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IMMUNOBIOLOGY
Inhibition of ␤-TrcP–dependent ubiquitination of p53 by HIV-1 Vpu promotes
p53–mediated apoptosis in human T cells
Sachin Verma,1 Amjad Ali,1 Sakshi Arora,1 and Akhil C. Banerjea1
1Laboratory
of Virology, National Institute of Immunology, New Delhi, India
HIV-1 viral protein U (Vpu) is involved in
ubiquitination and degradation of BM stromal cell Ag 2 and surface receptor CD4
through their recruitment to SCF␤-TrcP (Skp1/
Cul1/F-box) ubiquitin ligase (SCF) complex.
Here, we show that specific interaction of
wild-type Vpu protein with SCF complex
leads to inhibition of ubiquitination and proteasomal degradation of p53 protein in a
␤-TrcP–dependent manner. Successful interaction of SCF␤-TrcP complex with ␤-TrcP binding motif (DS52GNES56) present in Vpu is
essential because mutant Vpu possessing
specific alanine substitutions (DA52GNEA56)
in the ␤-TrcP binding motif not only failed to
stabilize p53 protein but was also unable to
inhibit ubiquitination of p53 protein. Furthermore, Vpu competes efficiently with the
interaction of p53 protein with the ␤-TrcP
subunit of the SCF complex and inhibits
subsequent ubiquitination of p53 proteins
in a dose-dependent manner. We also observed potent apoptotic activity in a p53 null
cell line (H-1299) that was cotransfected
with p53 and Vpu-expressing plasmids. Furthermore, MOLT-3 (human T-lymphoblast)
cells when infected with vesicular stomatitis
virus glycoprotein–pseudotypic HIV-1 possessing wild-type vpu gene exhibited maximum activation of p53/Bax proteins and
p53-mediated cell death. These findings
establish a novel function of Vpu in modulating the stability of p53 protein that
correlates positively with apoptosis during late stages of HIV-1 infection. (Blood.
2011;117(24):6600-6607)
Introduction
HIV type 1 (HIV-1) Vpu (viral protein U) is an accessory gene
exclusive to HIV-1 but not present in HIV-2 and in most SIVs.1-3
Studies in a macaque model have shown that Vpu from subtype B
plays a crucial role in massive loss of circulating CD4⫹ T lymphocytes,4-6 which can be modulated by replacing it with Vpu from
subtype C.7 The exact mechanism(s) underlying how Vpu makes
HIV-1 more pathogenic is only partially understood.8,9 Vpu is
involved in ubiquitination and degradation of antiretroviral restriction factor BM stromal cell Ag 2 (BST-2; also known as tetherin)
and surface receptor CD4 through their recruitment to SCF␤-TrcP
(Skp1/Cul1/F-box) ubiquitin ligase (SCF) complex.10-13 The key to
biologic function of Vpu is the presence of a highly conserved
6–amino acid (DS52GNES56) sequence that constitutes the ␤-TrcP
(␤-transducin repeat-containing proteins) binding motif.14 This
motif is constitutively phosphorylated and mediates interaction of
Vpu with ␤-TrcP, the substrate recognition subunit of SCF␤-TrcP
complex. Hence, Vpu acts as an adaptor linking its target proteins
(CD4 and BST-2) to a host E3 ubiquitin ligase SCF␤-TrcP complex,
which are otherwise not the natural substrates of this complex.
However, unlike natural substrates of ␤-TrcP, which are targeted
for degradation, Vpu itself is resistant to degradation and can form
stable complexes with ␤-TrcP.15 Thus, expression of constitutively
phosphorylated Vpu in infected cells leads to competitive inhibition of ubiquitination and subsequent proteosomal degradation of
many natural substrates of SCF␤-TrcP complex (␤-catenin, activating
transcription factor 4, and I␬␤-␣) and results in changes in the
profile of cellular proteins that may contribute to cytopathic effects
during HIV-1 infection.9,15,16 The inability to degrade I␬␤-␣ and to
activate NF-␬␤ on stimulation with TNF-␣ explains the TNF-␣–
sensitive phenotype of Vpu-expressing cells.9 Interestingly, more
recent data suggest that Vpu is also degraded by both ␤-TrcP–
dependent17 and –independent pathways,18 which in turn may have
a role in modulating biologic activities of Vpu.
The SCF␤-TrcP ubiquitin ligase complex controls the functions of
a wide spectrum of cellular proteins16,19-22; many of them are
involved in pathways crucial to HIV-1 pathology. Tumor suppressor protein p53 is reported to be a major player involved in
HIV-1–induced apoptosis.23-25 p53 protein has been shown to be an
important substrate of ␤-TrcP–dependent ubiquitination. Small
interfering RNA (siRNA)–mediated depletion of components of
SCF␤-TrcP complex or the expression of a dominant-negative form
of ␤-TrcP was shown to enhance the stability of p53 protein and
also to activate p53 downstream signaling events.22 Like most of
the natural ␤-TrcP substrates (I␬␤-␣, ␤-catenin, p65, and steroid
receptor coactivator 3), the I␬␤ kinase 2 (IKK2/IKK␤) also
phosphorylates p53 at serine 362/366 (Ser362/366) position directing it for ␤-TrcP–mediated ubiquitination in an Mdm-2 independent manner,22 which is a well-studied negative regulator (E3
ubiquitin ligase) of p53.26 Interestingly, multiple viral proteins
(Env and Vif) are known to trigger activation of p53 by different
mechanisms with possible involvement of Mdm-2 ubiquitin ligase.27-30 Sequestration of ␤-TrcP by Vpu however is speculated to
result in alteration of ␤-TrcP–dependent p53 ubiquitination that
may further potentiate p53 activation during HIV-1 infection. In the
present study, we demonstrate a novel effect of Vpu expression on
p53 metabolism. We show that Vpu stabilizes p53 and inhibits its
ubiquitination. This leads to enhancement of p53-mediated apoptosis during HIV-1 infection. Our findings suggest that Vpu (a
late-stage protein unique to the HIV-1 genome) potently inhibits
the ␤-TrcP–mediated pathway responsible for p53 degradation
during HIV-1 infection that results in higher cell death observed
during viral infection.
