By
From the * Unité d'Immunologie Virale and Laboratoire Rétrovirus et Transfert Génétique, Institut
Pasteur, 75724 Paris, Cedex 15, France; § Laboratoire Mécanismes Moléculaires du Transport
Intracellulaire, Centre National de la Recherche Scientifique UMR 144, Institut Curie, 75248 Paris
Cedex 05, France;
Laboratoire d'Immunologie Cellulaire, Centre National de la Recherche
Scientifique URA 625, CERVI, 75013 Paris, France; and ¶ Theodor Kocher Institute, University of
Bern, CH-3000 Bern 9, Switzerland
Ligation of CCR5 by the CC chemokines RANTES, MIP-1 or MIP-1
, and of CXCR4 by
the CXC chemokine SDF-1
, profoundly inhibits the replication of HIV strains that use these
coreceptors for entry into CD4+ T lymphocytes. The mechanism of entry inhibition is not
known. We found a rapid and extensive downregulation of CXCR4 by SDF-1
and of
CCR5 by RANTES or the antagonist RANTES(9-68). Confocal laser scanning microscopy
showed that CCR5 and CXCR4, after binding to their ligands, are internalized into vesicles
that qualify as early endosomes as indicated by colocalization with transferrin receptors. Internalization was not affected by treatment with Bordetella pertussis toxin, showing that it is independent of signaling via Gi-proteins. Removal of SDF-1
led to rapid, but incomplete surface
reexpression of CXCR4, a process that was not inhibited by cycloheximide, suggesting that
the coreceptor is recycling from the internalization pool. Deletion of the COOH-terminal, cytoplasmic domain of CXCR4 did not affect HIV entry, but prevented SDF-1
-induced receptor downregulation and decreased the potency of SDF-1
as inhibitor of HIV replication.
Our results indicate that the ability of the coreceptor to internalize is not required for HIV entry, but contributes to the HIV suppressive effect of CXC and CC chemokines.
Expression of CD4 is necessary but not sufficient for
productive infection of human cells with HIV (1, 2).
The existence of an additional recognition site was postulated several years ago (3), and it was recently shown that
some chemokine receptors fulfill such a function (5).
CXCR4 and CCR5 are the major HIV coreceptors (5,
10), although similar functions were also reported for
CCR2b and CCR3 (8, 9). It has been shown that the CC
chemokines, RANTES, MIP-1 Chemokines act via seven-transmembrane domain receptors that couple to heterotrimeric Gi-proteins. Their antiviral activity is thought to depend on competition for the
binding of the HIV envelope (Env) glycoprotein gp120 to
chemokines receptors (14, 15). It has been reported that
mere occupancy of HIV coreceptors by chemokines, in the
absence of Gi protein-mediated signaling, is sufficient for
inhibition of HIV infection. In fact, RANTES inhibits
HIV infection of cells treated with B. pertussis toxin, and a
CCR5 antagonist, RANTES(9-68), was shown to prevent
infection by primary NSI isolates (14, 16). The chemokines,
however, could also inhibit viral entry by downregulating
the expression of their receptors which may be endocytosed
upon ligand binding, as previously shown for the IL-8 and
MCP-1 receptors (17, 18). Therefore, we have studied this
process and the effect of receptor uptake on the HIV suppressive activity of chemokines. In this paper we describe a
rapid, profound downregulation of CXCR4 by SDF-1 DNA Expression Vectors, Cells, and Chemokines.
The CXCR4
WT expression vector contains the LESTR cDNA (19) cloned in
a pcDNA3 plasmid (InVitrogen, The Netherlands). The CXCR4
Indirect Immunofluorescence Staining.
CHO- and HeLa-CCR5-GFP cells were cultured on glass coverslips in 24-well plates. CEM
cells were seeded on coverslips coated with poly-L-lysine. The
cells were treated for 30 min at 37°C with 200 nM SDF-1 Cell Transfection, Flow Cytometry Analysis and Recording of Intracellular Ca2+ ([Ca2+]i) Changes.
