Identification of V3 Loop-binding Proteins as Potential Receptors
Implicated in the Binding of HIV Particles to CD4+
Cells*
Christian
Callebaut
,
Julià
Blanco
,
Nadia
Benkirane§,
Bernard
Krust
,
Etienne
Jacotot
,
Gilles
Guichard§,
Nabila
Seddiki
,
Josette
Svab
,
Elisabeth
Dam
,
Sylviane
Muller§,
Jean-Paul
Briand§, and
Ara G.
Hovanessian
¶
From the
Unité de Virologie et Immunologie
Cellulaire, ERS 572 CNRS, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15 and § Institut de Biologie Moléculaire
et Cellulaire, UPR 9021 CNRS, 15 rue Descartes,
67084 Strasbourg Cedex, France
 |
ABSTRACT |
The binding of human immunodeficiency virus (HIV)
type 1 particles to CD4+ cells could be blocked
either by antibodies against the V3 loop domain of the viral external
envelope glycoprotein gp120, or by the V3 loop mimicking pseudopeptide
5[K
(CH2N)PR]-TASP, which forms a stable complex with a
cell-surface-expressed 95-kDa protein. Here, by using an affinity
matrix containing 5[K
(CH2N)PR]-TASP and cytoplasmic
extracts from human CEM cells, we purified three V3 loop-binding
proteins of 95, 40, and 30 kDa, which after microsequencing were
revealed to be as nucleolin, putative HLA class II-associated protein (PHAP) II, and PHAP I, respectively. The 95-kDa cell-surface protein was also isolated and found to be nucleolin. We show that recombinant preparations of gp120 bind the purified preparations containing the V3 loop-binding proteins with a high affinity, comparable to the binding of gp120 to soluble CD4. Such binding is
inhibited either by 5[K
(CH2N)PR]-TASP or antibodies
against the V3 loop. Moreover, these purified preparations inhibit HIV entry into CD4+ cells as efficiently as soluble CD4. Taken
together, our results suggest that nucleolin, PHAP II, and PHAP I
appear to be functional as potential receptors in the HIV binding
process by virtue of their capacity to interact with the V3 loop of
gp120.
 |
INTRODUCTION |
HIV1 is an enveloped
virus that infects target cells by the fusion of viral and cellular
membranes. This fusion requires first the binding of HIV external and
transmembrane envelope glycoprotein complex to the CD4 receptor, and is
dependent on the presence of cofactors on the cell surface (for
reviews, see Refs. 1 and 2). The external envelope glycoprotein
contains the binding site for the CD4 receptor and an hypervariable
region of about 36 amino acids referred to as the V3 loop. The
transmembrane glycoprotein contains a potential fusion peptide at its
amino terminus that is implicated in the membrane fusion process. The
external and transmembrane glycoproteins (gp120-gp41 for HIV-1) are
associated in a noncovalent manner to generate a functional complex in
which the V3 loop plays a critical role (3).
Throughout the years, several potential cofactors of CD4 have been
proposed in order to explain why CD4 molecule is essential but not
sufficient for HIV entry and infection. Accordingly, several cell-surface proteins (reviewed in Ref. 4) and heparan sulfates (5, 6)
have been proposed to be implicated in the viral entry process. More
recently, however, convincing evidence was provided by several
laboratories to show that the G protein-coupled chemokine receptors,
such as fusin/CXCR4 and CCR5, serve as essential cofactors for the
entry of T cell (lymphotropic) and macrophage-tropic HIV-1 isolates,
respectively (7-13). Consequently, chemokines specific for CXCR4 and
CCR5 receptors inhibit efficiently T cell and macrophage tropic HIV-1
isolates, respectively. Interestingly, chemokines inhibit HIV entry
without affecting the binding of HIV particles to cells (14, 15).
Chemokines therefore block HIV infection by interfering at a
post-binding event, namely fusion between viral and cellular membranes.
