From the Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460
Received for publication, November 21, 2000, and in revised form, February 15, 2001
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ABSTRACT |
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The human immunodeficiency virus type 1 (HIV-1)
Vpu protein binds to the CD4 receptor and induces its
degradation by cytosolic proteasomes. This process involves the
recruitment of human The human immunodeficiency virus type 1 (HIV-1)1 is a complex
retrovirus, which, in addition to the prototypical Gag, Pol, and Env
products, requires the activity of a number of accessory proteins for
replication in vivo. Most if not all of the viral accessory
proteins exert multiple independent functions. This includes Vpu, which
not only regulates virus release from infected cells (1, 2) but also
induces degradation of the HIV-1 receptor CD4 in the endoplasmic
reticulum (3).
The best characterized function of Vpu is its ability to induce the
degradation of CD4 (for reviews, see Refs. 4 and 5). Structurally, this
activity is dependent on sequences present in the cytoplasmic domain of
Vpu and, in particular, requires the phosphorylation by casein kinase
II of two highly conserved serine residues at positions 52 and 56 (6).
CD4 degradation is initiated by physical interactions between the
cytoplasmic domains of Vpu and CD4 (7). Interestingly, phosphorylation
of the two serine residues in Vpu, while critical for CD4 degradation,
is not required for this initial binding (7). Instead, the
phosphoserine residues are involved in the recruitment of human Recent studies have confirmed the important role of TrCP in the
regulated degradation of cellular proteins. Indeed, the
SCFTrCP was shown to mediate the ubiquitination and
proteasome targeting of Among the known substrates of TrCP, Vpu is exceptional in that it does
not appear to be targeted for degradation itself but rather acts as an
adapter for proteasome targeting of CD4 (8). Also, in contrast to
NF- In HIV-infected cells, Vpu is synthesized from a bicistronic mRNA
also encoding the viral envelope (Env) protein gp160. Comparative analysis of Vpu and Env synthesis suggests that the two proteins are
synthesized at similar rates. However, unlike the Env protein, which is
packaged into virions and exported from the cell, Vpu remains largely
cell-associated and accumulates over time in infected cells. Based on
these observations, we speculated that the accumulation of Vpu in
HIV-infected cells could interfere with the normal physiological function(s) of TrCP by acting as a competitive inhibitor of TrCP. To
test this hypothesis, we characterized in this study the effects of Vpu
on TrCP-mediated degradation of IkB- We now present evidence that the stable interaction between Vpu and
TrCP in HIV-1-infected cells leads to impaired TrCP function. Indeed,
infection of the CD4-positive A3.01 cell line with the NL4-3 molecular
clone of HIV-1 but not with its Vpu-deficient variant resulted in an
inhibition of TNF- Cells and Plasmids--
Plasmids expressing the NL4-3
full-length molecular clone of HIV-1 or a Vpu-defective variant
NL4-3/Udel have been described previously (1, 23). Construction of the
Env-defective variant of pNL4-3, pNL43-K1, was previously described
(24). The subgenomic viral expression plasmid pNL-A1 used here for the
expression of Vpu was derived from a Vif-cDNA clone and encodes all
other viral genes except gag and pol (25). The
CD4 expression plasmid pHIV-CD4
For the construction of stable, inducible cell lines, CD4U and
CD4U2/6 chimeras were placed under the transcriptional
control of a tetracycline-repressible promoter as follows: First, the multiple cloning site of the pTRE plasmid
(CLONTECH, Palo Alto, CA) was modified by
oligonucleotide-directed PCR mutagenesis. The multiple cloning site of
the resulting vector, pTRS, contains 5' SacII,
EcoRI, NheI, BsrGI (compatible with
Acc65I), AflII, BamHI,
NotI, XhoI, and XbaI sites. Plasmids
pTRS-CD4U and pTRS-CD4U2/6 were constructed by inserting a
1542-bp EcoRI-Acc65I fragment from pHIV-CD4U or
pHIV-CD4U2/6, respectively, into the
EcoRI-BsrGI sites of pTRS.
For measuring NF- Antibodies--
For the detection of HIV-1-specific proteins, an
HIV-positive patient serum reacting with all major HIV-1 proteins was
used. A rabbit polyclonal antiserum directed against the cytoplasmic domain of Vpu (U2-3) was used for the identification of Vpu (28). The
T4-4 rabbit polyclonal antibody, directed against the ectodomain of
CD4, was obtained from the AIDS Research and Reference Reagent Program
and was originally contributed by R. Sweet (29). The T4-Cy rabbit
polyclonal antibody is directed against the cytoplasmic tail of the CD4
molecule and was previously described (30). Rabbit polyclonal
antibodies to I Construction of Tetracycline-inducible CD4U and
CD4U2/6 Cell Lines--
Approximately 2 × 106 HeLa Tet-off cells (CLONTECH, Palo
Alto, CA) were transfected with 30 µg of pTRS-CD4U or
pTRS-CD4U2/6 plasmid DNAs along with 2 µg each of the
hygromycin expression vector pTK-Hyg (CLONTECH)
using a standard calcium-phosphate transfection protocol as described
previously (30). Cells were maintained in G418 (1 mg/ml), tetracycline
(100 ng/ml), and hygromycin (200 µg/ml) selection medium for 2 weeks,
at which time individual drug-resistant clones were harvested.
Individual clones were tested for their ability to express CD4U or
CD4U2/6 in an inducible fashion by cultivating cells in
Dulbecco's modified Eagle's medium containing 10% tetracycline-free
fetal calf serum (CLONTECH) for 48 h. CD4U and
CD4U2/6 expression was assessed by Western blotting as
described below using the U2-3 anti-Vpu antibody. Following initial
selection, cells were maintained in the presence of the tetracycline
analogue doxycycline (10 ng/ml).
