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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2454-2462
RAPID COMMUNICATION
A CD4-Independent Interaction of Human Immunodeficiency Virus-1 gp120
With CXCR4 Induces Their Cointernalization, Cell Signaling, and T-Cell
Chemotaxis
By
Dorothée Missé,
Martine Cerutti,
Nelly Noraz,
Patrick Jourdan,
Jean Favero,
Gérard Devauchelle,
Hans Yssel,
Naomi Taylor, and
Francisco Veas
From the Laboratoire d'Immunologie Rétrovirale et
Moléculaire, Institut de Recherches pour le Développement,
Montpellier, France; the Centre National de la Recherche Scientifique
(CNRS), URA 2209, INRA, Saint Christol lez Alès, France; the
Institut de Génétique Moléculaire, CNRS UMR 5535, Montpellier, France; the Institut National de la Recherche
Médicale (INSERM), U 454, Montpellier, France; and the
Université de Montpellier II, USTL, INSERM U 431, Montpellier,
France.
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ABSTRACT |
The gp120 envelope glycoprotein of human immunodeficiency virus-1
(HIV-1) interacts with the CXCR4 chemokine receptor, but it is not
known whether gp120 activates CXCR4-mediated signaling cascades in the
same manner as its natural ligand, SDF1 . We assessed the effects of
wild-type gp120 and a mutant gp120 that interacts with CXCR4 but not
CD4 on CD4 /CXCR4+ cells and
CD4+/CXCR4+ cells, respectively. Under both
experimental conditions, the interaction of CXCR4 and gp120 resulted in
their CD4-independent cointernalization. Both molecules were
translocated into early endosomes, whereas neither protein could be
detected in late endosomes. Binding of gp120 to CXCR4 resulted in a
CD4-independent phosphorylation of Pyk2 and an induction of chemotactic
activity, demonstrating that this interaction has functional
consequences. Interestingly, however, whereas SDF1 activated the
ERK/MAP kinase pathway, this cascade was not induced by gp120.
Together, these results suggest that the pathology of HIV-1 infection
may be modulated by the distinct signal transduction pathway mediated
by gp120 upon its interaction with CXCR4.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ENTRY OF HUMAN immunodeficiency virus
(HIV) into target cells requires binding to CD4, as well as to one of
several coreceptors, which have been identified as chemokine receptors belonging to the G-coupled seven transmembrane protein
family.1 The tropism of HIV depends on the usage of the
chemokine receptor: CCR5 is primarily used by macrophagetropic
(M-tropic) HIV strains, whereas lymphotropic (T-tropic) HIV strains
primarily use CXCR4 as a coreceptor.2-7 However, dual
tropic HIV-1 strains have been described that interact with either type
of chemokine receptor. This has led to a novel classification that
identifies HIV-1 strains based solely on their coreceptor
usage.8
Upon interaction with their receptor, stromal-derived factor
1 (SDF1), and regulation-upon-activation, normal T expressed and
secreted (RANTES), the natural ligands of CXCR4 and CCR5, respectively,
are able to trigger signaling cascades, resulting in chemotaxis. In
addition, a number of chemokines, including RANTES, macrophage
inflammatory protein 1 (MIP1), MIP1 , and SDF1,
are able to inhibit HIV-1 infection after binding to their respective
receptors.9 Indeed, it has been demonstrated that interaction of SDF1 with CXCR4 leads to a rapid downmodulation of
this receptor,10-12 suggesting that this phenomenon might
be responsible, at least in part, for the ability of chemokines and possibly other ligands of chemokine receptors to inhibit HIV-1 infection.
The existence of a tri-molecular complex formed between CD4, gp120, and
CXCR4 at the cell surface has been reported.13,14 Although
the sequence of events leading to its formation has not yet been well
characterized, it has been proposed that the binding of the HIV surface
envelope gp120 to CD4 induces conformational changes,15,16
resulting in a high-affinity interaction with the
coreceptors.17-19 The interaction of gp120 with CD4 results in the endocytosis of the gp120-CD4 complex20-25 and the
concurrent stimulation of signaling molecules normally activated
through the CD4 receptor.23,26-28 However, some HIV strains
can infect cells via a CD4-independent pathway,29,30 and it
has recently been shown that gp120 can interact directly with
CXCR4.31,32 Binding of the HIV envelope to one its
coreceptors has been reported to induce the activation of the ERK/MAP
kinase pathway, as well as the phosphorylation of the tyrosine kinase
Pyk2.33,34 However, these studies did not exclude the
involvement of CD4 in the CXCR4 or CCR5-mediated signal transduction
cascade. It is therefore still unclear whether gp120 can mimic its
natural receptor ligand, SDF1, and induce a CD4-independent
signaling pathway upon binding to CXCR4.