Cells (8 × 106) were resuspended
in Dulbecco's modified Eagle's medium, supplemented with 10%
FCS, 1-4 µg of the appropriate DNA expression vectors, and 12 µg
of a non-coding carrier DNA plasmid. Electroporation was performed in 4-mm cuvettes at 220 V, 960 µF in a Bio-Rad Gene
Pulser. For flow cytometry, cells (5 × 105) were incubated for 1 h
with anti-CXCR4 and subsequently labeled with a secondary,
PE-conjugated goat anti-mouse IgG antibody (Southern Biotechnology, Birmingham, AL). After staining, cells were fixed in
1% paraformaldehyde-PBS containing 0.2% BSA and CXCR4 expression was analyzed with a FACScan® cytofluorometer (Becton
Dickinson, Mountain View, CA). CXCR4 expression in monocyte-depleted PBMC was studied on gated CD4+ cells. The cells
were incubated with anti-CXCR4 and labeled with the PE-conjugated secondary antibody followed by a FITC-conjugated anti-CD4 antibody (Leu3a; Becton Dickinson). Real-time recordings of [Ca2+]i were performed with an IMSTAR imaging system in
Fura-2 loaded cells as described previously (25). To remove cell-bound SDF-1 Viral Infections.
U373-CD4 LTRlacZ cells transfected with
either the CXCR4 WT or CXR4 After incubation with
SDF-1
CXCR4 downregulation induced by SDF-1 To investigate the fate of CXCR4 after downregulation,
CEM cells treated with SDF-1 Ligand-induced endocytosis of CXCR4 was assessed in
CEM cells by confocal laser scanning microscopy. In the
absence of chemokine, CXCR4 was mainly detected at the
cell surface. Exposure to SDF-1
It was previously
shown that endocytosis of IL-8 receptors requires an intact
COOH-terminal cytoplasmic domain (26). The role of the
corresponding domain of CXCR4 was, therefore, investigated using HeLa cells that were transiently transfected
with a vector expressing a CXCR4 cDNA deleted of the
last 41 amino acids (CXCR4
CHO cells transiently expressing either CXCR4 WT or
CXCR4 The capacity of the CXCR4
We have shown that CXCR4 and CCR5 are rapidly
down-regulated by endocytosis when the cells are exposed
to the appropriate chemokine ligand. The mechanism of
receptor internalization was ligand dependent, but was
clearly dissociable from chemokine-induced Gi-protein signaling. In fact, CXCR4 internalization was not affected by
Gi-protein inactivation with pertussis toxin, and CCR5 internalization was induced by the antagonist RANTES(9-68) which binds to CCR5 but does not activate Gi-proteins. On the other hand, no internalization was observed
in cells expressing CXCR4 deleted of its COOH-terminal, cytoplasmic domain, although Gi protein-dependent [Ca2+]i
changes were not impaired.
Deletion of the COOH-terminal domain does not affect
the ability of CXCR4 to act as a coreceptor for HIV entry.
If receptor endocytosis is prevented by truncation, however, SDF-1, and MIP-1
, which are
agonists for CCR5, inhibit entry of primary, non-syncytium-inducing (NSI) strains that are preferentially isolated
at early stages of the infection (11). The CXC chemokine,
SDF-1
, the ligand of CXCR4, inhibits cell fusion and infection by HIV strains of the syncytium-inducing (SI) phenotype that are usually isolated at late, symptomatic stages
of the disease (12, 13).
and of CCR5 by RANTES and RANTES(9-68) in different cells, and show that the HIV suppressive effect of chemokines is markedly reduced when receptor endocytosis does
not occur.