Immunoblotting--
Cells (5 × 106) were lysed
in 200 µl of CHAPS lysis buffer (50 mM Tris, pH 8.0, 5 mM EDTA, 100 mM NaCl, 0.5% CHAPS) and
25 µl of DOC buffer (2% deoxycholate in CHAPS lysis buffer)
supplemented with a protease/inhibitor mixture (CompleteTM;
Roche Molecular Biochemicals). Insoluble material was removed by
centrifugation, and the supernatant was combined with an equal volume
of sample buffer and boiled. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon membranes. Following transfer, membranes were blocked for 30 min with
5% dry milk in TN-T buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.3% (v/v) Tween 20). Membranes were briefly
rinsed with TN-TN wash buffer (0.05% IGEPAL CA-630 in TN-T buffer) and
then incubated for 1 h with the primary antibody in 3% bovine
serum albumin in TN-T. Blots were then washed once each for 5 min with TN-TN and TN-T and then reacted for 45 min in 3% bovine serum albumin/TN-T with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). Membranes were washed twice
each with TN-TN and TN-T (5 min each wash). Proteins were visualized by
ECL (Amersham Pharmacia Biotech). Bands were quantified after
densitometric scanning of the films using Fuji MacBAS software.
Analysis of NF- Electroporation of A3.01 Cells--
Productive HIV-1 infection
was initiated in CD4-positive A3.01 cells by electroporation of pNL4-3
or pNL4-3/Udel plasmid DNA. Approximately 5 × 106
cells in exponential growth phase were electroporated with 5 µg of
DNA in duplicate using a Bio-Rad Gene Pulser II with a capacitance extender set at 0.3 kV and 975 microfarads. Electroporated cells were
immediately transferred to a 12-well culture plate containing 1 × 106 fresh A3.01 cells in complete RPMI 1640 medium (total
volume, 2 ml). Ninety percent of the culture medium was replaced
12 h postelectroporation, and aliquots were collected every 2 days thereafter for the purpose of monitoring the infection kinetics by
reverse transcriptase assay.
Vpu Inhibits the TNF- Vpu Inhibits Degradation of Phosphorylated I
We also tested the ability of CD4U to regulate virus release. To avoid
an interference of HIV-1 Env with CD4U function due to the
formation of intracellular complexes between the Env and CD4
ectodomains (31), the Env-defective variant of pNL4-3, pNL43-K1, was
used in this experiment (Fig. 2B). HeLa cells were
transfected with Vpu-defective (Vpu (
To directly address the effect of Vpu on I
The inducible CD4U and CD4U2/6 lines were used to assess
the impact of Vpu on endogenous I CD4U-mediated Inhibition of I
To further support the notion that the effect of Vpu on NF- Vpu Modulates HIV-1-induced NF- HIV-1 is a human retrovirus that induces long term chronic
infection in affected individuals. The virus is highly pathogenic and
leads to the complete destruction of the immune system in the vast
majority of patients. Disease progression is controlled by a complex
interplay of viral and host factors, in particular cytokines that
regulate the immune response (for a review, see Ref. 35). Chronic HIV
infection results in the deregulation of numerous cellular functions,
including cytokine production, which can either be increased or
decreased (reviewed in Ref. 36). In HIV-1-positive individuals,
expression of the proinflammatory cytokines TNF- As one of the key regulators of cytokine expression, NF- The majority of the currently known HIV-1 isolates encode a complete
vpu gene (51). However, a number of HIV-1 isolates such as
HxB2 and HTLVIIIB/LAI/BRU, which have been widely used in tissue
culture studies, do not encode a functional Vpu protein (51). It is
therefore conceivable that the regulatory role of Vpu could have been
overlooked in cases where recombinant protein, subgenomic expression
vectors, or Vpu-deficient viruses were used (32-34, 48, 52-54). Thus,
our finding in the present study that Vpu negatively regulates the
activation of NF- The effect of Vpu on NF- Despite the fact that Vpu is unlikely to completely inhibit NF-TrCP (TrCP), a key member of the
SkpI-Cdc53-F-box E3 ubiquitin ligase complex that
specifically interacts with phosphorylated Vpu molecules. Interestingly, Vpu itself, unlike other TrCP-interacting proteins, is
not targeted for degradation by proteasomes. We now report that, by
virtue of its affinity for TrCP and resistance to degradation, Vpu, but
not a phosphorylation mutant unable to interact with TrCP, has a
dominant negative effect on TrCP function. As a consequence, expression
of Vpu in HIV-infected T cells or in HeLa cells inhibited TNF-
-induced degradation of I
B-
. Vpu did not inhibit
TNF-
-mediated activation of the I
B kinase but instead interfered
with the subsequent TrCP-dependent degradation of
phosphorylated I
B-
. This resulted in a pronounced reduction of
NF-
B activity. We also observed that in cells producing
Vpu-defective virus, NF-
B activity was significantly increased even
in the absence of cytokine stimulation. However, in the presence of
Vpu, this HIV-mediated NF-
B activation was markedly reduced. These
results suggest that Vpu modulates both virus- and cytokine-induced
activation of NF-
B in HIV-1-infected cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TrCP
(TrCP). TrCP is a key component of a recently characterized E3
ubiquitin ligase complex that regulates protein degradation through the ubiquitin-dependent proteasome pathway (8). This finding
was of particular significance, since there is evidence for the
involvement of the ubiquitin-proteasome machinery in Vpu-mediated CD4
degradation (9, 10). The role of TrCP in the SkpI-Cdc53-F-box protein (SCF) ubiquitin ligase complex is to select and recruit the proper substrate for polyubiquitination by the SCF. This is achieved by the
modular organization of TrCP, which allows for simultaneous interactions with the target protein on the one hand through its C-terminal WD repeats and the SCF on the other hand through
interactions between its F-box domain and SkpI (reviewed in Ref.
11).