We have previously demonstrated that a recombinant gp120 protein (gp120
 HX1), which is deleted of its amphipatic helix-1, is no
longer able to bind CD4, but retains its capacity to interact with
CXCR4.32 Using this gp120 mutant as well as a
CD4/CXCR4+ cell line, we have analyzed
the cellular localization of CXCR4 after interaction with gp120 and
addressed the question of whether the presence of CD4 is required for
gp120/CXCR4-mediated signal transduction. It is demonstrated here, for
the first time, that the CD4-independent interaction of HIV-1 gp120
with its coreceptor induces the cointernalization of these two
proteins, Pyk2 phosphorylation, and T-cell chemotaxis.
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MATERIALS AND METHODS |
Cells and culture conditions.
CD4 CHO-K1 and CD4 Jurkat cell
lines were obtained from the American Type Culture Collection (ATCC;
Rockville, MD) and grown in RPMI 1640 supplemented with 10% fetal calf
serum (FCS; Biomedia, Toulouse, France). The CHO-K1 cell line,
transfected with a CXCR4 expression vector (a generous gift of Marc
Parmentier, Euroscreen Co, Bruxelles, Belgium), was grown in HamF12
medium (Life Technologies, Cergy Pontoise, France) supplemented with
10% FCS and 400 µg/mL of G418. The human Th2 clone CB-828 was
generated using cloning and culture conditions as previously
described35 and was grown in Yssel's medium (Irvine
Scientific, Santa Ana, CA) supplemented with 1% AB+ human
serum.36 Cell surface expression of CXCR4 on the latter cells was induced by the addition of 100 U/mL of recombinant
interleukin-4 (rIL-4) in the culture medium for at least 1 week before
their use.37
Antibodies and reagents.
The following antibodies were used: the anti-CD4 monoclonal antibodies
(MoAbs) OKT4a (Ortho Diagnostic Systems, Ortho-Mune, Raritan, NJ) and
ST4 (a kind gift of Dr Pierre Gros, SANOFI Recherche, Montpellier,
France), the anti-CD3 UCHT1 MoAb (PharMingen, La Jolla, CA), the
anti-CXCR4 12G5 MoAb38,39 (kindly provided by James Hoxie,
University of Pennsylvania, Philadelphia, PA), as well as the
fluorescein isothiocyanate (FITC)- and biotin-conjugated anti-CXCR4 12G5 MoAb (R&D Systems, Oxon, UK). The anti-gp120 110.4 MoAb
(Genetics Systems, Redmond, WA), a rabbit anti-gp120
antiserum, was produced in our laboratory after immunization with a
recombinant HIV-1 IIIB gp120 (purchased at Intracel Corp, Cambridge,
MA). An FITC-conjugated anti-LAMP1 H4A3 MoAb (anti-CD107) and human transferrin coupled to Texas Red (both from Molecular Probes Europe BV,
Leiden, The Netherlands) were used for confocal microscopy studies. The
anti-active ERK MAPK polyclonal Ab (Promega, Madison, WI) and
anti-ERK-2 MoAb (Santa Cruz Laboratories, Santa Cruz, CA),
anti-Pyk2 MoAb (Transduction Laboratories, Lexington, KY), anti-phosphotyrosine 4G10 MoAb (Upstate Biotechnologies Inc, Lake Placid, NY), and horseradish peroxydase (HRP)-conjugated goat antirabbit or antimouse secondary Abs (Amersham, Arlington
Heights, IL) were used in signal transduction experiments. Stromal
derived factor-1 chemokine (SDF1) was purchased from R&D Systems.
Recombinant SUgp120 proteins.
Recombinant monomeric gp120 wild-type (wt) and gp120 HX1 proteins
were produced in baculovirus and purified as previously described.32 Briefly, a 1,414-bp fragment encoding gp120 wt (from amino acid V12 to R481) was amplified by polymerase chain reaction (PCR), using the plasmid pHXB2R (carrying the complete genome
of a clone of HIV-1 IIIB) as a template. The mutant gp120 HX1
protein was generated by a deletion of 26 amino-acid residues, corresponding to the putative helix-1 from the C1 region of gp120
wt. The soluble gp120 wt and gp120 HX1 proteins were produced in
insect SF9 cells infected by the respective recombinant baculovirus. Cell supernatants were collected 6 days postinfection and gp120 wt or
gp120 HX1 was concentrated and immunopurified with the anti-gp120
D7324 Ab (Aalto, Dublin, Ireland) linked to bromacetyl-sepharose.