Several groups have proposed that HIV-1 binding to CD4+
cells creates a high affinity interaction site for the cofactor CCR5/CXCR4, and that in this mechanism, the V3 loop plays an important role (7, 9, 15-19). However, no evidence has been provided for a
direct binding event between the chemokine receptors and the V3 loop.
Consequently, as these latter experiments were carried out by
investigating the binding of soluble gp120 to the cell surface, it
remains difficult to eliminate the possibility for the existence of
complementary interactions of gp120 with other cell-surface
proteins.
Previously, it has been considered that the expression of human CD4 in
human or heterologous cells is sufficient for the binding of HIV
particles to cells (20). However, we and others have recently
demonstrated that this latter conclusion is not correct and was due to
the use of soluble gp120 instead of HIV particles (21, 22). Indeed, HIV
particles, which do not infect CD4-negative human or heterologous
cells, have the capacity to bind such cells. Interestingly, monoclonal
antibodies directed against the V3 loop inhibit significantly HIV
binding to CD4
human or murine cells (22). On the other
hand, in the CD4+ HIV permissive human cell lines, HIV
binding is blocked by monoclonal antibodies directed against either the
gp120 binding site in CD4 or against the V3 loop (21, 22). It should be
emphasized that the inhibition of HIV binding in the presence of
anti-V3 loop antibodies is not the direct consequence of gp120 shedding
from HIV particles or of a decreased affinity of CD4 or gp120 for
binding to its surface counterpart (22). Consequently, anti-V3 loop antibodies bind HIV virions and prevent the binding of HIV to CD4+ cells, despite the fact that the CD4-binding domain of
gp120 remains accessible for a potential interaction with CD4 (21, 22).
These observations have indicated that the neutralizing effect of
anti-V3 loop antibodies is not a post-binding event as it has been
considered before, and have suggested the existence of other
cell-surface molecules besides CD4 for stable binding of HIV particles
to cells. Moreover, the inhibition of virus binding to
CD4
cells in the presence of anti-V3 loop antibodies have
pointed out the importance of the V3 loop in the HIV binding process. Accordingly, HIV binding appear to require both CD4 and cell-surface components interacting directly with the V3 loop (21, 22).
Recently, we reported that the pseudopeptide
5[K
(CH2N)PR]-TASP, which mimics the V3 loop, is a
potent inhibitor of HIV entry by a specific interaction with protein
components on the cell surface, other than the CD4 molecule (4, 23).
Furthermore, we demonstrated that 5[K
(CH2N)PR]-TASP
binds and becomes complexed with a cell-surface-expressed 95-kDa
protein (4). This pseudopeptide inhibitor blocks the binding of HIV
particles to CD4+ cells, as neutralizing antibodies against
the CD4 receptor or antibodies against the V3 loop (4, 22).
5[K
(CH2N)PR]-TASP inhibits infection of cells by HIV-1
or HIV-2 but not by SIV-mac (23) and has no effect on HIV-1 pseudotyped
with MMLV envelope proteins (results herein), thus demonstrating its
specific action on the HIV-envelope-mediated entry process. Here, by
using an affinity matrix containing either
5[K
(CH2N)PR]-TASP or a synthetic V3 loop peptide, we
report the isolation of nucleolin, PHAP II and PHAP I as three V3
loop-binding proteins (V3-BPs). The 95-kDa cell-surface protein, by
virtue of its capacity to bind 5[K
(CH2N)PR]-TASP and
form a stable complex with it (4), was also isolated and identified to
be nucleolin. The identity of nucleolin, PHAP II, and PHAP I was
confirmed by their respective reactivity with polyclonal antibodies
raised against synthetic peptides corresponding to the NH2
terminus of each one of them. These three proteins were demonstrated to
bind a synthetic peptide corresponding to the sequence of HIV-1 V3
loop; consequently, they were referred to as V3-BPs. Recombinant
preparations of gp120 were then used to demonstrate the specific
binding of gp120 to purified preparations containing the three V3-BPs.