-catenin as well as the NF-
B-inhibitory
molecule I
B-
(12-14). Similar to the TrCP-binding domain in Vpu
(15), the signal for recognition of both
-catenin and I
B by
TrCP includes a pair of conserved phosphorylated serine residues that
are arranged in a consensus
motif, DS[PO3]G
XS[PO3]
(where
represents a hydropholic residue) present in all three
proteins. Serine phosphorylation plays a major role in regulating the
stability of SCF target proteins. In the case of I
B-
, activation
of the I
B kinase complex following stimulation by cytokines such as
TNF-
results in the phosphorylation of two serine residues
(Ser32 and Ser36) and leads to the rapid
degradation of I
B-
by cytosolic proteasomes (16, 17). Similarly,
-catenin phosphorylation constitutes a signal for degradation and is
developmentally regulated by glycogen synthase kinase 3
(18).
B or
-catenin, Vpu is constitutively phosphorylated by the
ubiquitous casein kinase II (19), and, as a consequence, the vast
majority of Vpu present in infected cells is TrCP binding-competent at
all times. Both the constitutive Vpu phosphorylation as well as the
inability to degrade TrCP-bound Vpu are likely to contribute to
unusually stable complexes between the two proteins. Indeed, we have
previously reported that expression of Vpu and TrCP in vitro
in the presence of microsomal membranes resulted in efficient
redistribution of TrCP from its normal cytoplasmic fraction to the
Vpu-containing membrane fraction (8).
and the resulting activation of
NF-
B transcriptional activity. NF-
B is a transcriptional activator that under resting conditions resides in the cytoplasm in an
inactive complex with its inhibitor I
B-
. Degradation of IkB-
following stimulation of cells by growth factors, chemokines, or
inflammatory cytokines such as TNF-
leads to activation of NF-
B
and its translocation into the nucleus, where it induces target gene
expression. NF-
B plays a central role in the expression of numerous
genes encoding cytokines, chemokines, and factors involved in T-cell
activation and proliferation (reviewed in Refs. 17 and 20). In
addition, NF-
B has been implicated in the control of
antiapoptotic genes (reviewed in Refs. 21 and 22). Thus,
deregulation of NF-
B in HIV-infected cells will impact on a
multitude of normal cellular functions.
-induced I
B degradation. Using CD4/Vpu chimeric
molecules with biological activities indistinguishable from those of
authentic Vpu, we demonstrate that Vpu is required and sufficient to
block I
B degradation. Using a Vpu phosphorylation mutant, we confirm
that the ability of Vpu to block I
B degradation is linked to its
ability to bind TrCP, demonstrating that Vpu indeed acts as a
transdominant negative inhibitor of TrCP. Inhibition of I
B
degradation by Vpu was found to severely impair TNF-
-induced NF-
B
activation. Similarly, Vpu was found to significantly reduce HIV-induced activation of the NF-
B pathway. Taken together, these results suggest that Vpu has a transdominant negative effect on TrCP
function that impairs both HIV- and cytokine-mediated activation of
NF-
B.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bam and pCMV-CD4 expressing
full-length CD4 under the transcriptional control of the HIV-1 long
terminal repeat or the cytomegalovirus immediate early promoter,
respectively, were described before (7). Plasmid pcDNA-TrCP
F was
derived from pcDNA-TrCP, encoding the wild type human
TrCP
protein under the transcriptional control of the cytomegalovirus
immediate early promoter (8). pcDNA-TrCP
F bears a 148-amino acid
deletion (residues 32-179) that includes the F-box domain
described previously (8). CD4/Vpu chimeric proteins bearing the Vpu
transmembrane and cytoplasmic domains fused to the CD4 ectodomain (CD4U
and CD4U2/6) were engineered with a structure similar to
those previously described (26, 27). The chimeras were constructed in a
two-step PCR as follows. First, two PCR fragments, encoding the
CD4 ectodomain (amino acids 1-369) and Vpu (amino acids 2-81),
respectively, were generated using pHIV-CD4
Bam or pNL-A1 plasmid
DNAs as PCR templates. The primary PCR products were designed such that
the two fragments shared a 29-bp overlap, which allowed the two PCR
products to anneal during the second PCR. The second PCR was performed
using the outside primers from the first reaction and the two primary PCR products as templates. The resulting 1111-bp PCR product encoded the CD4 ectodomain fused to the Vpu TM and cytoplasmic domains. The
final PCR product was digested with MfeI and KpnI
(New England Biolabs, Beverly MA) and cloned into the corresponding
sites in pHIV-CD4
Bam. The pHIV-CD4U2/6 carrying serine
to alanine mutations at positions 52 and 56 of Vpu was generated in an
analogous reaction except that pNL-A1/U2/6 (6) plasmid DNA
was used as template for the primary PCR. Expression of CD4U and
CD4U2/6 from these plasmids was Tat-dependent.
For Tat-independent expression, CD4U and CD4U2/6 were
placed under the transcriptional control of the cytomegalovirus
immediate early promoter by subcloning a 1559-bp EcoRI-KpnI fragment from pHIV-CD4U or
pHIV-CD4U2/6 into pcDNA3.1(
) (Invitrogen, Carlsbad, CA).
B activity, the NF-
B indicator plasmid,
pNF
B-Luc, was employed (Stratagene, La Jolla, CA). pNF
B-Luc
expresses the luciferase gene under the control of an
NF-
B-dependent minimal promoter containing five NF-
B
binding sites.
B (anti-I
B) and phosphorylated I
B
(anti-P-I
B) were obtained from New England Biolabs.
B Activity: Luciferase Assay--
One day
prior to transfection, target cells were placed into six-well plates
(approximately 2 × 105 cells/well). Cells were
transfected using FuGENE (Roche Diagnostics Corp.) or LipofectAMINE
(Life Technologies, Inc.) transfection reagents. In general, a total of
4-5 µg of plasmid DNA (1 µg of pNF
B-luc reporter plasmid plus
3-4 µg of test plasmid(s) or filler DNA (pcDNA3.1(
)) was used
for transfection. Cells were harvested 20-24 h after transfection.
