From the CNRS, Faculté de Médecine Nord,
Boulevard Pierre Dramard, Marseille, 13015 France and ¶ Animal
and Microbial Sciences, University of Reading, Reading RG6 6AJ, United
Kingdom
Received for publication, June 3, 2002, and in revised form, September 4, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human immunodeficiency virus (HIV)
envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded
molecule that attaches HIV to the lymphocyte surface receptors CD4 and
CXCR4. Conformation changes within gp120 result from binding and
trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated
protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting
that redox changes within Env are required. Using a sensitive assay
based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface
enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI
inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120
exhibited CD4 but not CXCR4 binding capacity. In addition, PDI
inhibition did not impair gp120 binding to receptors. We
conclude that on average two of the nine disulfides of gp120 are
reduced during interaction with the lymphocyte surface after CXCR4
binding prior to fusion and that cell surface PDI catalyzes this
process. Disulfide bond restructuring within Env may constitute the
molecular basis of the post-receptor binding conformational changes
that induce fusion competence.
The mature human immunodeficiency virus
(HIV)1 envelope (Env) is
composed of the surface glycoprotein (gp) 120 and transmembrane gp41
subunits (1). HIV binding to CD4+ lymphocytes is
initiated by gp120 interaction with cell surface CD4, which creates a
high affinity binding site on gp120 for the coreceptor CXCR4.
Interactions between gp120 and the receptors lead to structural changes
within Env that eventually promote the unmasking of the fusion peptide
present on gp41, its insertion into the target cell surface and
HIV/cell membrane fusion (2-6).
Although structural changes within Env following interaction with the
cellular receptors are mostly attributed to the intrinsic properties of
the viral envelope, they occur in the context of proteolytic and other
catalytic cell surface activities that are indispensable for membrane
fusion to occur (7-9). One such activity is cell surface
protein-disulfide isomerase (PDI), which has been implicated in the
process of HIV infection (9, 10).
PDI is a member of the thioredoxin superfamily. It catalyzes reduction,
oxidation, and thiol-disulfide interchange reactions and has important
roles in the folding of secretory proteins in the biosynthetic pathway
(11, 12). On the surface of the cell, it has been shown to cause
structural modifications of exofacial proteins involved in biological
process (11, 13-15). Clustered on the lymphocyte surface in the
vicinity of CD4-enriched regions (9), PDI may influence the
conformation modifications that occur during the interaction of HIV Env
with the target cell surface receptors through a partial reorganization
of the network of the disulfide bonds of the viral protein.
Alternatively, it may influence the thiol-disulfide content of other
cell surface antigens involved in the HIV/cell fusion process.
Experimental data support both possibilities because PDI inhibition
interferes with the virus/lymphocyte fusion process post-CD4 binding
(9).
Besides the observation that PDI inhibition blocks the HIV-replicative
cycle (9, 10), the plausible involvement of redox changes of Env as
part of the HIV-lymphocyte interaction is suggested by the observation
that an unusually dense cluster of disulfide bonds occurs close to the
receptor binding surfaces (16). As the precise molecular basis of the
Env conformational changes that take place upon fusion remains
enigmatic, we undertook a detailed study of the relationship between
cell surface-associated receptor binding and the thiol-disulfide
content of HIV Env. We focused solely on the redox state of the surface
protein gp120 as it contains 9 of the 10 disulfide bonds of
EnvLai (17).
Here, using a sensitive thiol reagent, we show that on average two Env
disulfide bonds are reduced during interaction with the lymphocyte
surface immediately prior to fusion. We provide evidence that a
reductase activity belonging to the PDI family is involved. We propose
that disulfide bond restructuring constitutes the molecular basis of
the post-receptor binding conformational changes that induce fusion competence.
Reagents--
The impermeant thiol reagent
3-(N-maleimidylpropionyl)-biocytin (MPB) was purchased
from Molecular Probes (Eugene, OR). Reagents including thyroglobulin
(Tg), the PDI inhibitors 3,3',5 tri-iodothyronine (T3) (18) and
bacitracin (10) were purchased from Sigma. EnvGB8 and
EnvLai (reference batches) were supplied by the EVA Medical Research Council AIDS Reagent Program (Potters Bar, United
Kingdom) and the ANRS (Paris, France), respectively. Purified
recombinant soluble CD4 (sCD4) was supplied by the EVA Medical Research
Council AIDS Reagent Program and was labeled (30 µCi/µg) using
iodogen before purification by Sepharose G25 chromatography
as described previously (9). SDF1- Cell Infections and Env Production--
Human CD4+
lymphoid cells (CEM) (106 cells/ml) and CD4 MPB Labeling
Thiol Content of Purified Antigens--
Samples
containing either Tg or Env diluted in phosphate-buffered saline (PBS),
pH 7.4, were dot-blotted onto a nitrocellulose filter (Schleicher & Schuell). After blocking with PBS, 2% casein, filters were incubated
with MPB (0.1 mM, 30 min at 25 °C). After washing, they
were incubated with streptavidin-coupled peroxidase (1:500, Amersham
Biosciences) for 30 min. After washing, labeling was performed using
diaminobenzidine (Sigma), and spot intensity was quantified by
densitometry (PhosphorImager, Amersham Biosciences). In some
experiments, the antigen was incubated with MPB before blotting the
sample on the nitrocellulose filter, blocking with PBS, 2% casein, and
subsequent processing.