No binding was observed with the transmembrane envelope glycoprotein
gp41 of HIV-1, or with histone H3, which is rich in basic
positively-charged amino acids. Interestingly, monoclonal antibodies
directed against the V3 loop domain block the binding of gp120 to
V3-BPs. The binding of gp120 to V3-BPs is also inhibited by
5[K
(CH2N)PR]-TASP. These results, and the observation
that the V3-BPs block HIV entry as efficiently as soluble CD4, are
consistent with the implication of nucleolin, PHAP II, and PHAP I as
potential V3 loop receptors functioning in the process of HIV particle
binding to CD4+ cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Recombinant gp120 and gp41 (produced by the
baculovirus and Escherichia coli expression system,
respectively) of HIV-1 Lai (IIIB), and recombinant soluble CD4
(produced in CHO cells or by the baculovirus expression system) were
purchased from Neosystem Laboratories, Strasbourg, France. Other
recombinant preparations of gp120 corresponding to that of HIV-1
isolates, LAV (or Lai), MN (both from MicroGeneSys, Inc., Meriden, CT),
SF2, and the unglycosylated gp120-SF2 (Env 2-3; from Dr. K. Steimer;
Chiron Corp. were obtained through the AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, National Institutes of
Health. The gp120 MN and Lai (LAV) were produced in insect cells using
baculovirus, gp120 SF2 was produced in CHO cells, whereas the
unglycosylated gp120 SF2 was produced in the yeast. The concentration
of p24 was measured by p24 Core Profile ELISA (DuPont).
Cells--
CEM cells (clone 13) derived from human lymphoid cell
line CEM (provided by L. Montagnier, Institut Pasteur, France) were cultured in suspension medium RPMI 1640 (Bio-Whittaker, Verviers, Belgium). Peripheral blood mononuclear cells (PBMCs) from healthy donors were prepared by Ficoll Hypaque density gradient centrifugation, and suspended in RPMI 1640 medium containing 10% fetal calf serum (23). Human HeLa-CD4-LTR-
-gal cells (from Dr. F. Clavel, Institut Pasteur, Paris) were cultured in Dulbecco's medium (24). All cells
were cultured with 10% (v/v) heat-inactivated (56 °C, 30 min) fetal
calf serum.
Preparation of Cell Extracts--
Cells were first washed
extensively in PBS before lysis in buffer E (100 µl/3 × 107 cells) and the nuclei were pelleted by centrifugation
(350 g for 5 min). Buffer E contains 20 mM Tris-HCl, pH
7.6, 150 mM NaCl, 5 mM MgCl2, 0.2 mM PMSF, 5 mM
-mercaptoethanol, aprotinin
(1000 units/ml) and 0.5% Triton X-100. The nuclei-free supernatant was further centrifuged at 12,000 × g for 10 min, and the
supernatant was stored at
80 °C. Routinely, cell extracts were
prepared 24 h after cell passage.
The Production of HIV-1 Pseudotyped MMLV Virus--
HeLa cells
were cotransfected by electroporation with plasmids pNL4-3 defective
in env gene (25) and pE-4070A expressing amphotrope envelope
glycoproteins of MMLV (26). Electroporation (27) was performed at 200 V, 960 microfarads, using a 4-mm wide cuvettes in a Bio-Rad Gene
Pulser. The pseudotyped virus was recovered from the culture
supernatant after 48 h of culturing. Plasmids were kindly provided
by Dr. S. Le Gall (Institut Pasteur, Paris).