Submitted January 27, 2011; accepted April 8, 2011. Prepublished online as
Blood First Edition paper, April 26, 2011; DOI 10.1182/blood-2011-01-333427.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2011 by The American Society of Hematology
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BLOOD, 16 JUNE 2011 䡠 VOLUME 117, NUMBER 24
Methods
Plasmid constructs, proviral DNAs, and transient
siRNA-mediated knockdown
Vpu from subtype B (pNL4-3) and C (Indian Isolate 93IN905) HIV-1
(obtained from Acquired Immunodeficiency Syndrome Research and
Reference Reagent Program, Division of Acquired Immunodeficiency
Syndrome, National Institute of Allergy and Infectious Diseases [NIAID],
National Institutes of Health [NIH]) were amplified by PCR and cloned in
the mammalian expression vector pCMV-HA (Clontech) to generate Vpu
B-HA and Vpu C-HA constructs. The S52A and S56A mutations were
introduced by site-directed mutagenesis with Pfu Turbo polymerase in
pCMV Vpu B-HA to generate pCMV M-Vpu-HA (mutant Vpu) construct
with the use of overlapping primers with the following sequences: motif
forward, 5⬘-GGCAGAAGACGCAGGCAATGAGGCAGA-3⬘, and motif
reverse, 5⬘-GGCTCGCACTCATTGCCGCAGTCTTCT-3⬘.
The pNL4-3 HIV-1 clone as well as mutant pNL4-3 (Mt; having
Ser52/56 substituted with alanine 52/56 in Vpu open reading frame) or
pNL4-3⌬Vpu (having deletion in Vpu initiation codon) variants were kind
gifts from K. Strebel (NIH).9 We used the infectious molecular clone of
HIV-1 subtype C, pIndie as originally described by Mochizuki et al.31
Reporter virus pNL⌬GFP (obtained from NIH) was used to ensure uniform
infection in different cell lines. Viral stocks of pNL4-3 and its variants were
prepared by cotransfecting different proviral constructs (pNL4-3, Mt
pNL4-3, pNL4-3⌬Vpu, and pIndie) and plasmid-encoding vesicular stomatitis virus glycoprotein (VSV-G) into HEK 293T cells, followed by
collection of virion particles from culture supernatant fluid at 48 and
72 hours.9 The shRNA sequence against ␤-TrcP that was reported previously22 was cloned in RetroQ-Zs Green Vector. HA-p53 and GST-p53
expression plasmids were a kind gift from Yukiko Gotoh (Institute of
Molecular and Cellular Biosciences, University of Tokyo)32 and Sanjeev
Das (National Institute of Immunology)33 respectively. p53⌬I-expressing
plasmid was provided by Karen Vousden (Beatson Institute of Cancer
Research).34
Cell culture, transfections, and immunoblot analysis
HEK 293T (human embryonic kidney 293 cells), MCF-7 (breast cancer cell
line, p53 Wt), and H-1299 (nonsmall cell lung carcinoma, p53 null) cells
were maintained in DMEM (Gibco, Invitrogen) supplemented with glutamine, 10% FCS, 100 U/mL penicillin, and 100 ␮g/mL streptomycin
(Invitrogen) at 37°C with 5% CO2. MOLT-3 T cells (T-lymphoblastoid cell
line, p53 Wt) and K-562 cells (human erythroleukemia cell line, p53 null)
were maintained in RPMI (Gibco, Invitrogen) media supplemented with
glutamine, 10% FCS, 100 U/mL penicillin, and 100 ␮g/mL streptomycin
(Invitrogen) at 37°C with 5% CO2. Plasmid transfections were performed
with Lipofectamine 2000 (Invitrogen). Relative levels of different proteins
were compared by immunoblot analysis. Cells were lysed with RIPA
Lysis buffer (20mM Tris [pH 7.5], 150mM NaCl, 1mM
Na2ethylenediaminetetraacetic acid, 1mM EGTA, 1% NP-40, 1% sodium
deoxycholate, 2.5mM sodium pyrophosphate, 1mM ␤-glycerophosphate,
1mM Na3VO4, and 1 ␮g/mL leupeptin). Protein estimation was done with
the use of BCA Protein Estimation Kit (Pierce Biotechnology Inc). Proteins
were resolved with PAGE and transferred to Immobilon membrane
(Millipore). The membranes were blocked with 5% nonfat dry milk
(Sigma-Aldrich) in PBS, washed with PBS containing 0.1% Tween 20
(Merck) and incubated in the same buffer overnight at 4°C in the presence
of primary Ab (1:2000 dilution). The primary Abs used were anti-p53,
anti-GST, anti-His, anti-HA, anti-GAPDH, anti–␤-TrcP, anti–I␬␤-␣, antiBax (Santa Cruz Biotechnology), anti–Ser362/366 phosphorylated p53
(P-362/366 p53; Abcam) and anti-Ser15 phosphorylated p53 (P-15 p53;
Cell Signaling Technology). The membranes were washed with PBS
containing 0.1% Tween 20 and then incubated with secondary Ab of either
anti–mouse or anti–rabbit conjugated with HRP (1:10 000 dilution; Jackson
ImmunoResearch Laboratories Inc) in 5% nonfat dry milk in PBS with
0.1% Tween 20 at room temperature. The proteins of interest were detected
Vpu STABILIZES p53 VIA INHIBITING ␤-TrcP FUNCTION
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with EZ Western HRP substrate (Biologic Industries). GAPDH was used as
a loading control in all cases.