Where appropriate, cells were treated with TNF-
(20 ng/ml;
Calbiochem) for 5-20 min prior to cell lysis. For measuring NF
B
activation by luciferase assay, TNF-
-containing medium was replaced
with normal tissue culture medium after an initial 15-min stimulation
followed by a 5-h incubation. Cell extracts were prepared by lysis in
1× Reporter Lysis Buffer (Promega, Madison, WI). Cell lysates were
normalized for protein content, and luciferase activity was determined
using the Promega Luciferase Assay System. Samples were analyzed using
an Optocomp II Luminometer (MGM Instruments, Hamden, CT).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced Degradation of I
B in
HIV-infected T Cells--
In a first set of experiments, we assessed
the impact of Vpu expression on the TNF-
-induced degradation of
I
B in HIV-1-infected T cells. Infections were initiated in the CD4+
A3.01 cell line by electroporation of plasmid DNA encoding the
molecular clone NL4-3 or its Vpu-defective variant NL4-3/Udel. Virus
replication was monitored by measuring the viral reverse transcriptase
activity in the infected cultures (data not shown). Cells were
harvested 7 days after electroporation near the time of peak virus
production, and equal aliquots were subjected to treatment with TNF-
for 5 or 20 min or were left untreated (0 min). Cell lysates were then
analyzed by immunoblotting with an I
B-specific antibody (Fig.
1A, top
panel). To control for differences in infection rates and
relative protein concentrations, the same blot was subsequently reacted
with an HIV-positive patient serum (Fig. 1A,
bottom panel). I
B-specific bands were
quantified and plotted as a percentage of the amount recovered before
TNF-
stimulation (Fig. 1B, time 0). The response of
infected A3.01 cells to treatment with TNF-
, as measured by the
degradation of I
B-
, was markedly different in cells infected with
wild type versus Vpu-deficient HIV-1 (Fig. 1A,
top panel). In fact, in cells infected with the
Vpu-defective virus, TNF-
stimulation resulted in degradation of
over 80% of total I
B within 20 min of treatment (Fig.
1B, NL4-3/Udel). In striking contrast, TNF-
treatment of
cells infected with wild type NL4-3 failed to induce I
B degradation
during the 20-min induction time (Fig. 1B, NL4-3). This
difference in the cellular response to TNF-
treatment following
infection with wild type and Vpu-defective viruses could not be
attributed to different infection efficiencies, since the levels of
viral proteins expressed in the two cultures at the time of the
experiment were virtually identical (Fig. 1A,
bottom panel). These data therefore suggest that
accumulation of Vpu in T cells during a normal productive HIV-1
infection blocks I
B degradation following TNF-
stimulation.
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Fig. 1.
Vpu inhibits
TNF- -induced degradation of
I
B in infected A3.01 cells. A3.01 cells
were electroporated with pNL4-3 (NL4-3) or the Vpu-deficient
pNL4-3/Udel (NL4-3/Udel) plasmid DNA. A, seven days
postinfection, cells were harvested, and equal fractions were treated
with TNF-
(20 ng/ml) for 5 or 20 min. A control sample (0 min) was
left untreated. Cells were lysed in detergent buffer containing
protease inhibitors. Equal aliquots of each cell lysate were separated
by 12.5% SDS-polyacrylamide gel electrophoresis and transferred to
Immobilon membranes. I
B-
was revealed by immunoblotting using a
polyclonal antibody to I
B-
followed by ECL (top
panel). The same membrane was subsequently reacted with an
HIV-positive patient serum to detect viral proteins in the lysates
(bottom panel). Proteins are identified on the
right. B, bands corresponding to I
B-
were
quantified using the Fuji MacBAS software and plotted as a percentage
of the corresponding uninduced sample.
B-
in a
Dose-dependent Manner--
We next examined whether the
presence of Vpu alone could account for the inhibition of I
B-
degradation observed in Fig. 1. Presumably due to the presence of
cryptic splice signals within the Vpu-coding region, we were unable to
express detectable levels of Vpu from Tat- and Rev-independent vector
systems (data not shown). Interestingly, we found that insertion of the
fully spliced extracellular domain of human CD4 upstream of the Vpu
coding sequence resulted in efficient and stable expression of the
chimeric protein. We made use of this phenomenon by creating chimeric
molecules between the CD4 ectodomain and wild type Vpu or a Vpu mutant
(S52N/S56N) previously shown to be unable to interact with human
TrCP (8). The resulting constructs, termed CD4U and
CD4U2/6, respectively, showed high levels of
Rev-independent protein expression in HeLa cells (data not shown). To
verify that CD4U and CD4U2/6 are functionally comparable
with Vpu and Vpu2/6, respectively, we first assessed the
ability of the chimeras to induce degradation of CD4 (Fig. 2A). Due to the absence of the
CD4 cytoplasmic domain, which contains sequences critical for
TrCP-dependent degradation, CD4U chimeras were expected to
be resistant to TrCP-dependent degradation. Degradation of
CD4 was measured by comparing steady state levels of CD4 in the
presence or absence of Vpu, Vpu2/6, CD4U, or
CD4U2/6. HeLa cells were transfected with equal amounts of
pHIV-CD4
Bam (CD4) in combination with pHIV-CD4U (CD4U),
pHIV-CD4U2/6 (CD4U2/6), pNL-A1 (Vpu), or
pNL-A1/U2/6 (Vpu2/6), as indicated in Fig.
2A. Therefore, expression of CD4 and the chimeric CD4/Vpu
molecules was dependent on the expression of Tat from pNL-A1 or
pNL-A1/U2/6. Cell lysates were prepared 24 h after
transfection and analyzed by immunoblotting with antibodies to CD4
(Fig. 2A, anti-CD4) or Vpu (Fig. 2A, anti-Vpu).