Immunofluorescence and flow cytometry analysis.
Two hundred thousand T cells were resuspended in 50 µL of
phosphate-buffered saline (PBS), supplemented with 3% bovine serum albumin (BSA) and 0.02% NaN3 in the presence of the
relevant MoAbs. After 1 hour of incubation with agitation at 4°C,
the cells were washed twice in PBS-0.3% BSA-0.02% NaN3
and resuspended in PBS-0.3% BSA-0.02% NaN3 in the
presence of a 1/50 dilution of FITC-conjugated goat-antimouse Ab
(Caltag, Burlingame, CA). After an additional 1 hour of incubation with
agitation at 4°C, the cells were washed three times as described
above, resuspended in PBS, and analyzed by single-color flow cytometry
using a FACSort (Becton Dickinson, San Jose, CA) and LYSIS
II software. Each datum point represents the acquisition of 10,000 gated events.
Internalization assays.
Confocal microscopy studies were performed on transfected CHO-K1 cells
and the T-cell clone CB-828 (2 × 105 cells/mL). Cells
were preincubated for 1 minute at 37°C in an acid buffer (pH 3.0),
consisting of 50 mmol/L glycine and 100 mmol/L NaCl, to remove any
cell-bound proteins. To exclude a possible role of the CD4 in the
different cellular responses induced by gp120, two different
experimental procedures were used: either CD4+/CXCR4+ cells (CB-828) were incubated with
the mutant gp120 HX1 or CD4/CXCR4+
cells (CHO-K1) were incubated with gp120 wt. After washing with PBS-3%
BSA, the appropriate cell line was incubated for 1 hour at 37°C
with one of the gp120 proteins or SDF1 in PBS-3% BSA. Cells were
then washed, fixed in PBS-3.7% paraformaldehyde for 20 minutes, washed
again, incubated for 15 minutes in PBS-0.1 mol/L glycine to quench free
aldehydes, and permeabilized by incubation with 0.05% saponine in
PBS-0.2% BSA. Cells were incubated for 45 minutes at room temperature
with either the anti-CXCR4 12G5 MoAb or the anti-gp120 110-4 MoAb,
washed, and subsequently incubated with an FITC- or Texas
Red-conjugated goat antimouse IgG antibody (Caltag). To identify the
compartment in which gp120 proteins and CXCR4 were translocated after
their internalization, their localization was monitored together with
that of transferrin and LAMP1, markers of early and late endosomes,
respectively. For this purpose, transferrin was coupled to Texas Red,
and LAMP1 was detected with an FITC-conjugated anti-LAMP1 (anti-CD107)
MoAb. Before staining with iron-loaded human transferrin, cells were incubated in serum-free RPMI 1640 medium for 30 minutes at 37°C. Cells were slide-mounted in Mowiol and analyzed by confocal microscopy. Simultaneous double-fluorescence acquisition was performed at wavelengths of 488 and 588 nm. The images were assembled and printed directly using Adobe Photoshop software (Adobe Systems Inc, San Jose, CA).
Cell stimulations and immunoblot analysis.
Before stimulation, CD4 Jurkat cells were cultured
for 24 hours in RPMI-1640 medium, supplemented with 0.5% FCS. For
analysis of Pyk2 phosphorylation, 107 cells were
resuspended in 100 µL of serum-free RPMI 1640 medium and incubated at
37°C for 15 minutes before stimulation with either 100 µg/mL
anti-CD3 UCTH-1 MoAb, 10 µg/mL of each of the gp120 proteins, 25 nmol/L SDF1, or 10 µg/mL of the anti-CD4 ST4 MoAb for different
time periods. For analysis of ERK MAPK phosphorylation, 106
cells were preincubated on ice for 60 minutes in the presence of each
of the above-mentioned reagents and then transferred to 37°C for
the indicated times. Cells were lysed in a 1% NP40 buffer and
postnuclear supernatants were immunoprecipitated for 1 hour at 4°C
with a polyclonal anti-Pyk2 Ab, followed by collection on protein-A
sepharose beads (Pharmacia, Uppsala, Sweden).40-42 Immunoprecipitates or whole cell lysates were boiled, resolved on an
8.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred electrophoretically to nitrocellulose membranes. Membranes were incubated for 30 minutes in TBS (150 mmol/L
NaCl, 20 mmol/L Tris, pH 7.5) containing 5% BSA and 0.1% Tween 20 and
then incubated from 1 hour with either the anti-P-tyr 4G10 MoAb or an
anti-active MAPK Ab that recognizes the dually phosphorylated
T183 Y185 form of ERK1/2 (Promega). Blots were
washed in TBS containing 0.1% Tween 20 and incubated with
HRP-conjugated goat antirabbit or antimouse secondary antibodies.