Cyt vector was prepared by a PCR-based strategy, by deleting the last 41 amino acids that correspond to the COOH-terminal
intracytoplasmic domain of CXCR4. A PCR-synthesized CCR5
DNA insert deleted of the stop codon was fused to a red-shifted
variant of the wild-type Green Fluorescent Protein (GFP) in a
pEGFP plasmid (Clontech, CA). All the PCR products were sequenced by the dideoxy method. HeLa is a human epithelial cell
line. U373-CD4 LTR lacZ cell clone derived from the human
glioblastoma cell line U373-MG (20), was transduced with human CD4 and carries an integrated E. coli
-galactosidase reporter
gene driven by a HIV-1 LTR. U373-MG cells, contrary to CEM
and HeLa cells, do not express CXCR4 constitutively (21). HeLa-CCR5-GFP cells (clone P4-C5) were derived from HeLa-CD4 LTR lacZ (clone P4-2) (22) cotransfected with the CCR5-GFP
vector and a plasmid carrying a hygromycin-resistance cassette.
CHO is a chinese hamster epithelial cell line. The CHO-CCR5-GFP cell clone was established by transfecting the CCR5-GFP
vector that encodes a neomycin resistance gene. The CCR5-GFP receptor proved to be fully competent to support viral entry
and Env-mediated cell fusion by CCR5-tropic isolates (HIVJR-CSF,
HIVYU2, HIVADA), both in HeLa-CCR5-GFP cells or in CHO-CCR5-GFP transiently expressing CD4 (not shown). CEM is a
human lymphoblastoid CD4+ T cell line. Human peripheral
blood mononuclear cells (PBMC) were isolated by Ficoll gradient
centrifugation and depleted of monocytes by plastic adherence at
37°C. SDF-1
, RANTES, and the antagonist RANTES(9-68)
were chemically synthesized by Dr. I. Clark-Lewis (University of
British Columbia, Vancouver, Canada) (23).
,
RANTES, or RANTES(9-68). After incubation with ligands,
the cells were washed and fixed for 20 min in 3.7% paraformaldehyde-PBS, washed again in PBS, mounted in 133 mg/ml Mowiol (HOECHST), 33% glycerol, 133 mM Tris-HCl, pH 8.5, and
analyzed by confocal microscopy. After fixation, CEM cells were
incubated for 15 min in PBS and 0.1 M glycine to quench free aldehydes, and permeabilized by incubation with 0.05% saponin in
PBS supplemented with 0.2% BSA for 15 min. The cells were
then incubated for 45 min at room temperature with the anti-CXCR4 monoclonal antibody 12G5 (24), a kind gift of Dr. J. Hoxie (University of Pennsylvania Medical Center, Philadelphia,
PA), and labeled with a secondary, fluorescein-conjugated (FITC)
goat anti-mouse IgG antibody. For staining with iron-loaded human transferrin coupled to rhodamine (Tf-RITC), cells were deprived of transferrin for 30 min at 37°C in serum-free medium,
and then incubated for 30 min at 37°C with Tf-RITC along with
the appropriate chemokine or antagonist. Confocal laser scanning
microscopy and double-fluorescence analysis were performed
with a TCS4D confocal microscope (Leica, Nussloch, Germany) interfaced with Argon/Krypton lasers. Simultaneous double-fluorescence acquisitions were performed using the 488- and the
568-nm laser lines to excite FITC and RITC dyes using a 100×
oil immersion Plan APO objective (NA = 1.4). The fluorescence
was selected with the appropriate double-fluorescence dichroic
mirror and band pass filters, and measured with blue-green and
red side sensitive one photomultipliers.
, the cells were exposed for 1 min at 37°C to an
acid buffer, pH 3.0, consisting of 50 mM glycine and 100 mM
NaCl buffer (106 cells per ml), and then centrifuged and resuspended into the appropriate medium (17).
Cyt vectors were seeded in
microtiter plates (104 cells per well) in a final volume of 200 µl.