Expression of wild type Vpu from pNL-A1 (lane 1)
significantly reduced the steady state levels of CD4 when compared with
cells expressing the TrCP binding mutant Vpu2/6 (lane 2) although comparable levels of Vpu
protein were expressed (lanes 1 and 2,
anti-Vpu). Importantly, coexpression of CD4U (lanes 3 and 4) resulted in almost complete depletion of
the CD4 steady state levels even in Vpu2/6-expressing cells
(lane 4). This suggests that CD4U is able to
induce degradation of CD4 with similar efficiency than authentic wild
type Vpu. Coexpression of CD4U2/6 did not affect the
ability of wild type Vpu to induce CD4 degradation (compare
lanes 1 and 5, CD4). As expected,
coexpression of Vpu2/6 and CD4U2/6
(lane 6) did not affect CD4 levels and resulted
in CD4 steady state levels similar to those observed in cells
expressing Vpu2/6 only (compare lanes
2 and 6). These results demonstrate that CD4U but
not CD4U2/6 has the ability to induce CD4 degradation. These data also indicate that CD4U has retained the ability to interact
with
TrCP, a necessary event in the process of CD4 degradation.
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Fig. 2.
Molecular characterization of the CD4U and
CD4U2/6 chimeric proteins. A, CD4U and
CD4U2/6 are functionally equivalent to Vpu with respect to
their ability to induce CD4 degradation. Vpu and Vpu2/6
expression in this experiment was dependent on Tat and Rev, both of
which are encoded by the pNL-A1 construct. Expression of CD4 CD4U, and
CD4U2/6 was Tat-dependent as well. HeLa cells
were transfected with 5 µg of pHIV-CD4 Bam and equimolar amounts of
plasmids expressing Vpu (pNL-A1), Vpu2/6
(pNL-A1/U2/6), CD4U (pHIV-CD4U), or CD4U2/6
(pHIV-CD4U2/6). Cells were harvested 24 h post
transfection. Detergent extracts were loaded on a 12.5% gel and
transferred to Immobilon membranes. Immunoblotting was performed as in
Fig. 1 using the ECL system. CD4 and CD4U proteins present on the
membrane were revealed using a mixture of the T4-4 and T4-Cy polyclonal
antisera (anti-CD4). Wild type Vpu and the TrCP-binding mutant,
Vpu2/6, were detected on the same blot using the
Vpu-specific U2-3 antibody (anti-Vpu). The positions of CD4, Vpu, and
the slower migrating CD4U chimera are indicated on the left.
The plasmid constructs used in this experiment are schematically shown
at the bottom. B, CD4U supports efficient virus
release. HeLa cells were transfected with Vpu-defective (Vpu
(
)) or Vpu-expressing (Vpu (+)) variants of pNL4-3/K1 in
the presence or absence of pHIV-CD4U (CD4U) as indicated on
the right. The effect of CD4U on virus release was
determined by pulse/chase analysis. Cells were pulse-labeled for 30 min
in the presence of [35S]methionine and chased for up to
4 h. At each time point, viral proteins present in the cell and
medium fractions were recovered by centrifugation and lysed in
detergent buffer. Viral proteins were immunoprecipitated with an
HIV-positive human serum, separated by SDS-polyacrylamide gel
electrophoresis, and visualized by fluorography (not shown). HIV Gag
proteins were quantified, and particle release was calculated as the
ratio of viral proteins detected in the medium over the total amount of
viral proteins present in both the cell and medium fractions.
C, inducible expression of CD4U and CD4U2/6.
Stable HeLa cell lines expressing tetracycline-inducible CD4U or
CD4U2/6 chimeras were constructed as described under
"Experimental Procedures." For the experiment shown here, cells
(3 × 106 each) were seeded into 25-cm2
tissue culture flasks (1 flask/time point). Cells were grown in the
absence of doxycycline for 15, 20, or 40 h before cells were
harvested. An uninduced control culture (0 h) was grown in the presence
of doxycycline for 15 h before cells were harvested. Detergent
lysates from each time point were analyzed by immunoblotting for CD4U
or CD4U2/6 expression using a CD4-specific antibody. The
position of CD4U and CD4U2/6 in the gel is marked on the
right.
)) or Vpu-expressing (Vpu (+))
variants of pNL4-3/K1 in the presence of absence of pHIV-CD4U (CD4U),
and the effect of CD4U on virus release was determined by pulse/chase analysis as described in the legend to Fig. 2B. In the
absence of Vpu (Fig. 2B, Vpu (
)) expression of CD4U in
trans increased virus release by more than 3-fold (Fig.
2B, Vpu (
), CD4U (+)). Coexpression of Vpu and CD4U showed
only little synergistic effect on particle release, indicating that
CD4U alone is sufficient for near maximal virus release (Fig.
2B, Vpu (+), CD4U (+)). These data demonstrate that CD4U can
indeed enhance the release of virus particles in a manner similar to
wild type Vpu. Taken together, our results show that CD4U and
CD4U2/6 are functionally equivalent to Vpu and
Vpu2/6, respectively, both with respect to CD4 degradation and enhancement of virus release.
B metabolism, we
constructed stable cell lines expressing CD4U and CD4U2/6
under the control of an inducible tetracycline-repressed promoter
(Tet-off). Individual cell clones were screened for their ability to
express the chimeric proteins in an inducible fashion by removal of the doxycycline inhibitor and assessment of CD4U and CD4U2/6
expression over time by immunoblotting using a CD4-specific antiserum.
As shown in Fig. 2C, the two clones selected express CD4U
and CD4U2/6 with similar efficiency.