Immuno-reactive proteins were visualized using the enhanced
chemiluminescence assay (Amersham, Bucks, UK). For
reblotting with the anti-Pyk2 or the anti-ERK2 MoAbs, filters were
stripped as previously reported.41
T-cell chemotaxis.
Migration of CXCR4-expressing human T cells, in response to the
chemotactic activity of SDF1, gp120 wt, or gp120 HX1 proteins, was analyzed in Chemo Tx-96 Disposable Chambers with a 5-µm pore size
that separate the upper and lower compartments (Neuro Probe Inc,
Gaithersburg, MD), using the method described by Bacon and Schall.43 Expression of cell surface CXCR4 was assessed by
flow cytometry before the experiment. Twenty nine microliters of
Yssel's medium 1% human serum, containing increasing concentrations
of either SDF1, gp120 wt, or gp120 HX1 (0.1 nmol/L, 1 nmol/L, 10 nmol/L, 100 nmol/L, and 1 µmol/L) were added to the lower wells. Fifty thousand T cells were transferred directly in triplicate on the
filter sample sites (upper compartment) in a final volume of 25 µL.
After 1 hour of incubation at 37°C in a 5% CO2
incubator, cells that migrated through the filter were collected in the
lower compartment, resuspended in culture medium, and counted through 10 power fields of a Malassez hemocytometer. Spontaneous cell migration
was determined in the presence of medium alone or after the addition of
SDF1 to both upper and lower compartments. The number of
spontaneously migrating cells was subtracted from the total number of
cells present in the lower compartment after the addition of a
CXCR4-ligand to determine the actual extent of migration. Results are expressed as the ratio of migrating cells/total number of
cells × 100%.
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RESULTS |
CD4-independent internalization of cell-surface CXCR4 after interaction
with a gp120 mutant protein.
Interaction of CXCR4 with its natural ligand, SDF1, results in
internalization of the receptor-ligand complex from the surface of T
cells.10-12 To analyze whether cell surface expression of CXCR4 changes after binding to gp120, CXCR4-expressing T cells were
stimulated with a recombinant wild-type gp120 protein and the
expression of CXCR4 was analyzed by confocal laser scanning microscopy.
As shown in Fig 1, incubation of the human
T-cell clone CB-828 with similar concentrations of either gp120 (Fig 1B) or SDF1 (Fig 1D) for 1 hour at 37°C resulted in capping of CXCR4 at the cell surface and internalization of the receptor. To
determine whether the gp120-induced internalization of CXCR4 was
dependent on an interaction with CD4, the effect of gp120 HX1, a
mutant gp120 envelope protein that binds to CXCR4 but not to CD4, was
assessed (Fig 1C). Similar to the effects observed with the wild-type
protein, incubation of T cells with gp120 HX1 resulted in an
internalization of CXCR4, indicating that the gp120-mediated internalization of CXCR4 can occur in the absence of a gp120-CD4 interaction. Furthermore, we find that, upon incubation with gp120 proteins, there was a decrease of 35% in the level of CXCR4 receptor on the cell surface due to CXCR4 internalization (data not shown).
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| Fig 1.
CD4-independent internalization of cell-surface CXCR4
after interaction with gp120. Cell surface expression of CXCR4 on the
human Th2 clone CB-828 (A) was analyzed by flow cytometry after
staining with an isotype-matched control MoAb (a) and the
FITC-conjugated anti-CXCR4 12G5 MoAb (b). The Th2 cells (clone CB-828)
were incubated with either gp120 wt (10 µg/mL; B), a mutant gp120
 HX1 that associates with CXCR4 but not CD4 (10 µg/mL; C),
SDF1 (10 µg/mL; D), or medium alone (E) for 1 hour at 37°C.