24 h after transfection the cells were incubated with infectious supernatants obtained from MT4 cell cultures infected with the
HIV-1 molecular clone NL4-3 (HIV-1NL4-3) (40 ng of HIV-1p24
per well). SDF-1
or RANTES were added to the cells 20 min
before infection and were present during the whole culture period. Cultures were carried out in triplicate. Infected cells were
identified by staining for
-galactosidase. HIV-1(
Env)G is a pseudotyped HIV-1NL4-3 variant with a replacement of the Env protein with the Env (G) glycoprotein of the vesicular stomatitis virus
(VSV). To generate infectious supernatants of HIV-1(
Env)G,
plasmids encoding the HIV-1NL4-3 proviral DNA deleted of the
env gene and the Env (G) glycoprotein of VSV were cotransfected in HeLa cells. U373-CD4 LTRlacZ cells were infected
with HIV-1(
Env)G infectious supernatants (4 ng of HIV-1 p24
per well).
Chemokine Receptor Internalization.
, surface expression of CXCR4 in CEM cells (Fig.
1 a), monocyte-depleted PBMC (Fig. 1 b), or HeLa cells
(Fig. 1 c), was analyzed by flow cytometry using anti-CXCR4. Before analysis, SDF-1
-treated cells were washed
in an acidic glycine buffer, a procedure previously adopted
to dissociate IL-8 from its receptors (17). The washing removed receptor-bound SDF-1
that could interfere with
the binding of anti-CXCR4 (data not shown). As shown
in Fig. 1, a-c SDF-1
induced a profound decrease of cell
surface receptors in all three cell types. The downregulation of CXCR4 was specific for SDF-1
and was not observed when the cells were treated with RANTES, which
does not bind CXCR4 (Fig. 1, a-c). Upon treatment with
RANTES CXCR4 expression was comparable to that of
control cells that were not exposed to a chemokine but
washed with the glycine buffer. This indicates that the
washing with the acidic buffer did not modify the expression of CXCR4 or the ability of anti-CXCR4 to recognize
surface receptors.
Fig. 1.
Effect of SDF-1
stimulation on CXCR4 surface
expression. CEM lymphoblastoid
T cells (a), monocyte-depleted
PBMC (b), or HeLa cells (c) were
treated for 40 min at 37°C with
200 nM SDF-1
, 200 nM
RANTES, or medium alone
(UNTREATED). The cells were
then washed with acidic glycine
buffer, labeled at 4°C with anti-CXCR4 and a PE-conjugated
secondary antibody, and analyzed by flow cytometry. In b,
analysis was performed on gated
CD4+ cells (FITC-conjugated
anti-CD4). Control cells (CTRL)
were labeled with the secondary
antibody only. (d) Dependence
of CXCR4 downregulation on
SDF-1
concentration. CEM
cells were incubated for 40 min
at 37°C with increasing concentrations of SDF-1
, and surface
expression of CXCR4 was determined. PTX: before incubation with SDF-1
, the cells were
treated with 5 µg/ml pertussis
toxin for 90 min. (e) Time course of CXCR4 downregulation. Cells were pre-incubated at 4°C for 60 min with 200 nM SDF-1
or RANTES. After
washing, cells were cultured at 37°C for the indicated times in the absence of chemokines. ( f ) Re-expression of CXCR4. The cells were incubated at
37°C for 40 min with 200 nM SDF-1
in the presence or absence of 100 µg/ml cycloheximide, washed in acidic glycine buffer and further cultured for
up to 60 min at 37 °C with or without cycloheximide in the absence of SDF-1
. (d-f ) Relative surface expression of CXCR4 was analyzed by flow cytometry as described for a.