B turnover in the absence of other viral proteins. This was done by measuring the TNF-
-mediated degradation of I
B at various times following induction of CD4U and
CD4U2/6. Cells were grown in 25-cm2 tissue
culture flasks in the presence (0 h) or absence (15-40 h) of
doxycycline. Cells were harvested at the indicated time points, and
equal aliquots were stimulated with TNF-
(20 ng/ml) for 0, 5, or 20 min (Fig. 3A). Cell lysates
were then analyzed by immunoblotting first with antibodies to total
I
B (Fig. 3A, I
B), followed by reaction with antibodies
specific for phosphorylated I
B (Fig. 3A,
pI
B). I
B-specific bands were quantified by
densitometric scanning, and the relative amounts of I
B remaining at
the indicated times after TNF-
stimulation were used to calculate
the percentage of I
B degradation as a function of time (Fig.
3B). As expected, TNF-
treatment of doxycycline-repressed
CD4U or CD4U2/6 cells resulted in the rapid degradation of
I
B (Fig. 3A, I
B, 0 h). This was accompanied in
both cases by the transient appearance 5 min after TNF-
treatment of
a band corresponding to phosphorylated I
B (Fig. 3A,
pI
B, 0 h). Quantitation of total I
B in
uninduced cells revealed that in both cell lines 90-95% of the total
I
B protein was degraded within 20 min of TNF-
treatment (Fig.
3B, 0 h). Thus, both cell lines show a similar response
to TNF-
stimulation and have the same intrinsic ability to
phosphorylate and degrade I
B. Induction of CD4U2/6
expression following removal of doxycycline did not interfere with
TNF-
response, and the kinetics of I
B degradation following
TNF-
treatment remained virtually unchanged even after 40 h of
CD4U2/6 induction (Fig. 3, A and B,
CD4U2/6). In striking contrast, induction of CD4U resulted
in an increasing inhibition of TNF-
-induced degradation of I
B.
Forty hours after CD4U induction, only about 25% of total I
B was
degraded within the 20 min of TNF-
treatment (Fig. 3B,
CD4U). Thus, intracellular accumulation of CD4U resulted in the
dose-dependent inhibition of TNF-
-induced degradation of
I
B. Interestingly, the presence of CD4U had no apparent effect on
the TNF-
-induced phosphorylation of I
B (Fig. 3A,
pI
B). In fact, phospho-I
B appeared to
accumulate in response to rising levels of CD4U in the cells. This
suggests that Vpu does not affect TNF-
-induced activation of the
I
B kinase but instead blocks the subsequent
TrCP-dependent degradation of phosphorylated I
B-
.
View larger version (27K):
[in a new window]
Fig. 3.
Vpu inhibits the SCFTrCP-mediated
degradation of phosphorylated
I B-
in a
dose-dependent manner. A, CD4U and
CD4U2/6 cell lines were induced by the removal of
doxycycline as described for Fig. 2C. Cells were harvested
at the indicated time points, washed once in phosphate-buffered saline,
and divided into three equal fractions. One fraction was left untreated
(0 min). Cells from the other two fractions were treated with TNF-
(20 ng/ml) for 5 or 20 min, respectively. Cell lysates were then
subjected to immunoblot analysis using antibodies to total I
B
(I
B) or phospho-I
B (pI
B) followed by
ECL. B, I
B-
-specific bands were quantified as in Fig.
1B and used for the calculation of the degradation kinetics
(percentage degraded relative to time 0).
B Degradation Leads to Reduced
NF-
B Transcriptional Activity--
The results shown in Figs. 1 and
3 demonstrate a pronounced inhibition of I
B degradation following
cytokine stimulation in Vpu or CD4U-expressing cells. To assess the
impact of this phenomenon on the transcriptional activity of NF-
B,
we examined the effect of CD4U or CD4U2/6 on NF-
B
transcriptional activity using a luciferase reporter gene. The HeLa
CD4U- and CD4U2/6-inducible cell lines were transfected in
triplicate with the pNF
B-Luc reporter plasmid, and CD4U or
CD4U2/6 protein synthesis was induced by removal of doxycycline. At 48 h postinduction, cells were incubated in the presence or absence of TNF-
(10 ng/ml) for 15 min. The cytokine was
then removed, and cells were cultured for an additional 6 h to
allow for luciferase synthesis. The control cultures were treated in
the same fashion but remained in the presence of doxycycline throughout
the experiment. As shown in Fig.
4A, TNF-
treatment resulted
in a comparable activation of NF-
B in uninduced (Dox (+))
CD4U and CD4U2/6 cell lines. Induction of
CD4U2/6 expression (CD4U2/6, Dox (
)) did not
affect the TNF-
response in these cells and resulted in NF-
B
activation similar to that observed in uninduced cells (compare
CD4U2/6, Dox (+), and Dox (
)). In contrast, induction of
CD4U expression (CD4U, Dox (
)) led to a 3.5-fold reduction in the
amount of luciferase activity measured (compare CD4U, Dox (+), and Dox
(
)). Interestingly, CD4U but not CD4U2/6 also decreased
by 3.5-fold the basal level of NF-
B activity observed in the absence
of TNF-
stimulation (Fig. 4A, TNF (
)). Taken
together, these results show that inhibition of I
B degradation by
Vpu in infected T cells or stably transfected HeLa cells (Figs. 1 and
3) has a direct negative impact on transcriptionally active
NF-
B.
View larger version (15K):
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Fig. 4.
Vpu interferes with TrCP to inhibit
TNF- -induced activation of
NF-
B. A, inducible HeLa-CD4U
and CD4U2/6 cell lines were transfected with 1 µg of
pNF
B-Luc and maintained in the presence or absence of doxycycline
(Dox (+) or Dox (
)) for 48 h. Cells
were then incubated in the presence or absence of TNF-
(10 ng/ml)
for 15 min. Cells were cultured in the absence of TNF-
for an
additional 6 h, and the relative amount of luciferase activity was
obtained from triplicate measurements. B, HeLa cells were
transfected in triplicates with 1 µg of reporter plasmid
(pNF
B-Luc) and 4 µg of either pcDNA3.1(
) (mock),
pCMV-CD4 (CD4), pcDNA-CD4U (CD4U), or
pcDNA-TrCP
F (TrCP
F). Cells were
harvested 24 h post-transfection. Equal fractions of cells were
either treated with 10 ng/ml of TNF-
(TNF(+)) for 15 min or left
untreated (TNF(
)) and cultured for an additional 5 h at
37 °C. Cell lysates were normalized for equal protein content, and
luciferase activity was determined. Error bars
reflect the S.D.