After fixation and permeabilization, cells were stained with an
FITC-conjugated anti-CXCR4 12G5 MoAb and analyzed by confocal
microscopy as described in Materials and Methods.
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gp120 and CXCR4 cointernalize in early endosomes after the
CD4-independent interaction of gp120 with CXCR4.
To assess whether gp120 and CXCR4 cointernalized in a CD4-independent
manner, the CD4/CXCR4+ CHO-K1 cell line
was incubated with gp120 wt protein for 1 hour at 37°C and the
cellular localization of the CXCR4-gp120 complex was analyzed
(Fig 2C). Although a residual amount of
gp120 could still be detected at the surface of the cells, a
significant level of CXCR4 and gp120 proteins were colocalized within
the cells, demonstrating that internalization as well as colocalization
occurs independently of CD4. It is important to note that the
colocalization of the gp120-CXCR4 complex can be observed only in cells
in which endosomes are present in the microscopic confocal laser
section.
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| Fig 2.
CD4-independent internalization of the CXCR4-gp120
complex from the cell surface of a
CD4/CXCR4+ cell line. To assess CXCR4 (A)
and CD4 (B) receptor surface expression by single-color flow cytometry
analysis, cells were incubated for 1 hour at 4°C with either medium
alone (control cells; a), the anti-CXCR4 12G5 MoAb (A; b), or the
anti-CD4 MoAb OKT4a (B; b). Cells were stained with an anti-IgG mouse
FITC. To analyze the subcellular localization of CXCR4 and gp120,
CHO-K1 cells were incubated with 10 µg/mL of gp120 wt (C) or medium
alone (D) for 1 hour at 37°C. After fixation and permeabilization,
cells were stained with a rabbit anti-gp120 antiserum and a Texas
Red-conjugated antirabbit IgG as well as the FITC-conjugated anti-CXCR4
12G5 MoAb, such that the intracellular presence of gp120 was observed
as a red fluorescence and that of CXCR4 as a green fluorescence. The
superposition of the two fluorochromes (yellow) is indicative of a
colocalization of gp120 and CXCR4. This colocalization can only be
observed in cells in which endosomes are observed in the microscopic
confocal laser section.
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We next determined the subcellular localization of internalized CXCR4
and gp120 molecules, by comparing the localization of each of these
proteins with that of transferrin, a protein that is internalized and
transported to early endosomes upon endocytosis, and CD107 (LAMP1), a
lysosome-associated membrane protein that is present exclusively in
late endosomes and primary lysosomes. After incubation of
CD4/CXCR4+ CHO-K1 cells with gp120, both
CXCR4 (Fig 3A, B, and C) and gp120 (Fig 3D)
colocalized with transferrin, as detected by confocal laser microscopy.
Similarly, binding of SDF1 to CXCR4, used as a positive control,
resulted in a colocalization of CXCR4 and transferrin, after
endocytosis (Fig 3B). In contrast, neither gp120 nor CXCR4 could be
detected in late endosomes, as shown by the absence of colocalization
of either protein with LAMP1 (Fig 4). Taken
together, these results indicate that, after interaction of gp120 with
CXCR4, the resulting receptor-ligand complex is rapidly internalized
and translocated to early, but not late endosomes.
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| Fig 3.
CD4-independent internalization of CXCR4 and gp120 in
early endosomes. CD4/CXCR4+ CHO-K1 cells
were incubated in medium alone (A), in medium with 10 µg/mL of
SDF1 (B), or in medium with 10 µg/mL of gp120 wt (C and D) in the
presence of 125 µg/mL of Texas Red-conjugated transferrin for 1 hour
at 37°C. After fixation and permeabilization, cells
were stained with the FITC-conjugated anti-CXCR4 12G5 MoAb (A, B, and
C) or the anti-gp120 110-4 MoAb and an FITC-conjugated antimouse IgG
(D). The intracellular localizations of CXCR4 and transferrin (A, B,
and C) or gp120 and transferrin (D) were analyzed by confocal
microscopy. Yellow spots are indicative of the colocalization of
transferrin (red) with either CXCR4 (green) or gp120 (green) in early
endosomes.
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| Fig 4.