Cycloheximide chase assay
Untransfected (1.2 ⫻ 106) or transfected (2 ␮g of Wt or mutant Vpu) MCF-7
cells in a 35-mm dish were incubated for 36 hours. Subsequently, cycloheximide
(Sigma-Aldrich) was added to a final concentration of 100 ␮g/mL, and cells were
harvested after the indicated time points. The lysate was prepared, and immunoblotting was performed as described in the preceding paragraph.
GST pull-down assay
To study the effect of Vpu on the interaction between p53 and ␤-TrcP,
GST pull-down assay was performed. In 100-mm dishes HEK 293T
cells were transfected with 5 ␮g of GST-p53–expressing plasmid
pEBG-p53 in the presence or absence of Vpu-expressing plasmid in a
1:1 ratio. GST-expressing plasmid pEBG was used as control. Cells
were harvested 48 hours after transfection and suspended in 1 mL of
GST lysis buffer (50mM Tris [pH 7.5], 0.1M NaCl, 50mM ␤-glycerophosphate, 50mM NaF, 1mM Na3VO4, 50mM sodium pyrophosphate,
2mM EDTA, 1mM EGTA, 1% NP-40, 2 ␮g/␮L aprotinin, 1 ␮g/␮L
leupeptin, 0.7 ␮g/mL pepstatin, and 1mM phenylmethylsulfonyl fluoride). The samples were sonicated on ice and centrifuged, and 60 ␮L of
50% Glutathione Agarose beads (Sigma-Aldrich) were added. The
samples were incubated at 4°C for 4 hours while rotating; subsequently,
the beads were washed twice with lysis buffer and boiled in SDS loading
buffer. The proteins were separated on 12% SDS-PAGE and immunoblotted with anti-GST and anti–␤-TrcP Abs.
In vivo ubiquitination assay
For detection of ubiquitinated p53 protein, HEK 293T cells were grown in
100-mm dishes and transfected with 5 ␮g of 6⫻ His-ubiquitin expression
plasmid35 along with equal amounts of various Vpu-expressing plasmids.
To equalize the DNA amount pCMV-HA vector was used. After 36 hours of
transfections, 25 ␮M of MG-132 (Sigma-Aldrich) was added, and cells
were further incubated for 8 hours. Thereafter, cells were collected in PBS
and were resuspended in 1 mL of lysis buffer (6M guanidinium-HCl, 0.1M
Na2HPO4/NaH2PO4, 0.01M Tris, pH 8.0, 5mM imidazole, and 10mM
2-ME), sonicated, and centrifuged. Ni-NTA beads (50 ␮L) were added to
the supernatant fluid, and the mixture was incubated at room temperature
for 4 hours while rotating. Subsequently, the beads were washed for
5 minutes at room temperature with 750 ␮L of each of the following
buffers: 6M guanidinium-HCl, 0.1M Na2HPO4/NaH2PO4, 0.01M Tris, pH
8.0, and 10mM 2-ME; 8M urea, 0.1M Na2HPO4/NaH2PO4, 0.01M Tris, pH
8.0, 10mM 2-ME; 8M urea, 0.1M Na2HPO4/NaH2PO4, 0.01M Tris, pH 6.3,
10mM 2-ME (buffer A) plus 0.2% Triton X-100; buffer A and then buffer A
plus 0.1% Triton X-100. After the last wash ubiquitinated proteins were
eluted by incubating the beads in 75 ␮L of buffer containing 200mM
imidazole, 5% SDS, 0.15M Tris, pH 6.7, 30% glycerol, 0.72M 2-ME for
20 minutes at room temperature. The eluates were mixed in a 1:1 ratio with
2⫻ Laemmli buffer and separated the proteins on 8% SDS-PAGE followed
by immunoblotting with anti-p53 Ab.
SubG1 DNA content analysis and cell death assay
SubG1 DNA content analysis was performed for determination of apoptotic
population.36 Cells were harvested, fixed in ethanol, and stained with
propidium iodide (PI) staining solution (50 ␮g/mL PI, 50 ␮g/mL ribonuclease A, 0.1% saponin, and 5mM EDTA in PBS) for 30 minutes at 37°C
and analyzed on a BD FACSCaliber. To study the effect of p53 in
Vpu-mediated apoptosis, we transfected H-1299 cells (1.2 ⫻ 106 cells in
35-mm culture plates) with 2 ␮g of p53-expressing plasmid alone or in
combination with Vpu variants in a 1:1 ratio. We used the empty control
vector pCMV-HA to normalize equal amounts of DNA in each transfection.