B
activity is caused by the inhibition of TrCP-dependent
I
B degradation, we compared the effects of CD4U and a transdominant
negative mutant of TrCP, TrCP
F (8), on TNF-
-induced NF-
B
activity (Fig. 4B). HeLa cells were each transfected with
pNF
B-Luc reporter plasmid along with empty pcDNA3.1(
) vector
DNA (mock), pCMV-CD4 (CD4), pcDNA-CD4U (CD4U), or
pcDNA-TrCP
F (TrCP
F) expressing a TrCP F-box deletion mutant,
which was previously found to have a transdominant negative effect on
both CD4 and I
B degradation (8, 13). At 24 h post-transfection,
one set of cells was treated with TNF-
for 15 min followed by a 5-h
incubation at 37 °C (Fig. 4B, TNF (+)). A
second set of cells was left untreated (TNF (
)). Cells
were lysed and assayed for luciferase activity. As predicted, TNF-
treatment of mock-transfected cells resulted in a significant induction
of NF-
B-driven luciferase expression during the 5-h incubation
period relative to the unstimulated culture (Fig. 4B,
mock). Similarly, expression of wild-type CD4 did not affect
the cellular TNF-
response and resulted in an increase in luciferase
activity comparable with the mock control (CD4). In contrast,
expression of CD4U significantly reduced luciferase expression, further
confirming that the Vpu component of the CD4U chimera was responsible
for the repression of NF-
B activation described above. Consistent
with the results from Fig. 4A, CD4U expression in
unstimulated cells reduced the basal activity of NF-
B. The effect of
CD4U was strikingly similar to that of the transdominant negative TrCP
mutant, suggesting that the two proteins act at a common step in the
degradation cascade, i.e. the binding of TrCP to
phosphorylated I
B (Fig. 4B, TrCP
F). The similarity between the inhibitory effects exerted by CD4U and TrCP
F further supports our conclusion that CD4U acts as a competitive inhibitor of
the SCFTrCP.
B Activation--
Previous
reports indicate that HIV-1 infection of cells can result in a
constitutive activation of NF-
B (32-34). Our data indicate that Vpu
inhibits NF-
B activation in response to TNF-
treatment. We
therefore addressed whether this effect of Vpu is also observed in the
context of HIV-mediated activation of NF-
B. In a first set of
experiments, HeLa cells were transfected with the pNF
B-luc indicator
plasmid together with subgenomic constructs expressing all HIV-1
proteins except Gag and Pol (pNL-A1). As seen in Fig.
5A, transfection of pNL-A1 led
to a moderate 3-fold activation of NF-
B, as compared with the mock
control expressing no viral protein. However, expression of
pNL-A1/Udel, a Vpu-deficient variant of pNL-A1, led to a much higher
14-fold enhancement of NF-
B activity. In addition, in the presence
of the
TrCP-binding mutant of Vpu, Vpu2/6
(pNL-A1/U2/6), NF-
B activity was similar to that
observed following transfection of pNL-A1/Udel. Very similar results
were obtained using full-length HIV-1 constructs (Fig. 5B).
These data indicate that HIV-1 encodes proteins with the intrinsic
ability to enhance NF-
B activity but that Vpu acts as a negative
modulator of that activity. Vpu is therefore capable of interfering not
only with TNF-
-mediated but also with HIV-1-induced activation of
NF-
B. Moreover, Vpu acts on both stimuli through the same mechanism,
since in both cases mutation of the TrCP binding site abolished the
inhibitory activity of Vpu.
View larger version (17K):
[in a new window]
Fig. 5.
Vpu modulates HIV-1-induced
NF- B activation. A, HeLa cells
were transfected in quadruplicate sets with each 1 µg of reporter
plasmid (pNF
B-Luc) and 4 µg of either pcDNA3.1(
)
(mock) or pNL-A1-based subgenomic constructs as indicated.
Cells were harvested 24 h post-transfection. Luciferase activity
was determined on lysates normalized for equal protein content.
Error bars reflect the S.D. B, HeLa
cells were transfected in triplicate sets with 1 µg of reporter
plasmid (pNF
B-Luc) and 4 µg of pcDNA3.1(
) (mock)
or the NL4-3 full-length molecular clone constructs as indicated. Cells
were harvested 24 h post-transfection, and luciferase activity was
determined as described above. Error bars reflect
the S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IL-1
, IL-6, and
IL-8 was found to be increased, especially at late stages of the
disease (37-42). Conversely, expression of the immunoregulatory
cytokines IL-2 and IL-12 was gradually lost over time (43, 44).
B is likely
to play a role in the perturbation of cytokine production observed in
HIV-infected individuals. In fact, three main mechanisms have been
proposed to account for the observed persistent activation of NF-
B
in HIV-1-infected cells: hyperphosphorylation and degradation of
I
B-
caused by the constitutive activation of the I
B kinase (32, 33), inhibition by nuclear I
B-
of the I
B-
-mediated dissociation of NF-
B/DNA complexes, and, more recently, modulation of the NF-
B-interacting p300 transcriptional co-activator (45). Several HIV proteins have been shown to directly or indirectly affect
NF-
B regulation. These include Tat, envelope gp120, and the
accessory protein Vpr (42, 45-49). We now report that the HIV-1-specific Vpu protein is also involved in the regulation of
NF-
B activity by acting as a negative modulator of TNF-
- and
HIV-mediated hyperactivation of NF-
B. Our data suggest that Vpu
functions both in HIV-1-infected T cells and in transfected HeLa cells
by acting as a competitive inhibitor of
TrCP, thereby preventing the
efficient degradation of phosphorylated I
B. Although NF-
B
activation was chosen to illustrate the effect of Vpu on TrCP function,
it is likely that this activity of Vpu has an even broader impact on
cell function. Indeed, one would expect that Vpu can perturb with
similar efficiency the degradation of other substrates of TrCP such as
-catenin (50).