Absence of internalized CXCR4-gp120 complex in late
endosomes. CD4/CXCR4+ CHO-K1 cells were
incubated in the presence of either 10 µg/mL of gp120 wt (A and D),
10 µg/mL of gp120 HX1 (B and E), or medium alone (C and F) for
1 hour at 37°C. After fixation and permeabilization, the
intracellular presence of CXCR4 (red; A, B, and C), gp120 (red; D, E,
and F), and LAMP1 (green) was analyzed by confocal microscopy. Cells
were stained with either the biotin-conjugated anti-CXCR4 12G5 MoAb
followed by staining with Texas Red-conjugated streptavidin (A, B, and
C) or a rabbit anti-g120 antiserum followed by staining with a Texas
Red-conjugated antirabbit IgG (D, E, and F). Coexpression of LAMP1 was
assessed after staining of the cells with 10 µL of the anti-CD107
FITC-conjugated MoAb.
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Pyk2, but not the ERK/MAP kinase pathway, is activated by the binding
of gp120 to CXCR4, on CD4 T cells.
Interaction of CXCR4 with either SDF1 or gp120 triggers signaling
cascades, involving the phosphorylation of the Pyk2 protein tyrosine
kinase and the activation of the ERK/MAPK pathway.33,34 However, it is not clear whether binding of gp120 to CXCR4 can activate
these pathways in a CD4-independent manner. As shown in
Fig 5, stimulation of
CD4/CXCR4+ Jurkat cells with gp120 wt,
as well as with gp120 HX1 proteins, resulted in the
phosphorylation of Pyk2, albeit to a lower extent than that observed
upon stimulation with SDF1. Additionally, as expected, Pyk2
phosphorylation was induced by engagement of the TCR/CD3 complex
signaling cascade (Fig 5).
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| Fig 5.
Binding of gp120 to CXCR4 results in a CD4-independent
phosphorylation of Pyk2. Cell surface expression of CXCR4 and CD4 on
CD4 Jurkat cells was analyzed by flow cytometry (A),
after staining of the cells with the anti-CD4 OKT4a MoAb (a), the
anti-CXCR4 12G5 MoAb (b), or an isotype-matched control MoAb (c).
CD4 Jurkat cells were incubated with either gp120 wt (10 µg/mL), gp120 HX1 (10 µg/mL), SDF1 (100 nmol/L), or the
anti-CD3 UCHT-1 MoAb (10 µg/mL) for the indicated time periods at
37°C (B). Immediately after incubation, cells were lysed and Pyk2
was immunoprecipitated with an anti-Pyk2 Ab. The tyrosine
phosphorylation status of Pyk2 was analyzed by immunoblotting using an
anti-phosphotyrosine (anti-pTyr) MoAb. The immunoblot was stripped and
reblotted with an anti-Pyk2 Ab (anti-Pyk2) to ensure that equivalent
levels of Pyk2 were immunoprecipitated in each lane.
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We next assessed the ability of gp120 to activate the ERK/MAPK pathway
in CD4/CXCR4+ Jurkat cells by measuring
the dual phosphorylation of the ERK1 and ERK2 kinases on residues
T183 and Y185. Stimulation of cells with either
SDF1 or an anti-CXCR4 MoAb resulted in a weak, but significant,
phosphorylation of both ERK1 and ERK2 as compared with engagement of
the CD3 complex that resulted in a dramatic phosphorylation of these
two proteins. Because the kinetics of ERK phosphorylation differed
after CD3 and CXCR4 stimulation with maximal response at 3 and 15 minutes, respectively, the abilities of gp120 wt and gp120 HX1 to
induce ERK phosphorylation was assessed after both 5 and 15 minutes of
stimulation at 37°C. However, irrespective of the conditions,
neither gp120 wt nor gp120 HX1 was able to induce the
phosphorylation of ERK1/ERK2. The lack of a functional CD4 receptor on
the Jurkat cells used in this study was confirmed by the inability of
an anti-CD4 MoAb to induce the phosphorylation of ERK1 and ERK2
(Fig 6), whereas both proteins were
phosphorylated in CD4+ T cells after stimulation with this
MoAb (data not shown). Collectively, these data demonstrate that the
interaction of gp120 with CXCR4, in the absence of CD4, results in a
signaling cascade, which involves the phosphorylation of Pyk2, but does
not induce the activation of the ERK/MAPK pathway.
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| Fig 6.
Binding of gp120 to CXCR4 does not involve activation of
the ERK1/ERK2 MAP kinase pathway.