Two to 3 days after transfection cells were harvested, fixed in ethanol, and
stained with PI for SubG1 DNA content analysis. Cell death was measured
according to the earlier published procedures.37,38 The FACS data were
analyzed by WinMDI 2.8 software (Joseph Trotter, Scripps Research
Institute).
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Figure 1. Expression of Vpu B-HA or Vpu C-HA in MCF-7 cells
results in the accumulation of p53 and P-362/366 p53. (A) Equal
amounts (2 ␮g) of Vpu B, Vpu C, or M Vpu-HA were transfected
into MCF-7 cells by Lipofectamine 2000 for 48 hours. Cell lysates
were subjected to immunoblotting with indicated Abs. (B) MCF-7
cells were transfected with siRNA against GFP or ␤-TrcP, and
immunoblot analysis was done. (C) Increasing amounts (0.5, 1,
and 2 ␮g) of Vpu B-HA was transfected into MCF-7 cells.
(D) Cycloheximide (CHX) chase to check half-life of endogenous
p53, MCF-7 cells either untransfected or transfected with M-Vpu
HA or Vpu B-HA, were treated with 100 ␮g/mL CHX and harvested at the indicated time points for immunoblotting. (E) After
quantification, the signals obtained in panel D were used to
calculate the p53/GAPDH ratios that were plotted with respect to
treatment period. (F) Equal amounts of Vpu B, Vpu C, or M
Vpu-HA were transfected into MCF-7 followed by treatment with
1 ␮g/mL doxorubicin for 12 hours. Immunoblotting analysis was
performed with anti-p53, anti–I␬␤-␣, anti–␤-TrcP, anti-HA, anti–P362/366 p53, and anti-GAPDH Abs. GAPDH was used as a
loading control. This is a representative result obtained from
3 independent experiments.
Infection by HIV-1 pNL4-3 or HIV-1 mutants
MOLT-3 T cells were infected with pNL4-3, Mt pNL4-3, or pNL4-3⌬Vpu
or mock infected (as a control) in the presence of 4 ␮g/mL Polybrene
(Sigma-Aldrich) for 4 hours at 37°C. Infection was accomplished by
incubating the cells for 4 hours with equal amounts of infectious virus
(1 MOI) assessed by ␤-galactosidase staining with the use of HIV-1
indicator Tzmbl cells.39 The infected cells were harvested 48 hours after
infection and subjected to immunoblotting with indicated Abs. Another set
of cells was stained with PI (10 ␮g/mL) for determining the total cell death.
To determine whether Vpu potentiated p53-mediated apoptosis in HIVinfected cells, we treated a parallel set of infected cells with 30␮M
Pifithrin-␣ (Santa Cruz Biotechnology), which is a small molecule inhibitor
of p53 transcriptional activity.29,40 Briefly, after 24 hours of infection, the
samples were divided into 2 equal sets, one set was treated with Pifithrin-␣
and the other set served as an untreated control. The cell death was
determined by PI staining.
Results
Vpu expression leads to cellular accumulation and stabilization
of p53 and P-362/366 p53
Vpu is a competitive inhibitor of ␤-TrcP function and is known to
stabilize natural ␤-TrcP substrates. To test whether Vpu expression
can inhibit ␤-TrcP–dependent degradation of p53 protein, we
compared relative p53 protein levels in MCF-7 cells on transfect-
ing Vpu variants. This cell line expresses Wt p53 protein constitutively. Expression of Wt subtype B and C Vpu led to cellular
accumulation of p53 (Figure 1A lanes 3-4). I␬␤-␣, which is another
␤-TrcP substrate known to be stabilized on Vpu expression,15 was
also elevated in Wt Vpu-expressing cells (Figure 1A lanes 3-4).
The observed effect of Wt Vpu on p53 level was ␤-TrcP dependent,
because the mutant Vpu B (M-Vpu), in which the 2 serine residues
were mutated to alanine in the ␤-TrcP binding motif, failed to show
any stabilization of p53 protein (Figure 1A lane 2). All the 3 Vpu
constructs directed synthesis of comparable levels of intracellular
Vpu protein that was detected by immunoblotting with the use of
anti-HA Ab (Figure 1A lanes 2-4). The GAPDH protein was used
as a loading control for each lane that remained unchanged. We
further confirmed this finding by knocking down ␤-TrcP with
specific siRNA (Figure 1B lane 2, explained in the legend), which
also led to accumulation or stabilization of p53 protein (Figure 1B
lane 2). The cellular expression of Wt Vpu B also up-regulated p53
protein in a dose-dependent manner (Figure 1C lanes 2-4). The
cycloheximide (CHX) chase assay was performed to study the
effect of Vpu expression on the turnover of p53 protein (Figure 1D).
After 8 hours of chase period, the level of p53 was reduced by
⬃ 80% in untransfected MCF-7 cells as well as in M-Vpu–
expressing cells (Figure 1D and quantitation shown in Figure 1E).