B does not necessarily conflict with the previously
reported activation of NF-
B in HIV-infected cells. Indeed, it is
clear from our own results in both HeLa and A3.01 cells that the
expression of full-length HIV-1 does lead to a net increase in NF-
B
activity despite the presence of Vpu. However, this increase in NF-
B
activity is small compared with the activation seen in the absence of
Vpu (Fig. 5).
B is dose-dependent and is
mediated through inhibition of the TrCP-dependent
degradation of I
B-
. Unlike most other viral proteins, Vpu is not
packaged into virions or secreted but gradually accumulates in the
cell. As a result, Vpu-mediated inhibition of NF-
B activation can be
expected to be minor early in infection but to become more and more
pronounced as the viral life cycle progresses. Nevertheless, our data
obtained in HIV-infected T-cells (Fig. 1) or CD4U-inducible cell lines (Fig. 3) suggest that even when maximal amounts of Vpu are expressed, total inhibition of I
B degradation is not achieved, and NF-
B activity is not completely inhibited (Fig. 4). There are several possible explanations for this observation. First, it is possible that
degradation of I
B is regulated by redundant cellular mechanisms. For
example, TrCP2, a WD-F-box protein closely related to
TrCP, was
recently found to target I
B for degradation (55). Although unlikely,
we cannot formally rule out the possibility that the residual NF-
B
activity observed in the presence of Vpu is due to an inability of the
latter to bind and interfere with the function of TrCP2. Second, it is
possible that due to its location in cellular membranes, Vpu is unable
to prevent the nuclear degradation of newly synthesized I
B (56) and
therefore might be unable to block the sustained activation of a small
pool of NF-
B. Finally, it is possible that NF-
B can be activated
through TrCP-independent mechanisms, such as the reported dissociation
of I
B-NF-
B complexes following tyrosine phosphorylation of
I
B (57).
B
activity in vivo, its effects on cell function could
nevertheless be profound. Indeed, studies performed in transgenic mice
expressing a dominant negative variant of I
B-
indicate that T-
and B-cell development can be dramatically perturbed when levels of
active NF-
B are lowered below a critical physiological threshold
(58, 59). Also, there is a body of evidence indicating that NF-
B plays a central role in regulating cellular apoptosis (reviewed in Ref.
22). However, it is still debated whether HIV-1 infection and the
ensuing constitutive activation of NF-
B can effectively protect
cells from apoptosis. In some cases, activation of NF-
B has been
shown to prevent the natural tendency of HIV to induce apoptosis (60),
while it is also well documented that HIV infection can prime cells for
apoptosis (61, 62). The complex interplay between NF-
B, TNF-
,
PKR, and other factors determines whether or not a cell will be able to
overcome an apoptotic signal (22). Nevertheless, it is possible that
the gradual inhibition of NF-
B activity by Vpu during the course of
an infection contributes to HIV-induced apoptosis. This notion is
supported by the recent findings that NF-
B confers resistance
against Fas-mediated apoptosis (63) and that Vpu increases
susceptibility of human immunodeficiency virus type 1-infected cells to
Fas killing (64). Based on the results described in the current study,
we propose the model outlined in Fig. 6.
In cells expressing Vpu, phosphorylation of I
B by various stimuli
does not lead to the normal degradation of I
B and activation of
NF-
B transcriptional activity. This is due to the fact that I
B
degradation requires the activity of
TrCP, which, in Vpu-expressing
cells, is competitively inhibited through interactions with the
cytoplasmic tail of Vpu. Normal cellular substrates of
TrCP are
rapidly ubiquitinated and degraded, thereby releasing
TrCP from the
complex. In contrast, Vpu is not targeted for degradation but instead
accumulates in virus-producing cells and forms stable complexes with
TrCP. Both the accumulation of Vpu in virus-producing cells and the
stability of Vpu-TrCP complexes are likely to contribute to the
efficiency of Vpu-mediated inhibition of
TrCP activity, which
becomes more and more pronounced in late stages of virus
replication.
View larger version (31K):
[in a new window]
Fig. 6.
Model of Vpu interference with cellular TrCP
function. The ability of Vpu to competitively inhibit TrCP
function is based on the fact that Vpu is constitutively phosphorylated
at two serine residues (Ser52 and
Ser56), which are part of a TrCP binding motif.
However, unlike normal cellular substrates of TrCP, Vpu is not targeted
for degradation following binding to TrCP. Therefore, the intracellular
accumulation of Vpu during the course of infection leads to a
progressive inhibition of TrCP-dependent degradation of
I B and thus to a gradual inhibition of NF-
B. Details of the model
are explained under "Discussion."
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Alicia Buckler-White for assistance with oligonucleotide synthesis and DNA sequencing, Karen Kibler for helpful discussions, and Sandra Kao for expert technical support.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Intramural AIDS Targeted Antiviral Program (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: NIH/NIAID, 4/312, 4 Center Dr., MSC 0460, Bethesda, MD 20892-0460. Tel.: 301-496-3132; Fax: 301-402-0226; E-mail: kstrebel@nih.gov.
Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M010533200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
TrCP, human TrCP;
SCF, SkpI, Cdc53,
F-box protein;
TNF, tumor necrosis factor;
PCR, polymerase chain
reaction;
bp, base pair;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
IL, interleukin.
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