Thiol Content of Surface-associated Env--
CEM cells (5 × 107) were either treated using 1 mM
bacitracin or mock-treated for 2 h. They were then incubated for
2 h at 37 °C in CO2 atmosphere with gp120 (30 µg/500 µl) produced by baby hamster kidney 21 cells infected using
VV vectors. Cells were washed and treated with NaN3 (0.1%)
to inhibit further surface remodeling (20). MPB was added to the cell
pellet (0.3 mM, 30 min at 25 °C). Excess reagent was
blocked using glutathione (0.6 mM, 10 min at 25 °C), and
the remaining sulfhydryl groups in the system were quenched with
iodoacetamide (1.2 mM, 10 min at 25 °C) (24). Cells were
washed twice and incubated in acid buffer (MES/HCl 10 mM,
NaCl 150 mM, pH 3.0) for 10 min to dissociate surface-bound
Env as described previously (25, 26). The eluate was adjusted at pH 7.0 using NaOH, and Env was immunoprecipitated for 4 h at 4 °C
using D7324 covalently coupled to CNBr-Sepharose CL4B (Amersham
Biosciences) as described previously (27). Gp120 present in the
original concentrated cell supernatant or in medium corresponding to
supernatant containing gp120 subsequently to incubation with CEM cells
(see above) was processed similarly. After elution from Sepharose-bound
D7324 using 1% SDS, the purified envelope samples were dot-blotted
onto nitrocellulose filter and processed as above to determine the
corresponding thiol content. In parallel, dot-blot quantitation of the
amount of gp120 present in the eluate after immunoprecipitation using
D7324 was achieved by incubation with a pool of anti-HIV-1 polyclonal
human IgG and staining using appropriate anti-IgG antibodies coupled to
peroxidase and diaminobenzidine (19). Spot intensity was quantified by densitometry to enable gp120 quantitation by comparison with a standard
curve obtained with a known reference batch of recombinant gp120Lai. The protein content of the eluate was also
assessed using a microquantitation assay (Pierce). Together, these
assays allow MPB reactivity to be related to the amount of
immunopurified Env to determine the thiol content per molecule of gp120.
Labeling of Surface-associated PDI--
We examined the
association of PDI with the outer cell membrane as described previously
(28). CEM cells (107) were washed, resuspended in 0.5 ml of
PBS containing 0.2 mM of MPB, and incubated for 30 min at
room temperature to label the thiols of cell surface proteins.
Quenching was performed using glutathione (0.4 mM, 10 min
at 25 °C) and iodoacetamide (0.8 mM, 10 min at
25 °C). The cells were washed, sonicated in lysis buffer (ice-cold
PBS containing 0.5% Triton X-100, 10 µM leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 10 µM
aprotinin), and incubated with streptavidin-agarose (Sigma) for 1 h at 4 °C in PBS containing 0.5% Triton X-100, 10 µM
leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 10 µM aprotinin. The beads were washed with PBS, and bound
proteins eluted with SDS sample buffer, resolved on 10% SDS-PAGE, and
transferred to nitrocellulose membrane. Anti-PDI polyclonal antibodies
(SPA-890, 1/1000) and anti-rabbit antibodies coupled to peroxidase
(1/2000, Sigma) were used for staining (15).
Thiol Content of Surface-associated Proteins--
We assessed
the thiol content associated with the native lymphocyte surface.
Uninfected cells (3 × 106) treated or not using PDI
inhibitors were incubated with MPB (0.1 mM, 30 min at
25 °C), and excess reagent was quenched with glutathione and
iodoacetamide as described above. After washing, cells were incubated
with streptavidin peroxidase and then were incubated with the
corresponding substrate before optical density reading.
Alternatively, after cell lysis using PBS 1% SDS, the lysates were
processed for dot-blot as described above. They were also analyzed by
SDS-PAGE (10%), and after blotting, they were processed using
streptavidin peroxidase as described above. The strips were scanned to
quantitate MPB labeling.