CD4/CXCR4+ Jurkat cells were preincubated
in medium alone or in the presence of gp120 wt (10 µg/mL), gp120
HX1 (10 µg/mL), SDF1 (25 nmol/L), the anti-CXCR4 12G5 MoAb
(10 µg/mL), the anti-CD3 UCHT-1 MoAb (10 µg/mL), or the anti-CD4
ST4 MoAb (10 µg/mL) for 60 minutes on ice. Stimulations were then
performed at 37°C for the indicated time periods before lysis.
ERK1/ERK2 activation was assessed using a polyclonal anti-active MAP
kinase Ab. The immunoblot was then stripped and reblotted with an
anti-ERK2 MoAb.
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gp120 induces a chemotactic response in T cells in a CD4-independent
manner.
The demonstration that gp120 can induce a CXCR4-mediated signaling
response prompted us to investigate whether this interaction, like the
binding of SDF1 to its receptor, would result in chemotaxis. Stimulation of CXCR4+ Th2 cells with either the wild-type
or mutant gp120 protein resulted in a similar dose-dependent
chemotactic response, showing typical bell-shape curves
(Fig 7). The maximal chemotactic responses
induced by both gp120 wt and gp120 HX1 proteins was observed at a
concentration of 100 nmol/L, with a migration of approximately 22% of
all cells in a representative experiment. Because equivalent results
were observed with the two gp120 proteins, these data indicate that the
interaction of gp120 with CD4 does not modulate the capacity of gp120
to mediate a chemotactic response through the CXCR4 receptor. A typical
bell-shaped chemotactic response was also induced by SDF1, with a
maximum migration of 72% observed after the addition of 10 nmol/L
SDF1. Although the SDF1-induced migration was higher than that
observed with gp120 proteins, our results indicate that the gp120
protein of the T-tropic HIV-1 IIIB strain is able to induce a
significant CD4-independent chemotactic response in human T cells.
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| Fig 7.
HIV-1 IIIB gp120 induces a chemotactic response in T
cells in a CD4-independent manner. Migration of the human Th2 cell
clone CB-828 in response to stimulation with increasing amounts (from
101 nmol/L to 103 nmol/L) of SDF1 ( ),
gp120 wt ( ), or gp120 HX1 () was analyzed in an in
vitro migration assay as described in Materials and Methods.
The data depicted are representative of one of three independent
experiments. Each point represents the mean ratio of migrated
cells/total cells ± standard deviation (SD) from a representative
experiment performed in triplicate. The number of spontaneously
migrating cells was subtracted from the total number of cells present
in the lower compartment.
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DISCUSSION |
In the present work, we demonstrated that the surface (SU) subunit of
the gp120 from the T-tropic HIV-1 IIIB strain induced CD4-independent
cellular responses after binding to CXCR4, the fusogenic coreceptor of
HIV-1. Indeed, SDF1, the natural ligand of the CXCR4 chemokine
receptor,44,45 as well as gp120 both induced cell
migration. To assess the importance of the gp120-CXCR4 interaction in
the absence of the CD4 receptor, the experiments in the present work
were performed using a gp120 HX1 mutant that has been previously
shown to interact with CXCR4 but not CD4.32 Additionally,
equivalent results were obtained when the effects of wild-type gp120
were assessed on CD4/CXCR4+ cells.
Confocal laser scanning analysis demonstrated that the CXCR4 receptor
was internalized after interaction with gp120 in a CD4-independent manner. gp120 was internalized and colocalized with CXCR4 within early
endosomes, as shown by the colocalization of both proteins with an
early endosome specific marker, transferrin. Our finding that neither
CXCR4 nor gp120 was present within late endosomes suggests that CXCR4
may be recycled and re-expressed at the cell surface. In fact, we have
observed that CXCR4 is rapidly recycled (within 30 minutes) after its
gp120- or SDF1-induced internalization (data not shown).