In Wt Vpu B–expressing cells, the p53 levels remained almost
unchanged after 8 hours of CHX chase (Figure 1E), confirming that
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BLOOD, 16 JUNE 2011 䡠 VOLUME 117, NUMBER 24
Vpu STABILIZES p53 VIA INHIBITING ␤-TrcP FUNCTION
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Figure 2. Effect of Vpu expression on ␤-TrcP interaction with and ubiquitination of p53. (A) HEK 293T cells were cotransfected with plasmids encoding GST-p53 and
either Vpu B, Vpu C, or M Vpu-HA for 48 hours as described in “Cell culture, transfections, and immunoblot analysis.” Cell lysates were used to pull down GST-p53–associated
proteins with the use of Glutathione beads, and the resulting eluent was used to measure GST-p53–bound ␤-TrcP by immunoblotting with anti–beta]-TrcP Ab. Total cellular
␤-TrcP levels were not altered on Vpu expression. (B) HEK 293T cells were cotransfected with His-ubiquitin (His-Ub) and either Vpu B, Vpu C, or M Vpu-HA. After 36 hours cells
were treated with MG132 for 8 hours followed by lysis in denaturation buffer, and then total ubiquitinated proteins were pulled down with the use of Ni-NTA beads, and p53
ubiquitination was checked by immunoblotting with anti-p53 Ab. (C) HEK 293T cells were cotransfected with His-Ub, and an increasing dose (0.5, 1, and 2 ␮g) of either M-Vpu
or Wt Vpu B-HA and p53 ubiquitination was checked as described in panel B.
Vpu protein increases half-life of p53 protein by decreasing its rate
of degradation. We conclude that Wt Vpu expression led to
stabilization of p53 protein via inhibition of ␤-TrcP function. We
next examined the cellular levels of P-362/366 p53, which is the
substrate form of p53 protein recognized by ␤-TrcP, using specific
phospho-Ab (Abcam) by immunoblot analysis (Figure 1F). As
expected, control untransfected cells and M Vpu–expressing cells
showed barely detectable levels of phosphorylated p53 (Figure 1F
lanes 1-2). However, expression of Wt Vpu resulted in significant
up-regulation of phosphorylated p53 (Figure 1F lane 4). Upregulation of phosphorylated p53 was also observed when cells
were treated with IKK2 activator doxorubicin (Figure 1F lane 3).
Interestingly, highest levels of phosphorylated p53 were observed
when Vpu-expressing MCF-7 cells were treated with doxorubicin,
showing an additive effect of Vpu and doxorubicin on the levels of
phosphorylated p53 (Figure 1F lane 5). These data suggest that
phosphorylated p53 gets accumulated because of inactive SCF␤-TrcP
complex rendered by Vpu interaction. An internal control (GFP)
was always included to normalize transfection efficiency, and equal
loading was confirmed by GAPDH levels for all transfection-based
experiments.
Wt Vpu inhibits association of p53 with ␤-TrcP and its
subsequent ubiquitination
Having established that Vpu inhibits ␤-TrcP–mediated degradation
of p53, we next investigated the molecular mechanism underlying
this phenomenon. Degradation of cellular proteins by SCF␤-TrcP
complex involves binding of natural substrates with WD40 repeat
domain of ␤-TrcP, and Vpu is known to compete with natural
substrates for binding with ␤-TrcP.16 We therefore evaluated
whether Vpu affects intracellular interaction of ␤-TrcP with p53
protein by pull-down assay. As shown (Figure 2A) expression of M
Vpu had little or no effect on ␤-TrcP binding with p53 (Figure 2A
lane 2) and is the same as in control cells (Figure 2A lane 1).
However, expression of both Wt Vpu B and C proteins inhibited
␤-TrcP binding with p53 (Figure 2A lanes 3-4). The total ␤-TrcP
levels were not altered by Vpu expression. Because binding of
natural substrate with ␤-TrcP leads to its ubiquitination, we next
evaluated whether Vpu expression inhibited p53 ubiquitination.
Both Wt Vpu B and Vpu C inhibited ubiquitination of p53
(Figure 2B lanes 3-4). However, cells transfected with M Vpu
showed p53 ubiquitination profile identical to that of untransfected
cells (Figure 2B lanes 1-2). Furthermore, we performed a doseresponse study of inhibition of p53 ubiquitination with Wt Vpu B
and M Vpu. Wt Vpu B transfections (Figure 2C lanes 5-7) resulted
in significant inhibition of p53 ubiquitination in a dose-dependent
manner, confirming the competitive nature of inhibition of SCF␤-TrcP
function by Vpu. In contrast M Vpu (Figure 2C lanes 2-4) did not
inhibit p53 ubiquitination in all 3 doses tested. These results show
that Vpu competitively inhibits association of ␤-TrcP with p53
protein and its subsequent ubiquitination in a dose-dependent
manner.
Vpu potentiates apoptotic activity of exogenous p53 in p53 null
(H-1299) cells
We further analyzed the functional effect of Vpu-mediated inhibition of p53 ubiquitination on biologic activity of both Vpu and p53.