Env Treatment with Env was treated with CD4 Binding Assay
The CD4 binding assay was performed as described previously
(29). Env (100 ng) bound to D7324-coated microtiter plates was incubated with 125I CD4 (2 × 104
cpm/well) for 2 h. After washing, radioactivity was counted. Background binding was measured as the signal generated by similar plastic wells lacked Env. Unlabeled CD4 (150 nM) was used
to determine nonspecific binding. To assess that the
CXCR4 Binding Assay
The CXCR4 binding assay was performed as described previously
(19, 20). Env (2 µg/50 µl) was added to living CEM cells (3 × 106) for 2 h at 37 °C in culture medium. Cells were
then treated by 0.1% NaN3 for 10 min and further incubated
with 125I SDF1- Titration of Thiol--
To measure small changes in the thiol
content of Env, a quantitative assay was developed using MPB, a
membrane-impermeant compound coupled to biotin whose reaction with the
thiols of proteins can be detected using streptavidin peroxidase. To
establish the assay, we employed Tg as this glycoprotein exhibits a
similar "cysteine to molecular weight" ratio to
gp120Lai (17, 30). Preliminary experiments showed that (i)
all the disulfide bonds of Tg are reduced using 1%
Using these conditions, we verified that MPB reacted with native Tg in
a dose-dependent manner and determined that its thiol content per molecule was 13 (Fig. 1B). Commercially
available gp120Lai (Fig. 1B) and
gp120GB8 (data not shown) exhibited a low reactivity with
MPB, which corresponded to 0.5-1 thiol/molecule. Thus, in native
folded and secreted gp120, essentially all Cys residues are involved in
disulfide bonds. The specificity and linearity of dosage observed for
Tg were also observed with gp120 (Fig. 1B).
Relationship between the Thiol Content of gp120 and Its Receptor
Binding Capacity--
We then determined the relationship between the
thiol content of Env and its capacity to bind lymphocyte receptors. Env
was treated with various concentrations of
Development of Env Disulfide Bonds during the Course of Membrane
Fusion--
MPB was first used to biochemically demonstrate the
presence of PDI on the CEM cell surface. Cells were incubated with MPB or a mock. Samples were washed, lysed, and incubated with
streptavidin-agarose before the eluates were separated by SDS-PAGE and
blotted with anti-PDI antibodies (28). A single band was detected
migrating with the apparent molecular mass of PDI that was
significantly enriched by the MPB labeling when compared with the
mock-treated sample (Fig. 4A).
Thus, PDI on the CEM cell membrane is accessible to the exogenous
reagent. We next examined the capacity of bacitracin to alter the
reductive activity associated on the CEM cell surface. Treatment with 1 mM bacitracin reduced MPB reactivity by 50% (Fig. 4B). This result is consistent with inhibition of surface
PDI and is similar to the reduction in the thiol pool of the cell surface obtained using anti-PDI antisense phosphorothioates (34). For
specificity, cell preincubation with thiol reagents was shown to
dramatically reduce MPB reactivity.
We then addressed the thiol content of Env after its interaction with
either CD4 or both CD4 and CXCR4. Gp120 was incubated with CEM cells as
follows: (i) cells and gp120 incubated without prior incubation with
SDF1-
The thiol content of the immunopurified samples was determined as
before, and the amount of gp120 was assessed to determine the
thiol/gp120 ratio. The thiol content of gp120 associated with the
lymphocyte surface in conditions where both CD4 and CXCR4 were
accessible was found to be 4-fold higher than that of either immunopurified Env from the original supernatant or of gp120
present in the cell supernatant of the
SDF+/CD4+ sample (Table
I). In the
SDF+/CD4
The significance of lymphocyte surface PDI activity on the redox
changes observed for lymphocyte-associated Env was addressed using 1 mM bacitracin. The inhibitor prevented the increase of the
thiol content observed in the SDF
Bacitracin reacts with the redox active CXXC
sequences of PDI to block the catalyst (10, 13) and is a specific and
potent PDI inhibitor (34). T3 is a weaker inhibitor that exerts its activity through binding the PDI/substrate interaction domain (18). To
independently confirm both the PDI dependence of Env reduction and its
physiopathological relevance, we did experiments using 200 µM T3, a dose that inhibits PDI activity (18), and gp12089
To confirm this data using a biological system mimicking the
HIV/lymphocyte interaction, we made use of EnvLAI expression on
the surface of CD4+ human lymphocytes. In this system, cell
densities of greater than 106 cells/ml permit syncytium
formation, whereas those below 105 cells/ml do not (9, 23).
MPB labeling was done on the surface of the same number of
Env-expressing cells cultured at either of the two densities, and cell
surface Env was isolated as before. The thiol content and the amount of
purified gp120 were assessed to determine the thiol/gp120 ratio. We
found that Env associated with the surface of the dense culture
(syncytium forming) had an MPB reactivity that was 3-fold higher than
that associated with the surface of cells maintained at low cell
density (Table I). Because various PDI inhibitors prevent syncytium
spread in this system (9), we conclude that changes in the thiol
content of lymphocyte surface-associated Env are obligatory for fusion.
HIV Env is an unusually highly disulfide-bonded molecule with 9 of
a total of 10 disulfide bonds occurring within the outer membrane
domain of gp120 (17). Gp120 is functionally complex as it interacts
with at least three ligand surfaces to trigger HIV entry: 1) CD4, the
primary receptor, 2) CCR, the secondary receptor, and 3) gp41, the
fusogenic partner (3, 6). In addition, in the course of fusion, this
heavily glycosylated protein (35) must be sufficiently pliable to not
sterically inhibit the conformational changes required for virus-cell
fusion (2, 5). Here, we demonstrate that the later stages of the
process leading to fusion are enabled by the reduction of disulfide
bonds of Env by a lymphocyte surface-associated reductase activity.