[View Larger Version of this Image (24K GIF file)]
was concentration dependent, and the maximal effect was reached
at 200 nM as shown in CEM cells (Fig. 1 d). Pretreatment
of the cells with pertussis toxin under conditions that completely inhibited Ca2+ mobilization by SDF-1
(data not
shown) did not prevent the decrease of cell surface receptor
expression (Fig. 1 d). This indicates that the observed downregulation of CXCR4 was not dependent on signaling by
Gi-proteins. The downregulation was rapid. It was already
detectable 5 min after addition of SDF-1
and progressed for ~20 min (Fig. 1 e). Further incubation in the presence
of chemokine up to 60 min did not lead to an additional
decrease of the number of surface-detectable receptors.
and washed with the acidic
buffer were further incubated at 37°C in the absence of the
chemokine. Surface re-expression of CXCR4 was detected
within the first 15 min (Fig. 1 f ), but no further increase in
receptor density was observed for up to 60 min. Re-expression was not dependent on de novo protein synthesis since
it was not affected by the presence of cycloheximide. These
results suggest that after binding of SDF-1
CXCR4 is internalized and re-expressed at the cell surface by a recycling
mechanism. Recycling is likely to account for the fact that
the expression of CXCR4 never decreased below 10-25%
even at high SDF-1
concentrations and prolonged incubation times.
induced a dramatic redistribution of the staining that is consistent with the intracellular accumulation of the receptor. This effect was specific
for SDF-1
and was not observed when the cells were exposed to RANTES (Fig. 2 a, CXCR4). The subcellular
distribution of internalized CXCR4 was studied by simultaneous labeling with anti-CXCR4 (green) and human transferrin coupled to rhodamine (Tf-RITC, red ) which is taken
up into early endosomes. As shown in Fig. 2 a (CXCR4+
Tf-RITC), both markers were largely colocalized, as revealed by the yellow spots, indicating a significant accumulation of internalized CXCR4 in early endosomes. Similar
experiments were performed to study the ligand-induced
endocytosis of CCR5. Since anti-CCR5 antibodies for immunodetection were not available, we used cell clones permanently expressing CCR5 fused to the fluorescence marker
GFP (CHO-CCR5-GFP and HeLa-CCR5-GFP). Addition of RANTES to either clone induced receptor endocytosis (Figs. 2, b and c, CCR5). Colocalization with Tf-RITC, in analogy to the above experiments, show that
CCR5-GFP accumulates preferentially in early endosomes, as indicated by the clusters of yellow spots in the juxtanuclear region (Fig. 2 b, CCR5+Tf-RITC). The occurrence
of internalized CXCR4 or CCR5 that do not colocalize with
Tf-RITC (green spots) might reflect the transfer of receptors
to late endosomes. The CCR5 antagonist RANTES(9-68),
which was previously shown to block infection by monocytotropic HIV isolates despite its inability to elicit Ca2+ mobilization and chemotaxis (16), was as effective as RANTES as
inducer of CCR5-GFP internalization (Fig. 2, b and c).
This is in agreement with the observation that the SDF-1
-dependent internalization of CXCR4 was not affected
by pertussis toxin and confirms that chemokine receptor
endocytosis does not require signaling via Gi-proteins.
Fig. 2.
Internalization of
CXCR4 (a) and CCR5 (b and c)
were analyzed by confocal laser
scanning microscopy in CEM
(a), CHO-CCR5-GFP (b), and
HeLa-CCR5-GFP (c) cells after
exposure to 200 nM SDF-1, RANTES or RANTES(9-68)
for 30 min at 37°C. -, untreated
cells; CXCR4, cells labeled with
anti-CXCR4 and a FITC-conjugated secondary antibody; CCR5,
autofluorescence of CCR5-GFP; CXCR4+, Tf-RITC and
CCR5+Tf-RITC, simultaneous
detection of Tf-RITC (red ) and either CXCR4 or CCR5-GFP
( green). Yellow spots indicate
colocalization of chemokine receptor and Tf-RITC.
[View Larger Versions of these Images (22 + 45 + 19K GIF file)]
Cyt). HeLa cells were chosen because they constitutively express CXCR4, and allow
to perform simultaneous analysis of SDF-1
effects on endogenous and transfected CXCR4. Cotransfection of either CXCR4 WT or CXCR4
Cyt with a GFP reporter vector permitted to distinguish transfected from nontransfected
cells. Incubation with SDF-1
induced a profound downregulation of CXCR4 WT, but did not affect the surface
expression of the COOH-terminally truncated receptor,
CXCR4
Cyt (Fig. 3, a and b). As expected the endogenous CXCR4, in nontransfected cells which are identified
by the lack of GFP fluorescence, was also markedly down-regulated by SDF-1
(Fig. 3 a). PMA had a similar effect.