Antibodies--
The mAb CC98 directed against human nucleolin
(28) was generously provided by Dr. N.-H. Yeh, Graduate School of
Microbiology and Immunology, National Yang-Ming Medical College,
Shih-Pai, Taiwan, Republic of China. The mAb specific to human CD4 and
reacting with the gp120 binding domain was kindly provided by Dr. E. Bosmans (clone CB-T4-2; Eurogenetics, Tessenderlo, Belgium). The mAb
specific to human CD45 (clone HB10AB2IB6) was generously provided by
Dr. R. Siraganian (NIDR/NIH, Bethesda; Ref. 29). mAbs N11-20 (also referred to as 110-H), 110-C, 110-D against gp120 Lai, and mAb 41-A
against gp41 Lai, were provided by Dr. J. C. Mazie, Hybridolab, Institut Pasteur. Other mAbs specific to gp120 included mAbs 110-4 and
110-1 provided by Genetics Systems, Seattle, WA (30, 31), mAb ADP390
(from Drs. J. Cordell and C. Dean) provided by MRC AIDS Directed
Program Reagent Repository (32), mAbs AD3 (from Drs. K. Ugen and D. Weiner), V3-21 (from Dr. J. Laman), and b12 (from Drs. D. Burton and
C. Barbas), obtained through the AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, NIH (33-35).
Synthesis of TASP Constructs and the Biotin-labeled V3 Loop
Peptide--
Synthesis and characterization of the different unlabeled
or biotin-labeled TASP constructs were as described previously (4, 23).
The V3 loop sequences corresponded to that from the HIV-1 Lai
NCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNIS, and HIV-1 Ba-L
NCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHCNLS isolate. The V3 loop peptides
were synthesized using classical Fmoc chemistry. The biotin was added
at the NH2 terminus of the peptide. The loop structure was
generated by air oxidation at pH 8, in a 10% Me2SO water
solution under vigorous stirring for 4 days at room temperature. The
final products were more than 90% pure. Fluorescence-activated cell
sorting (FACS) analysis was carried out as described previously
(4).
Purification of the V3-BPs--
Avidin-agarose (3 ml; ImmunoPure
immobilized avidin, Pierce) in PBS was incubated (18 h, 4 °C) in the
presence of the biotin-labeled 5[K
(CH2N)PR]-TASP (20 µM) before washing extensively in PBS-EDTA (PBS
containing 1 mM EDTA). Cytoplasmic extracts (material
corresponding to 2 × 109 CEM cells; Ref. 4) were
added to the affinity matrix and after 2 h of incubation at
4 °C, the samples were washed extensively with PBS-EDTA. The
purified proteins were eluted by 1 M NaCl solution containing 20 mM Tris-HCl, pH 7.6, 50 mM KCl, 1 mM EDTA, 1 mM PMSF, 5 mM
-mercaptoethanol, and 20% (v/v) glycerol. The eluted sample was
dialyzed overnight against PBS containing 0.1 mM EDTA and 1 mM PMSF and aliquots stored at
80 °C. For
microsequencing, the purified proteins (100 µg) were analyzed by
SDS-PAGE, and the different protein bands were visualized after a
slight staining with Amido Black. The respective bands were excised
from the gel and digested with endo-lysine C, which cleaves peptides
adjacent to lysine residues. The peptides were purified by HPLC
(DEAE-C18) using a gradient of acetonitrile/trifluoroacetic acid 0.1%.
The microsequencing was carried out by the Micro-Sequencing Laboratory at Institut Pasteur.
Purification of the Cell-surface-expressed p95 for
Microsequencing--
Twenty four h after passaging, CEM cells
(109 cells) were washed extensively with PBS before
incubation (4 °C, 30 min) in 10 ml of FACS buffer with 15 µM biotin-labeled 5[K
(CH2N)PR]-TASP. Cells were then washed in FACS buffer (2 × 100 ml), and
nucleus-free cell extracts were prepared using buffer E (3 ml). Such
extracts were first diluted in PBS (12 ml) prior the addition of 1 ml
of avidin-agarose to capture the biotin-labeled TASP complexed to p95.