Two articles suggested that changes in the redox status of the
disulfides of Env post-synthesis may occur in relation to HIV entry
into the target cell (9, 10). Ryser et al. (10) reported that HIV infection of human lymphocytes was markedly inhibited by
5,5'-dithiobis-2-nitrobenzoic acid (DTNB), bacitracin, and antibodies directed against PDI. More recently, we showed that these
inhibitors altered the HIV receptor-dependent gp41-mediated fusion process per se at a post-CD4 binding step (9).
Neither study, however, provided proof that the inhibition of PDI
impaired thiol/disulfide changes within Env that could be correlated
with abrogation of fusion. Here, using systems that address the
interaction of Env with an authentic lymphocyte surface in the context
of the various catalytic activities that lead to fusion (7-9), we clarified this issue and add three important new dimensions to previous
studies: (i) the development of a sensitive assay for the quantitation
of discrete changes in the thiol content of proteins, (ii)
identification of the minimum disulfide bond integrity required for Env
functional binding, and (iii) the stage of fusion at which thiol
emergence occurs.
The relationship between the integrity of the disulfides of gp120 and
its capacity to interact with viral receptors was examined initially.
We found that a population of mature gp120 species exhibiting on
average one reduced bond was capable of CD4 binding, whereas CXCR4
binding did not tolerate any alteration in the redox state of Env. We
also found that bacitracin had no direct effect on Env binding to
CXCR4. These data indicate that the presence of thiols on Env until
CXCR4 binding occurs is inconsistent with the development of the
process leading to fusion.
We then examined the redox state of gp120 as part of its interaction
with a CD4+ human lymphocyte surface. Firstly, we found
that the thiol content of gp120 after interaction with a native cell
surface displayed about 4 thiols per molecule compared with <1 thiol
in the original population of soluble antigen. This observation was
done both for a gp120 derived from a laboratory-adapted HIV strain or
from a dual-tropic primary isolate. Using high concentrations of
SDF1- We previously reported that PDI is clustered at the lymphocyte surface
in the vicinity of CD4-enriched regions and that some colocalization
occurs (9). We showed here that PDI is labeled on the cell surface by
the impermeant thiol probe MPB, further demonstrating its accessibility
on the outer membrane of the lymphocyte cell line. As both PDI-specific
inhibitors and anti-PDI antibodies inhibit HIV/lymphocyte fusion
post-CD4 binding (9), it seems probable that a member of this class of
catalysts may be the mediator of Env reduction. A direct evidence
supports this conclusion because the use of PDI inhibitors prevented
the increase in thiol content of lymphocyte surface-associated gp120 in
the SDF Because the increase in the thiol content of monomeric soluble gp120
after its interaction with the cell surface was similar to that
observed for oligomeric gp120 associated with syncytia, we conclude
that changes in redox state do not depend on Env oligomeric status or
on the presence of gp41. Our capacity to detect an increase in the
thiol content of Env associated with syncytia is consistent with a
report demonstrating that the surface of fusing cells is heavily
enriched in fusion-competent Env species (37).
Our study shows that a lymphocyte surface-associated PDI-related
reductase activity assists the cleavage of, on average, two disulfide
bonds within Env subsequent to its interaction with CXCR4 and that it
is obligatory for fusion (Fig.
5A). We speculate that the Env
reduction process and the disruption of the Env disulfide network it
promotes assist post-receptor binding Env conformational changes and
are necessary for acquisition of conformation competent to trigger
membrane fusion (4, 6). Indeed, if the surface-associated form of PDI
can act as a redox-driven chaperone to unfold proteins after disulfide
bond reduction, as has been shown for its endoplasmic reticulum
counterpart (38), it may directly catalyze the conformational changes
occurring within Env required to ultimately unmask the gp41 fusion
peptide. Recently, the reduction of the second domain of CD4 was
reported to be an obligatory step in CD4-dependent fusion
(39). Our results raise the possibility that redox changes observed
within CD4 may be the consequence of thiol/disulfide interchanges
occurring within the CD4·CXCR4·Env complex after Env
reduction by PDI.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was purchased from
Peprotech (London, United Kingdom) and was labeled (150 µCi/µg)
using lactoperoxidase before purification by Sepharose G25
chromatography as described previously (19, 20). Sheep polyclonal
antibody D7324 (Aalto, Dublin, Ireland) (19) is an anti-peptide
antibody directed against the C terminus of gp120 (APTKAKRRVVQREKR
sequence). Rabbit polyclonal antibody SPA-890 (Stressgen) (15) is
directed against bovine PDI. Vaccinia virus (VV) 9-1 (kindly provided
by M. P. Kieny) (21) and VBD3 (kindly provided by R. Collman
and R. Doms through the National Institutes of Health AIDS Program)
(22) are VV vectors encoding the native form of EnvLai and
Env89-6, respectively. Following secretion into the cell
supernatant, the viral antigens were concentrated and processed for
binding experiments as described in previously (19).
baby hamster kidney 21 cells (106 cells/ml) were
cultured and infected as described previously (9, 23). For Env
expression, cells were infected using VV vectors (CEM cells: 3-5
plaque-forming units/cell; baby hamster kidney 21 cells: 5-10
plaque-forming units/cell) in serum-free medium to enable further
supernatant concentration (30 times) using the Ultrafree 15 device
system (30-kDa cutoff; Millipore, St Quentin en Yvelines, France).