Expression of p53 leads to apoptosis because of activation of
proapoptotic protein Bax,41 whereas Vpu sensitizes cells for
apoptosis by inhibiting NF-␬␤ activation.9,15 To address the
question of whether Vpu induces apoptosis by the activation of p53
protein, we used a p53-null cell line (H-1299) and exogenously
expressed both proteins followed by measurement of subG1 DNA
content containing apoptotic population by PI staining (Figure 3A)
according to previously reported protocol.36 The proportion of
apoptotic cells was ⬍ 5% in M Vpu–transfected as well as
untransfected cells (Figure 3A samples 1-2). Cells transfected
either with p53 or Wt Vpu B or Vpu C constructs showed moderate
levels of apoptosis (Figure 3A samples 3-5, between 18% and
30%). However, when both p53 and Wt Vpu B or C were
expressed, there was a synergistic increase in the apoptotic
population (Figure 3A samples 7-8, between 72% and 75%). In
contrast, cells transfected with p53 and M Vpu showed apoptotic
population close to what was observed in cells transfected with p53
construct alone (Figure 3A sample 6, 30%). These results clearly
establish the physiologic consequences that result from inhibitory
influence of Vpu on p53 degradation. Bax is an immediate
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BLOOD, 16 JUNE 2011 䡠 VOLUME 117, NUMBER 24
Figure 3. The functional effect of Vpu expression on p53-mediated apoptosis. (A) H-1299 (p53 null) cells were cotransfected with plasmids encoding p53 and either Vpu
B, Vpu C, or M Vpu-HA. Then after 72 hours, cells were harvested, fixed, and stained with PI, and subG1 DNA content was determined by FACS to see relative proportion of
apoptotic population that is summarized below the histograms. (B.) H-1299 cells were cotransfected with p53wt and Vpu B, Vpu C, or M Vpu-HA, and endogenous Bax levels
were checked by immunoblotting analysis with the indicated Abs. (C.) H-1299 cells were cotransfected with indicated plasmids, and cell lysates were subjected to immunoblot
analysis. This is a representative result obtained from 3 independent experiments.
downstream target gene known to get up-regulated in p53mediated apoptotic pathway, cellular levels of which is directly
controlled by p53. We therefore checked cellular levels of Bax in
H-1299 cells after cotransfection of p53- and Vpu-expressing
constructs. As shown (Figure 3B), we failed to detect any
expression of Bax protein in untransfected H-1299 cells (Figure 3B
lane 1), but its expression was induced in cells transfected with p53
(Figure 3B lane 2). Wt Vpu B and C constructs were able to
significantly up-regulate Bax protein (Figure 3B lanes 4-5).
Furthermore, to rule out the possibility of involvement of Mdm-2
ubiquitin ligase for p53 degradation, we performed a parallel set of
transfections for 48 hours with plasmid encoding Mdm-2–binding
mutant form of p53 protein (p53⌬I).34 As shown (Figure 3C) the
Bax levels increased substantially in cells coexpressing p53⌬I and
Wt Vpu B or C (Figure 3C lanes 3 and 4) in comparison to cells
expressing M Vpu and p53⌬I (Figure 3C lane 2). Hence, we
conclude that Vpu augments apoptotic activity of p53 in a
synergistic and ␤-TrcP–dependent but Mdm-2–independent manner.
Vpu contributes to p53-dependent apoptosis in HIV-1–infected
MOLT-3 cells
Next, we investigated whether the observed phenomenon of p53
induction and p53-dependent apoptosis by exogenously expressed
Vpu contributes to apoptotic potential of HIV-1 during infection in
a biologically relevant T-cell line (MOLT-3). To address this
question, MOLT-3 T cells expressing Wt p53 were infected with an
equal MOI (1) of the following VSV-G–pseudotyped viruses:
pNL4-3 (Wt-HIV), pNL4-3⌬Vpu, and Mt pNL4-3 for 48 hours.
Thereafter, the cells were stained with PI and subjected to FACS
analysis to determine the extent of cell death, and the results are
shown in Figure 4A left panels. As expected the control cells
showed only background activity (8%). The Mt pNL4-3 and
⌬Vpu-HIV constructs exhibited 45% and 41% cell death, respectively. In comparison, HIV harboring the Wt Vpu B (pNL4-3)
exhibited 78% cell death under exactly similar conditions. A
parallel set of experiments was performed with the use of the same
viral constructs in the presence of p53 inhibitor Pifithrin-␣ to
determine the cytotoxic contribution of p53 alone in these cells,29,40
and the results are shown in Figure 4A right panels. Interestingly,
the extent of inhibition of cell death in the presence of Pifithrin-␣
was also highest in Wt HIV–infected cells (Figure 4A sample 8,
46% from 78% in sample 4) than in cells infected with either Mt
HIV (Figure 4A sample 6, 43% from 45% in sample 2) or ⌬Vpu
HIV (Figure 4A sample 7, 32% from 41% in sample 3). These data
confirm that Vpu independently contributes substantially to p53dependent apoptosis during HIV-1 infection. Next, we wanted to
compare the ability of HIV-1 subtypes B and C in mediating
p53-mediated apoptosis. For this purpose MOLT-3 (p53 Wt) and
K-562 (p53 null) cells were infected with an equal MOI (1) of pNL
4-3 (subtype B) and pIndie (subtype C). We initially ensured that
pseudotyped viruses were equally infectious for both the cell lines
with the use of the pNL⌬GFP reporter virus. The extent of cell
death was determined by PI staining shown in Figure 4B, and the
results are shown in Figure 4B. We always observed more cell
death (almost double) in MOLT-3 (Figure 4 B samples 2 and 3,
68% and 72%) than in K-562 cells (Figure 4 B samples 5 and 6,
30% and 31%). Thus, HIV-1 subtypes B and C were equally
potent in causing cell death, and p53 contributed substantially to
the cell death.