Additionally, other groups have shown that, after the rapid
downmodulation of CXCR4 upon binding of SDF1, there is a subsequent
re-expression of CXCR4 at the cell surface.11,12 The
internalization of CXCR4 does not seem to be required for productive
HIV-1 infection, because HIV-1 can infect cells with a CXCR4 deletion
that inhibits its internalization.10,11 However, the
presence of CXCR4 at the cell surface may be important for HIV-1
infection, because its downmodulation after interaction with SDF1
results in a decrease in HIV infection.10,11 In this
context, the interaction of soluble gp120, shed from infected cells,
with CXCR4 may inhibit HIV-1 infection,32 by both
downmodulating CXCR4 at the cell surface and competing for receptor
binding. Additionally, downmodulation of surface CD4 molecules after
association with the HIV-1 envelope has been found to decrease the
efficiency of subsequent HIV-1 infection.46
The evolution and enhancement of lymphocyte infection as well as the
subsequent physiopathological effects on HIV-1 infected lymphocytes
likely results from several CXCR4-specific biological phenomena. First,
at least in some instances, there is an increase in CXCR4 expression at
the cell surface after T-cell activation. Specifically, IL-4 treatment
has been shown to result in an increase in CXCR4 expression on Th2
cells, allowing them to be more efficiently infected by
HIV-1.37 Second, we now show that gp120 can itself induce a
chemotactic response in Th2 cells. Thus, the presence of soluble
T-tropic gp120 during the late phase of HIV infection may participate
in the recruitment (through chemotaxis) of uninfected cells to the
lymph nodes. This, in turn, would increase the possibility that these
cells would be infected by HIV-1 and accelerate the development of
acquired immunodeficiency syndrome (AIDS). Of note, this hypothesis has
been previously suggested for the M-tropic HIV-1 viruses in which
chemotaxis of macrophages is induced by the interaction of gp160 with
the CCR5 chemokine receptor.47
Several groups have shown that the natural ligand of CXCR4, SDF1, as
well as gp120 triggers activation of the ERK/MAP kinase pathway34,37 and Pyk2 phosphorylation.33
However, in the context of the previous studies, the ability of gp120
to trigger a specific signal through CXCR4 could not be ascertained,
because signals could also be transduced through the CD4 receptor.
Phosphorylation of the Pyk2 protein tyrosine kinase is known to be
induced by ligand binding to G-protein-coupled receptors, and its
activation is clearly dependent on changes in osmolarity induced by the
mobilization of Ca2+ from internal stores. In addition,
Pyk2 can function as an upstream mediator of the ERK/MAPK and/or
JUN/MAPK signaling pathways.40 In the present work, we
demonstrate that the binding of HIV-1 IIIB gp120 to CXCR4 triggers Pyk2
phosphorylation in a CD4-independent manner. These data are supported
by previous work showing that gp120-induced phosphorylation of Pyk2 in
CD4+ cells is partially inhibited by the anti-CXCR4 12G5
MoAb.33 Interestingly, however, we did not observe any
phosphorylation of ERK1/2 MAPK after stimulation with gp120, even
though SDF1 clearly induced ERK1/2 phosphorylation in a
CD4-independent manner. Thus, gp120 and SDF1 appear to
differentially activate CXCR4-mediated signaling cascades. This is the
first demonstration that interaction of different CXCR4 ligands induces
distinct transduction pathways.
The ability of HIV-1 IIIB gp120 to bind the CXCR4 chemokine receptor
and induce Pyk2 phosphorylation and chemotaxis suggests that this
association plays a major role in the activation status of the cells
and may contribute to the evolution of HIV infection. Additionally, the
physiological changes induced by the binding of gp120 to CXCR4 on
target cells may induce alterations in intracellular structures that
favor HIV infection through cell-to-cell contact.48,49 An
understanding of the processes involved in SU binding and fusion of HIV
envelopes will allow the development of new strategies to better
control HIV infection.
| |
ACKNOWLEDGMENT |
This study was made possible with the generosity and kindness of our
colleagues to whom we are indebted and grateful for their scientific
and technical input. We especially thank Marc Sitbon for reagents and
insightful discussions; Marc Parmentier for generously providing
plasmids and cell lines; James Hoxie for the 12G5 MoAb; François
Traincard for anti-gp120 MoAbs; Christophe Duperay, Nicole
Lautrédou, Bernard Geoffroy, Arnaud Dupuy d'Angeac, and Ilias
Stefas for helpful discussions and technical assistance; and Jeanne
Anne Ville for her continuous encouragement.
| |
FOOTNOTES |
Submitted November 18, 1998; accepted January 27, 1999.
Supported by the Institute of Research for Development, the Centre
National de la Recherche Scientifique (CNRS), the World Health
Organization, Sidaction-France, the Institut National de la Santé
et de la Recherche Médicale (INSERM), ARC, and the AFM.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Francisco Veas, PhD, Laboratoire
d'Immunologie Rétrovirale et Moléculaire, Institut de
Recherches pour le Développement, CNRS-URA2209, 911 Av.
Agropolis, 34032 Montpellier, France; e-mail: veas{at}mpl.ind.fr.
| |
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ABSTRACT
INTRODUCTION