G, Paya CV (1999) Ikappakappa mediates
NF-kappaB activation in human immunodeficiency virus-infected cells. J Virol
21. Algeciras-Schimnich A, Belzacq-Casagrande AS, Bren GD, Nie Z, Taylor JA, et
al. (2007) Analysis of HIV Protease Killing Through Caspase 8 Reveals a Novel
Interaction Between Caspase 8 and Mitochondria. The Open Virology Journal
22. Oakley JD, Taher MM, Hershey CM, Aggarwal PC, Estwani IB, et al. (2003)
Triggering of apoptosis is not sufficient to induce human immunodeficiency
virus gene expression. IUBMB Life 55: 415–427.
23. Takasawa R, Nakamura H, Mori T, Tanuma S (2005) Differential apoptotic
pathways in human keratinocyte HaCaT cells exposed to UVB and UVC.
Apoptosis 10: 1121–1130.
24. Chang DW, Xing Z, Capacio VL, Peter ME, Yang X (2003) Interdimer
processing mechanism of procaspase-8 activation. Embo J 22: 4132–4142.
25. Nie Z, Bren GD, Vlahakis SR, Schimnich AA, Brenchley JM, et al. (2007)
Human immunodeficiency virus type 1 protease cleaves procaspase 8 in vivo.
J Virol 81: 6947–6956.
26. Bren GD, Whitman J, Cummins N, Shepard B, Rizza SA, et al. (2008) Infected
cell killing by HIV-1 protease promotes NF-kappaB dependent HIV-1
replication. PLoS ONE 3: e2112.
Caspase 8 and HIV replication
PLoS ONE | www.plosone.org7 March 2009 | Volume 4 | Issue 3 | e4875