Finally, we examined the cellular levels of p53 and Bax protein
in MOLT-3 cells infected with the 3 subtype B viruses mentioned in
the previous paragraph. Moderate elevation in the levels of total
cellular p53 protein was observed after Wt HIV infection, but a
significant up-regulation was observed for Bax protein compared
with the other 2 viruses (Figure 4C lane 4). Intracellular expression
of Vpu protein was monitored in each case, and as expected only
Wt and Mt HIV virus directed the synthesis of authentic Vpu
proteins (Figure 4C lanes 3-4). GAPDH protein was monitored as
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BLOOD, 16 JUNE 2011 䡠 VOLUME 117, NUMBER 24
Vpu STABILIZES p53 VIA INHIBITING ␤-TrcP FUNCTION
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Figure 4. Vpu contributes to p53-mediated apoptosis during infection. MOLT-3 T cells or K-562 cells were infected with an MOI of 1 with indicated VSV-G–pseudotyped
HIV-1 virus. (A) Total cell death was measured after 48 hours of infection of MOLT-3 cells by PI staining by FACS: PI (FL-2H), (left) live cells (PI⫺), (right; region R1): dead cells
(PI⫹). p53-mediated apoptosis was inhibited by treating cells with 30␮M Pifithrin-␣ for 24 hours (samples 5-8) as described in “Infection by HIV-1 pNL4-3 or HIV-1 mutants.”
Percentage of cell death is indicated for each sample. (B) Total cell death (region R1) was measured 48 hours after infection of MOLT-3 and K-562 cells with indicated viruses.
After 48 hours of infection cell lysates were prepared, and immunoblotting was done with indicated Abs (C-D). Cells were evaluated for the extent of HIV-1 infection by
intracellular p24 staining with primary p24 rabbit Ab (NIH) followed by secondary anti–rabbit Ab (phycoerythrin conjugated). (E) Representative pictures showing comparable
infection efficiency of the various subtype B viruses in MOLT-3 cells by intracellular p24 staining.
internal control, which remained unchanged. We conclude that
HIV-1 infection in T cells results in moderate elevation of total
intracellular p53 but a significant elevation in the Bax protein
levels, both of which were mediated by Vpu. We then wanted to
determine whether P-15 p53 levels correlated with Bax levels,
because it is known to get specifically up-regulated under stress
conditions, including viral infection.23 Remarkably, the intracellular levels of biologically more active P5 p5342 correlated with the
Bax expression levels (Figure 4D).
Discussion
Exploitation of cellular pathways that govern apoptosis plays a
critical role in pathophysiologic outcomes of viral infection and is
achieved by direct or indirect interaction of viral proteins with key
components of highly regulated cell survival and death pathways.
The outcome of this interaction depends on the viral life cycle
where the viral genes are expressed in a temporal manner during
the course of virus replication.43 Early suppression of apoptotic
pathways in an infected mammalian cell is desired because it will
result in more virus production and subsequently the release at a
later stage because of apoptosis and cell lysis.
HIV-1 has evolved several mechanisms to modulate apoptotic pathways differentially during viral life cycle. Most of the
HIV-1–encoded proteins are known to have either proapoptotic
or antiapoptotic properties or both, for example, Gp120 as well
as protease are known to be proapoptotic, whereas Tat, Nef, and
Vpr proteins have been shown to possess both antiapoptotic and
proapoptotic activities.44,45 Because some of these proteins (Tat,
Nef) are expressed early in HIV-1 infection and some are
expressed late (Gp120, Vpu, Vpr), the ultimate outcome with
respect to apoptosis will vary at different stages of virus life
cycle. Clearly, HIV-1 uses multiple genes to modulate apoptotic
machinery to its advantage.
The tumor suppressor protein p53 is one of the dominant
proapoptotic transcriptional factors elicited specifically in HIV1–infected cells and not in uninfected lymphocytes.24,25 The
exact contribution of p53 protein in HIV-1 replication has been
controversial because it has been shown to play a positive as
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6606
BLOOD, 16 JUNE 2011 䡠 VOLUME 117, NUMBER 24
VERMA et al
well as a negative role in HIV-1 replication.46,47 Besides,
multiple viral proteins are known to alter cellular p53 levels and
its phosphorylation status to manipulate cell survival and death
pathways.27-29,48 Cellular expression of Vif and Tat, as well as
Env is known to trigger p53 activation and associated downstream events. Nef, however, is a potent inhibitor of p53
functions, binds to p53 protein, and targets it for degradation.48
In summary, overall p53 activity is modulated differentially by
viral proteins. Although Vpu is known to influence a number of
key cellular events, its possible involvement with p53 function
was not reported earlier.
A recent report identified ␤-TrcP as a novel regulator of p53
protein stability and biologic activity.22 Because Vpu is a
general inhibitor of ␤-TrcP function,16 we investigated a potential role of Vpu on the stability of p53 protein and its ensuing
physiologic consequences. We clearly show that Vpu upregulates p53 in a cell line that constitutively expresses Wt p53
(MCF-7). The same was observed when Vpu and p53 were
cotransfected in a p53-null cell line (H-1299). The results are in
agreement with the known functions of Vpu. It exploits
SCF␤-TrcP complex for degradation of CD4 and BST-2 and
simultaneously causes the accumulation of natural ␤-TrcP
substrates. The increase in the stability of p53 protein in the
presence of Vpu suggests that the accumulation is because of
inhibition of p53 degradation. In addition, inhibitory influence
of Vpu on interaction between p53 and ␤-TrcP further supports
the hypothesis that Vpu inhibits p53 degradation by exploitation
of SCF complex needed for ␤-TrcP–mediated ubiquitination.
The ubiquitination profile of p53 protein in the presence of Wt
and Mt Vpu clearly establish the inhibitory role of Vpu in p53
ubiquitination. Having shown the effect of Vpu expression on
p53 metabolism, we further investigated the physiologic consequences of p53 accumulation during virus infection. Vpu is a
late-stage viral protein, presence of which has been shown to
correlate with massive loss of CD4⫹ T lymphocytes in macaque
models. The p53 protein was also found to be elevated in the late
stage of viral infection that is associated with increased
apoptosis.23,25 On the basis of our findings it can be safely
argued that Vpu is an important contributor to this phenomenon.