It downregulated CXCR4 WT (Fig. 3 b) as well as the endogenous CXCR4 (not shown), but not CXCR4
Cyt
(Fig. 3 b). These results are in agreement with a previous report showing downregulation of CXCR4 by PMA in
human T lymphocytes (27), and suggest that phosphorylation of serines and threonines in the COOH-terminal region are involved in internalization. It has been shown that
phosphorylation of the COOH-terminal domain is essential for arrestin-mediated uptake of seven-transmembrane domain receptors via clathrin-coated pits (28, 29).
Fig. 3.
Effect of deletion of
the COOH-terminal cytoplasmic domain of CXCR4. (a)
HeLa cells were transiently transfected with the CXCR4 WT or
the CXCR4 Cyt expression
vector, along with a plasmid encoding the reporter gene GFP
(pEGFP). 24 h later, the cells were incubated for 45 min at
37°C in the presence or absence
of 300 nM SDF-1
, labeled with
anti-CXCR4, and analyzed by
flow cytometry. Expression of
GFP allows to distinguish transfected (GFP+) and nontransfected
(GFP
) cells. After transfection
with CXCR4 WT or CXCR4
cyt, SDF-1
-dependent downregulation of the endogenous
and the transfected receptor were
monitored in GFP
and GFP+
cells, respectively. (-) HeLa cells
were transiently transfected with
the CXCR4 WT or CXCR4
Cyt expression vector, along
with the pEGFP plasmid. 24 h
later, the cells were incubated for
45 min at 37°C with 300 nM
SDF-1
or with 20 ng/ml PMA,
labeled with anti-CXCR4 and
surface expression of CXCR4
was analyzed by flow cytometry
in GFP+ cells. (c) CHO cells
were transfected with either the
CXCR4 WT or the CXCR4
Cyt expression vectors and
were loaded 48 h later with
Fura-2. CHO control cells were
transfected with vector DNA alone (pcDNA3). Recordings of
[Ca2+]i changes after stimulation
with 200 nM of SDF-1
are
shown.
[View Larger Version of this Image (32K GIF file)]
Cyt were used to assess receptor signaling. As
shown by the changes in the cytosolic free Ca2+ concentration in response to SDF-1
, the wild-type and the COOH-terminally truncated receptor were equally capable of eliciting a response indicating that the COOH-terminal domain of
CXCR4 is not required for Gi-protein coupling (Fig. 3 c).
This result is in agreement with a previous report indicating
that deletion of the COOH-terminal domain of the IL-8 receptors did not affect signal transduction (26).
Cyt molecule to act as coreceptor for HIV-1 entry was studied in the
human, U373-CD4 LTRlacZ astrocytoma cell clone which
does not express endogenous CXCR4 and carries an integrated
-galactosidase reporter gene driven by the HIV-1 LTR. Both CXCR4
Cyt and the wild-type receptor
could be expressed with similar efficiency, as assessed by
FACS® analysis and by scoring of HIV-1 infected cells. It was,
therefore, possible to study the role of receptor occupancy
and receptor internalization as mechanisms for the HIV suppressive activity of SDF-1
, using U373-CD4 LTRlacZ
cells that were transiently transfected with CXCR4
Cyt
or CXCR4 WT. HIV infection was inhibited by SDF-1
in a concentration-dependent manner in cells expressing the truncated or the wild-type receptor, confirming that
both bind the chemokine and can act as HIV coreceptors.