-Mercaptoethanol
-mercaptoethanol (0-3%, 10 min at
25 °C) before dot-blotting, MPB labeling, and further processing as described above. For binding experiments, the sample was treated with
-mercaptoethanol and then with iodoacetamide (2.5:1
iodoacetamide:
-mercaptoethanol ratio, 30 min at 25 °C) to prevent
further reoxidation of the thiol groups (29). After lyophilization to
remove
-mercaptoethanol, the sample was processed for binding to CD4
and CXCR4 as described below.
-mercaptoethanol treatment did not modify the subsequent capacity of
Env to bind antibody-coated wells, the binding of 125I Env
to microtiter plates was investigated for each
-mercaptoethanol concentration used as reported previously (29).
(5 × 104 cpm) for
1 h at 25 °C in buffer (RPMI 1640 medium, 10 mM
HEPES, 5% bovine serum albumin, and 0.1% NaN3).
Cell-associated and free radioactivity were separated using the
dibutylphtalate/bis(2-ethylhexyl)phtalate two-phase system. Unlabeled
SDF1-
(200 nM) was used instead of Env to determine
nonspecific binding.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol
and (ii) similar results were obtained when incubation with MPB was
performed prior to or after immobilization of the protein onto
nitrocellulose. Densitometry of reduced Tg (0.2-200 ng) blotted and
processed using MPB produced a linear dose response between 2 and 40 ng. Taking into account its molecular mass (330 kDa) and Cys
content (122 Cys residues/molecule) (30), we determined the thiol
content for each protein sample. This provided a standard curve and
allowed the development of an assay detecting thiols with an absolute sensitivity of 0.3 pmol (Fig.
1A) and a signal/background
ratio of 50.
View larger version (13K):
[in a new window]
Fig. 1.
Thiol dosage. A,
standard curve. Samples containing increasing amounts (determined as
described under "Results") of thiols associated with
-mercaptoethanol-treated Tg were blotted onto a nitrocellulose
filter and stained using MPB. Labeling was performed using
streptavidin-coupled peroxidase and diaminobenzidine. Spot intensity
was quantified by densitometry and used to determine a standard curve
(n = 6, means ± S.D. are presented).
B, MPB reactivity of Env and Tg. Increasing amounts of
either gp120 (Env) or Tg treated (MSH Tg) or not
treated (Tg) by
-mercaptoethanol were blotted onto a
nitrocellulose filter and processed as described above
(n = 3, means ± S.D. are presented).
-mercaptoethanol, and the
corresponding thiol content was determined (Fig.
2A). Alternatively, after
reaction with
-mercaptoethanol, the sample was incubated with
iodoacetamide, the reducing agent was removed by lyophilization, and
the resulting Env was assayed for CD4 binding as described previously
(29). We observed that reduction of one disulfide bond was tolerated
but the reduction of more than one prevented CD4 binding (Fig.
2B).
View larger version (15K):
[in a new window]
Fig. 2.
Thiol content and CD4 binding
properties. A, reduction of gp120 by
-mercaptoethanol. Gp120 was treated by increasing concentrations of
-mercaptoethanol. The thiol content of each sample
(SH/gp120) was then determined using MPB.
B, Env binding to CD4. Env samples (100 ng) presenting
various thiol contents (SH/gp120) were incubated with 125I
CD4. Bound radioactivity was counted (n = 2, means ± S.D. are presented).
-Mercaptoethanol-treated Env was tested for its CXCR4 binding
capacity (19, 20). Env and the natural CXCR4 ligand, SDF1-
, share a
binding site on CXCR4 that allows Env binding to be measured indirectly
through competition with SDF1-
(31), although low receptor affinity
limits maximum inhibition using Env to ~50% (19, 32, 33). The
reduction of a single disulfide bond impaired the capacity of gp120 to
compete with 125I SDF1-
binding at the lymphocyte
surface (Fig. 3A). The
treatment of the cell surface by bacitracin did not block SDF1-
binding inhibition by Env (Fig. 3B), indicating that Env
binding to CXCR4 did not require PDI activity. As a control,
125I SDF1 binding was inhibited by SPC3 (10
5
M), a V3-derived peptide that interacts with the binding
site of Env on CXCR4 (19, 20, 33).
View larger version (22K):
[in a new window]
Fig. 3.