To study additive or synergistic influence of Vpu on p53mediated apoptosis, we used a p53-null cell line. The total
apoptotic cells (as measured quantitatively by subG1 DNAcontaining cells) in Mt Vpu- and p53-transfected cells were
found to be additive. In contrast, cells expressing Wt Vpu and
p53 protein exhibited synergistic effect with respect to apoptosis. These data functionally complement our earlier results of
Vpu-mediated p53 accumulation. Because p53-mediated apoptosis involves Bax up-regulation, we measured the Bax and p53
protein levels in Wt and Mt Vpu-expressing cells. The Bax level
was highly elevated in Wt Vpu-expressing cells that further
confirmed that Vpu mediated accumulated p53 protein has an
important functional consequence in promoting Bax-mediated
apoptosis. A similar pattern was observed with Mdm-2 binding
mutant of p53, indicating the Mdm-2–independent nature of this
phenomenon. The higher Bax activation in samples containing
Mdm-2 binding p53 mutant is most probably because of its high
transactivation potential as reported previously.34 Finally, we
demonstrate the role of Vpu in up-regulating p53, which, in turn,
results in augmenting p53-mediated cell death in HIV-1–
infected cells. It is important to note that we observed maximum
cell death in Wt HIV-1 (pNL 4-3) infected MOLT-3 cells and on
Pifithrin-␣ treatment, the same cells exhibited maximum reduc-
tion with respect to cell death. The importance of p53 in causing
cell death was evaluated by infecting MOLT-3 cells (p53 Wt)
and K-562 cells (p53 null) with HIV-1 subtypes B or C. As
expected, under identical conditions higher cell death was
observed in MOLT-3 cells than in K-562 cells, establishing the
role of p53 in augmenting HIV-1–mediated apoptosis. We
observed that the proapoptotic Bax levels correlated with P-15
p53 and not total cellular p53 in infected cells. It is predominantly this phosphorylated p53 form that gets up-regulated on
stress conditions or on viral infection.23 These findings further
established the important contribution of Vpu in up-regulating
p53 levels during HIV-1 infection.
Taken together, in this study we demonstrate that Vpu inhibits
p53 ubiquitination and degradation by inhibiting ␤-TrcP function,
thereby leading to its enhanced stability, cellular accumulation with
concurrent increase in apoptosis. Earlier studies9,15 clearly have
shown that Vpu causes apoptosis by inhibiting activation of
NF-␬␤–induced antiapoptotic gene expression. The sub-G1 DNA
content analysis presented in Figure 3A (samples 3 and 4) verified
that this pathway is indeed active and that stabilization of p53 by
Vpu further enhances apoptosis (Figure 3A samples 7 and 8).
Recently HIV-1 Vif was shown to promote p53 activation by
inhibition of Mdm-2–mediated pathway,29 but our work establishes
that another late accessory protein, Vpu, competitively blocks the
␤-TrcP–mediated pathway for p53 degradation. Therefore, by
exploiting SCF␤-TrcP complex, Vpu enables HIV-1 to completely
hijack ␤-TrcP/p53/Mdm-2 axis to achieve higher p53-mediated
apoptotic potential. These results show an important implication of
inhibition of p53 ubiquitination by Wt Vpu that correlates positively toward promoting apoptosis.
Acknowledgments
We thank Sanjeev Das (NII, India) for GST-p53 expression
plasmid, Yukiko Gotoh (Institute of Molecular and Cellular Biosciences, The University of Tokyo) for HA-p53 expression plasmid,
Karen Vousden (The Beatson Institute of Cancer Research) for
p53⌬I expression plasmid, Dimitris Xirodimas (University of
Dundee) for His-Ubiquitin expression plasmid. We thank Klaus
Strebel (NIH) for generously providing HIV-1 clone pNL4-3 and
its Vpu variants and Udaykumar Ranga (JNCASR, India) for
subtype C pIndie HIV-1 plasmid. We also thank Aalia Shahr Bano
for technical help.
This work was supported by the Department of Biotechnology,
Government of India (A.C.B. and NII, New Delhi). Several
reagents were obtained from the Acquired Immunodeficiency
Syndrome Research and Reference Reagent Program, Division of
Acquired Immunodeficiency Syndrome, NIAID, NIH.
Authorship
Contribution: S.V. and A.C.B. designed the research; S.V. and A.A.
performed the research; S.V. analyzed and interpreted the data; and
S.V., A.A., S.A., and A.C.B. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Akhil C. Banerjea, Laboratory of Virology,
National Institute of Immunology, JNU Campus, New Delhi110067, India; e-mail: [email protected] or [email protected].
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BLOOD, 16 JUNE 2011 䡠 VOLUME 117, NUMBER 24
Vpu STABILIZES p53 VIA INHIBITING ␤-TrcP FUNCTION
6607
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2011 117: 6600-6607
doi:10.1182/blood-2011-01-333427 originally published
online April 26, 2011
Inhibition of β-TrcP−dependent ubiquitination of p53 by HIV-1 Vpu
promotes p53−mediated apoptosis in human T cells
Sachin Verma, Amjad Ali, Sakshi Arora and Akhil C. Banerjea
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