However, the efficacy of SDF-1
as an inhibitor of infection was markedly lower in cells expressing CXCR4
Cyt
(Fig. 4). In cells with the wild-type receptor SDF-1
has
two effects that decrease infectability, competition for HIV
binding and a drastic reduction of receptor numbers by rapid endocytosis. Both mechanisms operate at the same
time suggesting that the full antiviral effects of SDF-1
is a
combination of competition for receptor binding by the
virus and receptor endocytosis. In cells bearing the COOH-terminally truncated receptor downregulation cannot occur, and protection by SDF-1
is less efficient because receptor density remains high. Findings similar to those in
the U373-CD4 LTRlacZ glioblastoma cells were obtained
in HeLa-CD4 LTRlacZ cells, where SDF-1
was less efficient as an inhibitor of HIV replication when CXCR4
Cyt was expressed (data not shown). The selectivity of
SDF-1
as an inhibitor of CXCR4-dependent infection is
indicated by two lines of evidence. RANTES was unable
to block entry of the CXCR4-dependent HIVNL4-3 viral
clone in U373-CD4 LTRlacZ cells, and SDF-1
did not
inhibit infection by an Env-deleted HIVNL4-3 clone pseudotyped with the Env (G) protein of VSV (Fig. 4).
Fig. 4.
SDF-1-dependent inhibition of HIV infection in cells expressing wild-type or COOH-terminally truncated CXCR4. U373-CD4
LTRlacZ were transfected with either CXCR4 WT or CXCR4
Cyt
expression vector, plated in 96-well plates (104 cells per well) and infected
with the HIV-1NL4-3 strain or the pseudotyped HIV-1(
env)G, in the
presence of the indicated concentrations of SDF-1
or RANTES. HIV-1(
env)G-infected cells were treated with 500 nM SDF-1
. Cell cultures
were carried out in triplicates. Surface expression of CXCR4 WT or
CXCR4
Cyt was assessed 24 h after transfection by FACS® analysis and
amounted to 482 and 533 fluorescence units, respectively. HIV infection
was revealed 24 h later by staining for
-galactosidase activity and scoring
of positive cells. The numbers of HIV-infected cells per well in the absence of chemokines were 168 and 232 (mean of five experiments) for
cultures transfected with CXCR4 WT and CXCR4
Cyt, respectively.
In cultures infected with HIV-1(
env)G the average number of infected
cells per well was 280. Shown are the percentages of infected cells in the
presence of chemokines. Infection with HIV-1NL4-3 or HIV-1(
env)G in
the absence of chemokines was set to 100%.
[View Larger Version of this Image (27K GIF file)]
is clearly less efficient as an inhibitor of HIV
replication. Our present results show that the ability of the
receptor to internalize is not required for HIV entry, but
may contribute to the HIV suppressive effect of chemokine
ligands by reducing the density of coreceptors. Viral entry
is a complex phenomenon in which gp120 attachment to
CD4 creates a high-affinity binding site for the coreceptor, leading to membrane fusion (30, 31). Chemokines thus appear to exert two types of anti-HIV activities, competition
for HIV-1 binding, and downregulation of coreceptor surface expression. Both processes are functionally linked since
receptor occupancy triggers internalization, and are likely
to synergize with each other.
Address correspondence to Fernando Arenzana-Seisdedos, Unité d'Immunologie Virale, Institut Pasteur, 75724 Paris, Cedex 15, France.
Received for publication 21 March 1997 and in revised form 22 April 1997.
A. Amara and S. Le Gall contributed equally to this work.We thank E. Perret for help with confocal laser scanning analysis, I. Clark-Lewis for the generous gift of synthetic chemokines, and B. Moser for helpful discussions. Critical reading of the manuscript by A. Trautmann is acknowledged.
This work was supported by Agence Nationale de Recherche sur le SIDA (ANRS), Fondation pour la Recherche Médicale, a Concerted Action of the European Union (program BIOMED, project ROCIO), and Grant 31-39744.93 of the Swiss National Science Foundation (to M. Baggiolini). A. Amara and S. Le Gall were supported by fellowships from Ensemble contre le SIDA and ANRS, respectively (France). M. Montes is supported by a fellowship from COLCIENCIAS (Colombia).
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