These suspensions were incubated at 4 °C for 2 h, and then washed batchwise with PBS (6 × 60 ml). Finally, the
avidin-agarose pellet was resuspended in 2 ml of 2-fold concentrated
electrophoresis sample buffer (125 mM Tris-HCl, pH 6.8, 2 M urea, 1% SDS, 0.1% bromphenol blue, 150 mM
-mercaptoethanol, and 20% glycerol, (v/v)) and heated at 95 °C
for 5 min. Three 250-µl aliquots of the purified preparation were
analyzed by SDS-PAGE using a 7.5% polyacrylamide slab gel. The p95
band was transferred to a polyvinylidene difluoride sheets (Bio-Rad)
before microsequencing the NH2 terminus.
Production of Polyclonal Antibodies against Nucleolin, PHAP II,
PHAP I, and CXCR4--
Peptides corresponding to the NH2
terminus of nucleolin (residues 1-26), PHAP II (residues 1-23), PHAP
I (residues 1-20), and CXCR4 (residues 1-27) were synthesized
according to conventional Fmoc chemistry on a multichannel peptide
synthesizer. The peptides were conjugated to ovalbumin through the
cysteine residue added at the COOH termini of each peptide. Rabbits
were immunized five times at 2-week intervals by intramuscular
injections with the coupled material (150 µg of
peptide/injection/animal). The first injection was performed in the
presence of complete Freund's adjuvant (Difco, West Molesey, UK), and
the following injections were done with incomplete Freund's adjuvant.
The antisera were titrated by enzyme-linked immunoabsorbent assay
(ELISA) for the production of antibodies by monitoring reactivity with
the respective peptides.
ELISAs to Show the Binding of gp120 to V3-BPs--
The
microtiter plates were coated with the purified preparation of the
V3-BPs at different concentrations (12.5-200 ng/ml before incubation
with either gp120 (1 ng/ml), gp41 (2 ng/ml), or histone H3 (5 ng/ml).
After extensive washing, the binding of different reagents was
monitored using specific antibodies: mAb 110-D (1 µg/ml) specific for
residues 381-394 of gp120 of HIV-1, mAb 41-A (1 µg/ml) specific for
gp41, and mAb specific for histone H3 (2 µg/ml) (36). As control
antibodies, we used mAb OKT4A specific for CD4 and a rabbit antiserum
against CXCR4 (at 1:200 dilution).
Biosensor Measurements--
For real time binding experiments, a
BIAcoreTM biosensor system (Pharmacia Biosensor, AB,
Uppsala, Sweden) was used. Experimental procedures were as described
previously (36, 37). The purified preparation of the V3-BPs (100 ng/ml)
was immobilized on the sensor chips to characterize the binding with
the peptides 5[K
(CH2N)PR]-TASP and 5(KPR)-TASP, or the
gp120 preparations (each at 800 nM).
Assay of HIV Entry in HeLa CD4+ Cells--
The HIV-1
entry was monitored indirectly in HeLa-CD4-LTR-
-gal cells containing
the bacterial lacZ gene under the control of HIV-1 LTR.
HIV-1 entry and replication results in the activation of the HIV-1 LTR
leading to the expression of
-galactosidase (24). Cells were plated
at 104 cells/well in 96-well plates, and at 24 h later
cell monolayers were infected with HIV-1 Lai (a dose containing 20 ng
of p24). At 48 h, cell monolayers were washed with PBS before
lysis of cells in 100 µl/well of buffer-L containing 0.1% Nonidet
P-40 (v/v), 60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl,
10 mM MgSO4, 1 mM EDTA, and 50 mM
-mercaptoethanol. After 10 min of incubation at room
temperature, 100 µl of the reaction mix containing 10 mM
phosphate buffer at pH 7.4, 10 mM MgCl2, 10 mM
-mercaptoethanol, and 600 mM chlorophenol
red-
-D-galactopyranoside was added in each well. The
96-well plate was then incubated at 37 °C, and the
-galactosidase
activity was measured at 10-min intervals in a microplate reader using
a 570 nM filter.
 |
RESULTS AND DISCUSSION |
Inhibition of HIV Infection by 5[K
(CH2N)PR]-TASP
Is Specific to the HIV Envelope Glycoproteins--
We have previously
demonstrated that 5[K
(CH2N)PR]-TASP binds specifically
to the surface of CEM cells and blocks the binding of HIV particles to
cells (4, 23). Consequently, HIV-1/Lai infection is inhibited by
5[K
(CH2N)PR]-TASP as drastically as by the addition of
AZT, the well known reverse-transcriptase inhibitor of HIV (Fig.