Thiol content and CXCR4 binding
properties. A, Env binding to CXCR4. Living CEM cells
were incubated at 37 °C (i) with Env samples (1 µg/50 µl)
presenting various thiol contents (SH/gp120); (ii) with unlabeled
SDF1- (200 nM) (it determines the background signal
level); or (iii) in control conditions
(SDF1
/Env-, maximum signal). 125I
SDF1-
was then added for 1 h in buffer supplemented with 0.1%
NaN3, and the radioactivity associated with the cell pellet
was counted (n = 2, means ± S.D. are presented).
B, effect of bacitracin. CEM cells were preincubated
(+BCT) or not (
BCT) with bacitracin for 2 h at 37 °C. Gp120 (3 µg) (Env) was then added.
Alternatively, cells were incubated with either unlabeled SDF1-
(200 nM) (it determines the background signal level) or SPC3
(10
5 M) (SPC3), an anti-HIV
V3-derived peptide that binds the Env binding site on CXCR4. Labeled
SDF1-
was then added, and cells were processed as described above.
Maximum 125I SDF1-
binding inhibition was determined in
the absence of unlabeled CXCR4 ligands (No lig.)
(n = 2).
View larger version (34K):
[in a new window]
Fig. 4.
A, association of PDI with the
CEM cell surface. CEM cells were incubated with MPB (+) or
not ( ). Cell lysates were incubated with streptavidin-agarose.
Proteins associated with the gel were resolved on SDS-PAGE and Western
blotted using anti-PDI antibodies. B, MPB labeling of the
lymphocyte surface. CEM cells were treated with either bacitracin
(BCT) (0.3, 1, and 3 mM) or thiol reagents (1 mM DTNB; 1 mM
n-ethylmaleimide (NEM)). Cells maintained
in control conditions were also studied (C). After washing,
cells were incubated with MPB and then with streptavidin peroxidase.
The activity associated with the cell surface was assessed using
orthophenylene diamine (n = 4, a representative
experiment is shown). C, immunoprecipitation of gp120
associated with the lymphocyte surface. After incubation of CEM cells
with gp120 and washing, the envelope protein was acid-dissociated from
the cell surface, purified using Sepharose-coupled D7324 antibody, and
analyzed by SDS-PAGE and protein staining. D, inhibition of
gp12089
6 disulfide bond cleavage following lymphocyte
binding. CEM cells were treated using T3 (T3) (100 µM), bacitracin (BCT) (1 mM) or
mock-treated (C). They were then incubated with
gp12089
6 before MPB labeling. Env was then
acid-dissociated and immunoprecipitated before thiol content assessment
(SH/gp120). The thiol content of secreted
gp12089
6 (Scrtd) was determined in
parallel.
and sCD4 (SDF
/CD4
); (ii)
cells preincubated with SDF1-
(2 × 10
6
M to block CXCR4) before incubation with gp120
(SDF+/CD4
); and (iii) cells preincubated with
SDF1-
and Env preincubated with sCD4 (0.5 ×10
6
M) before the addition of gp120 to cells
(SDF+/CD4+). Concentrations of sCD4 and
SDF1-
were saturating (19, 27). After incubation with Env, thiol
labeling of the cell surface components was performed using MPB. In
samples described in (i) and (ii), labeled Env was recovered from the
lymphocyte surface by an acid wash (25, 26) and captured using the
C-terminal peptide antibody D7324 coupled to Sepharose, resulting in
the recovery of pure intact gp120 (Fig. 4C). In sample
described in (iii), the cell medium containing Env that did not bind
the cell surface was similarly incubated with D7324-Sepharose.
sample, there was no significant
change in the thiol content of cell-associated Env when compared with
gp120 in the original supernatant.
Thiol content of Env following interaction with a human lymphocyte
surface
/CD4
condition (Table I), whereas it neither interfered with Env binding to
CD4 (9) and CXCR4 (see above) nor the reaction of MPB with thiols of
proteins (data not shown).
6, an envelope derived from a dual-tropic primary isolate (22). We observed that Env89
6 was reduced upon interaction with the cell surface and that bacitracin and, to a lesser
extent, T3 inhibited Env89
6 reduction (Fig.
4D). We conclude that the thiol content of Env increases
after interaction with a lymphocyte surface competent for CXCR4 binding
and that this change depends upon a surface PDI activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and/or sCD4, we obtained conditions that specifically impaired Env binding to either one or both surface antigens (19, 27). A
comparison of the data obtained when cells were preincubated or not
with SDF1-
showed that changes in the redox status of gp120 required
the availability of the binding site for Env on surface CXCR4.
Secondly, Env associated with the surface of the dense
(syncytium-forming) culture infected using VV9-1 had a thiol content
that was 3-fold higher than that associated with the surface of cells
maintained at low cell density. These results and our previous report
that PDI inhibition prevented syncytium spreading but not CD4 binding
(9) allow us to conclude that changes in the thiol content of Env are a
requirement of the fusion reaction.
/CD4
sample. Our data also show that
the oxidizing nature of the cell surface still permits the persistence
of thiols resulting from disulfides reduction of proteins, in agreement
with a recent report (36).