1). The monoclonal antibody CB-T4 against
the gp120 binding domain in CD4 also inhibits HIV-1 infection, whereas
the control monoclonal antibody against the cell-surface-expressed antigen CD45 has no apparent effect (Fig. 1A). In order to
demonstrate the specificity of 5[K
(CH2N)PR]-TASP in
respect to the HIV-envelope glycoprotein mediated infection of
CD4+ cells, we investigated infection with an HIV-1
pseudotyped virus harboring amphotropic MMLV envelope proteins.
Infection of CEM cells by this pseudotyped HIV is inhibited by AZT but
not by the anti-CD4 mAb CB-T4 (Fig. 1B), as the viral
binding and entry in this case is mediated by the MMLV envelope
proteins (26). Interestingly, 5[K
(CH2N)PR]-TASP
exerted no significant effect on the infection with this pseudotyped
virus (Fig. 1B). These results therefore indicate that the
action of 5[K
(CH2N)PR]-TASP is specific to the
infection mediated by the HIV envelope glycoproteins. This is
consistent with our previous data showing that
5[K
(CH2N)PR]-TASP is active against HIV-1 and
HIV-2 isolates but not against the simian immunodeficiency virus
SIV-mac (23).

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Fig. 1.
The action of
5{K (CH2N)PR]-TASP is specific to the HIV envelope
glycoproteins. CEM cells (2 × 105) were first
incubated (37 °C, 15 min) with 5 µg/ml mAbs (the symbols for
anti) anti-CD4 (CBT4; (22) and anti-CD45 (29), or AZT (5 µM), or 5{K (CH2N)PR]-TASP (at 1 and 5 µM) before infection either with the HIV-1 Lai isolate
(0.2 synchronous dose; Ref. 23) in A or the HIV-1
pseudotyped MMLV virus (at a dose corresponding to 95 ng of p24/ml; see
"Experimental Procedures") in B. Virus production was
then monitored by measuring the concentration of p24 in culture
supernatants at 5 and 2 days after infection for the experiment
described in A and B, respectively. The mean ± S.D. of duplicate samples is given. The sample Control
represents infections without any prior treatment of cells. Note: the
infection by the HIV-1 pseudotyped MMLV is a one-cycle infection.
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|
5[K
(CH2N)PR]-TASP Affects the Interaction of a
Synthetic V3 Loop Peptide with the Surface of CEM Cells--
Recent
observations have suggested that the V3 loop in gp120 plays an
important role in the HIV binding process, by interacting with
cell-surface proteins other than CD4 and the chemokine receptors (22).
In view of this, we synthesized a biotin-labeled V3 loop peptide
corresponding to the sequence of the HIV-1 Lai isolate. At 25 µM V3 loop peptide, a significant labeling of cells was obtained by FACS analysis (Fig. 2). Such
labeling by the biotin-labeled V3 loop peptide was significantly
reduced in the presence of 25 µM unlabeled
5[K
(CH2N)PR]-TASP, suggesting that the V3 loop and 5[K
(CH2N)PR]-TASP interact with similar cell-surface
ligands. However, it should be noted that the affinity of the V3 loop
peptide to bind CEM cells is probably severalfold lower compared with 5[K
(CH2N)PR]-TASP. Indeed, significant labeling of CEM
cells could be obtained at 0.5 µM biotin-labeled
5[K
(CH2N)PR]-TASP (see Ref. 4 in Fig. 4). In contrast
cell labeling with the the biotin-labeled V3 loop peptide requires
25-50-fold higher concentrations.

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Fig. 2.