View larger version (36K):
[in a new window]
Fig. 5.
A, HIV/lymphocyte interaction. The main
steps of the HIV/lymphocyte interaction process are shown. Based on the
data presented here, the step where a reductase activity belonging to
the PDI family takes place is indicated. B, Gp120 structure.
The image was rendered from the coordinates of the three-dimensional
structure of gp120 (16) using RASMOL (40). The molecular surfaces
involved in binding the primary receptor, CD4, and the secondary
receptor, CXCR4, are shown. Cys residues are shown in cyan,
and disulfide linkages are shown in red. Of particular note
is the cluster of disulfide bonds that occurs at the base of variable
loop 4 (as indicated) and in close proximity to the CCR binding site.
Disulfide bond reduction at this location would be consistent with the
increase in thiol dosage documented in the text.
Our data do not address which disulfide bonds undergo reduction
post-CXCR4 binding, but we note that a cluster of bonds occurs in gp120
at the base of the V4 region important for Env bioactivity (16).
Disulfide bond reduction at this location could reduce a number of
bonds at one time and could bring about considerable conformational
change (Fig. 5B).
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. Alexandre Mezghrani for helpful discussions and critical readings of this article and to Pr. Roberto Sitia for much helpful advice on the analysis of PDI. Pr. Yves Laszlo is acknowledged for providing computer facilities. We thank Drs. Edward Berger, Robert Doms, Marie-Paule Kiény, Marie-Jeanne Papandréou, Hervé Rochat, and Jean-Marc Sabatier for the gift of reagents, discussions, or support. R. Barbouche acknowledges the kind support of Drs. Aghleb Bartegi and Ahmed Hellal and the help found at the Institut Supérieur de Biotechnologie de Monastir (Monastir, Tunisia). The help of the ANRS, of the United Kingdom EVA Medical Research Council AIDS Reagent Program (Dr. H. Holmes) and of the National Institutes of Health AIDS Reagent Program (Dr. Y. Akyel) is acknowledged.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Agence Nationale de Recherche sur le SIDA (ANRS) (to E. F.) and a grant from the Medical Research Council (to I. M. J).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.
§ Fellow of Bourse Scientifique de Haut Niveau from the ANRS.
To whom correspondence should be addressed. Tel./Fax:
33-491-69-88-47; E-mail: fenouillet.e@jean-roche.univ-mrs.fr.
Published, JBC Papers in Press, September 5, 2002, DOI 10.1074/jbc.M205467200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HIV, human immunodeficiency virus; Env, envelope; gp, glycoprotein; PDI, protein-disulfide isomerase; MPB, 3-(N-maleimidylpropionyl)biocytin; Tg, thyroglobulin; VV, vaccinia virus; CD4+ lymphoid cells, PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; SDF, stromal-derived factor; sCD4, soluble CD4; DTNB, 5,5'-dithiobis-2-nitrobenzoic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Einfeld, D. (1996) Curr. Top. Microbiol. 214, 133-176 |
2. | Sattentau, Q. J., and Moore, J. P. (1991) J. Exp. Med. 174, 407-415[Abstract] |
3. | Doms, R. W., and Peipert, S. C. (1997) Virology 235, 179-190[CrossRef][Medline] [Order article via Infotrieve] |
4. | Chan, D. C., and Kim, P. S. (1998) Cell 93, 681-684[Medline] [Order article via Infotrieve] |
5. |
Wyatt, R.,
and Sodroski, J.
(1998)
Science
280,
1884-1888 |
6. | Berger, E. A., Murphy, P. M., and Farber, J. M. (1999) Annu. Rev. Immunol. 17, 657-700[CrossRef][Medline] [Order article via Infotrieve] |
7. | Kido, H., Fukutomi, A., and Katunuma, N. (1991) FEBS Lett. 286, 233-236[CrossRef][Medline] [Order article via Infotrieve] |
8. | Avril, L. E., Di, Martino-Ferrer, M., Pignede, G., Seman, M., and Gauthier, F. (1994) FEBS Lett. 345, 81-86[CrossRef][Medline] [Order article via Infotrieve] |
9. | Fenouillet, E., Barbouche, R., Courageot, J., and Miquelis, R. (2001) J. Infect. Dis. 183, 744-752[CrossRef][Medline] [Order article via Infotrieve] |
10. | Ryser, H. J., Levy, E. M., Mandel, R., and DiSciullo, G. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4559-4563[Abstract] |
11. | Ferrari, D. M., and Soling, H. D. (1999) Biochem. J. 339, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
12. | Gilbert, H. F. (1998) Methods Enzymol. 290, 26-50[Medline] [Order article via Infotrieve] |
13. | Mandel, R., Ryser, H. J., Ghani, F., Wu, M., and Peak, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4112-4116[Abstract] |
14. | Couet, J., De, Bernard, S., Loosfeld, H., Saunier, B., Milgrom, E., and Misrahi, M. (1996) Biochemistry 35, 14800-14805[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Mezghrani, A.,
Courageot, J.,
Mani, J. C.,
Pugniere, M.,
Bastiani, P.,
and Miquelis, R.
(2000)
J. Biol. Chem.
275,
1920-1929 |
16. | Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., and Hendrickson, W. A. (1998) Nature 393, 648-659[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Leonard, C.,
Spellman, M. W.,
Riddle, L.,
Harris, R.,
Thomas, J.,
and Gregory, T. J.
(1990)
J. Biol. Chem.
265,
10373-10382 |
18. | Guthapfel, R., Gueguen, P., and Quemeneur, E. (1996) Eur. J. Biochem. 242, 315-319[Abstract] |
19. |
Papandréou, M. J.,
Barbouche, R,
Guieu, R,
Kieny, M. J.,
and Fenouillet, E.
(2002)
Mol. Pharmacol.
61,
186-193 |
20. | Barbouche, R., Papandreou, M. J., Miquelis, R., Guieu, R., and Fenouillet, E. (2000) FEMS Microbiol. Lett. 183, 235-240[CrossRef][Medline] [Order article via Infotrieve] |
21. | Kieny, M. P., Lathe, R., Riviere, Y., Dott, K., Schmitt, D., Girard, M., Montagnier, L., and Lecocq, J. (1988) Protein Eng. 2, 219-225[Abstract] |
22. | Doranz, B. J., Rucker, J., Yi, Y., Smyth, R. J., Samson, M., Peiper, S. C., Parmentier, M., Collman, R. G., and Doms, R. W. (1996) Cell 85, 1149-1158[Medline] [Order article via Infotrieve] |
23. | Barbouche, R., Decroly, E., Kieny, M. P., and Fenouillet, E. (2000) Virology 273, 169-177[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Stathakis, P.,
Lay, A. J.,
Fitzgerald, M.,
Schlieker, C.,
Matthias, L. J.,
and Hogg, P. J.
(1999)
J. Biol. Chem.
274,
8910-8916 |
25. | Pelchen-Matthews, A., Armes, J. E., and Marsh, M. (1989) EMBO J. 8, 3641-3649[Abstract] |
26. |
Kozak, S. L.,
Kuhmann, S. E.,
Platt, E. J.,
and Kabat, D.
(1999)
J. Biol. Chem.
274,
23499-23507 |
27. | Fenouillet, E., Clerget-Raslain, B., Gluckman, J. C., Guetard, D., Montagnier, L., and Bahraoui, E. (1989) J. Exp. Med. 169, 807-822[Abstract] |
28. |
Jiang, X. M.,
Fitzgerald, M.,
Grant, C. M.,
and Hogg, P. J.
(1999)
J. Biol. Chem.
274,
2416-2423 |
29. | Papandréou, M. J., Idziorek, T., Miquelis, R., and Fenouillet, E. (1996) FEBS Lett. 379, 171-176[CrossRef][Medline] [Order article via Infotrieve] |
30. | Malthiery, Y., and Lissitzky, S. (1987) Eur. J. Biochem. 165, 491-498[Abstract] |
31. | Zhou, H., and Tai, H. H. (1999) Arch. Biochem. Biophys. 369, 267-276[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Doranz, B. J.,
Orsini, M. J.,
Turner, J. D.,
Hoffman, T. L.,
Berson, J. F.,
Hoxie, J. A.,
Peiper, S. C.,
Brass, L. F.,
and Doms, R. W.
(1999)
J. Virol.
73,
2752-2761 |
33. | Barbouche, R., Feyfant, E., Belhaj, B., and Fenouillet, E. (2002) AIDS Res. Hum. Retroviruses 18, 201-206[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Zai, A.,
Rudd, M. A.,
Scribner, A. W.,
and Loscalzo, J.
(1999)
J. Clin. Invest.
103,
393-399 |
35. | Fenouillet, E., Gluckman, J. C., and Jones, I. M. (1994) Trends Biochem. Sci. 19, 65-70[Medline] [Order article via Infotrieve] |
36. | Donoghue, N., Yam, P. T., Jiang, X. M., and Hogg, P. J. (2000) Protein Sci. 9, 2436-2445[Abstract] |
37. |
Finnegan, C. M.,
Berg, W.,
Lewis, G. K.,
and DeVico, A. L.
(2001)
J. Virol.
75,
11096-11105 |
38. | Tsai, B., Rodighiero, C., Lencer, W. I., and Rapoport, T. A. (2001) Cell 104, 937-948[Medline] [Order article via Infotrieve] |
39. | Matthias, L. J., Yam, P. T., Jiang, X. M., Vandegraaff, N., Li, P., Poumbourios, P., Donoghue, N., and Hogg, P. J. (2002) Nat. Immunol. 3, 727-733[Medline] [Order article via Infotrieve] |
40. | Sayle, R. A., and Milner-White, E. J. (1995) Trends Biochem. Sci. 20, 374[CrossRef][Medline] [Order article via Infotrieve] |