The capacity of the V3 loop peptide to bind a
cell-surface ligand is inhibited partially by the pseudopeptide
5[K (CH2N)PR]-TASP inhibitor. CEM cells were
incubated with 25 µM of the biotin-labeled V3 loop
peptide Lai in the absence (peak V3 Loop/B*) or presence of
25 µM unlabeled 5[K (CH2N)PR]-TASP
(peak 5(K PR)-TASP + V3 Loop/B*) before processing for
FACS analysis under the experimental conditions as described previously
(4). Peak C gives the fluorescence of cells incubated with
the unlabeled 5[K (CH2N)PR]-TASP construct (25 µM). The ordinate gives the relative cell
number, whereas the abscissa gives the relative fluorescence
intensity. Under similar experimental conditions, the control
biotin-labeled 5[QPQ]-TASP construct does not give a positive signal
(4).
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|
Purification of Several Proteins from Crude Cell Extracts Using the
Biotin-labeled 5[K
(CH2N)PR]-TASP--
Several
experiments were carried out to optimize the experimental conditions
for the purification of 5[K
(CH2N)PR]-TASP-binding proteins from crude cell extracts using the biotin-labeled TASP construct coupled to avidin-agarose. No proteins were recovered by the
avidin-agarose in the absence of the biotin-labeled
5[K
(CH2N)PR]-TASP or when cell extracts were incubated
with the biotin-labeled control 5[QPQ]-TASP construct (data not
shown). Thus, the biotin-labeled 5[K
(CH2N)PR]-TASP
coupled to avidin-agarose represents an efficient affinity matrix for
the purification of 5[K
(CH2N)PR]-TASP-binding proteins. Fig. 3 shows the profile of the
purified proteins revealed by staining the SDS-PAGE gel with Coomassie
Blue, and by ligand blotting using either the biotin-labeled
5[K
(CH2N)PR]-TASP or the biotin-labeled V3 loop
peptide. By using this experimental procedure, four major proteins of
95, 60, 40, and 30 kDa (p95, p60, p40, and p30, respectively) were
purified. By ligand blotting, each one of these proteins was shown to
bind 5[K
(CH2N)PR]-TASP and the V3 loop peptide, thus
pointing out that they are V3-BPs.

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Fig. 3.
Purification of the V3-BPs. Nucleus-free
cell extracts were purified by the affinity matrix constructed using
the biotin-labeled 5[K (CH2N)PR]-TASP and
avidin-agarose (see "Experimental Procedures"). Aliquots from the
purified preparation (panels V3 Loop-BPs for V3 loop-binding
proteins), were analyzed by SDS- PAGE using a 12.5% polyacrylamide
slab gel. A part of the gel was stained with Coomassie Blue to reveal
the protein bands (lanes 1 and 2), and the other
part of the gel was processed for ligand blotting using the
biotin-labeled 5[K (CH2N)PR]-TASP (5 µM;
lane 3) or the biotin-labeled V3 loop peptide (25 µM; lane 4) as described previously (4). The
biotin was revealed using streptavidin-horseradish peroxidase complex
and light-based enhanced chemiluminescence reagents as recommended by
the manufacturer (Amersham Pharmacia Biotech). The numbers
on the left (200, 97, 68,
43, and 29) show the position of molecular mass
(in kDa) protein markers (lane 1). Material corresponding to
10 and 3 µg of protein was analyzed in lanes 2 and
3/4, respectively.
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Identification of the V3-BPs: p95/Nucleolin, p40/PHAP II, and
p30/PHAP I--
The four proteins purified from cell extracts (Fig. 3)
were analyzed by microsequencing after digestion with endo-lysine C, which cleaves peptides adjacent to lysine residues. The peptides were
purified by HPLC, and some of the peaks were processed for microsequencing (see "Experimental Procedures"). The amino acid sequences obtained from the different peptides indicated that p95 is
nucleolin (38), p40 is PHAP II, and p30 is PHAP I isoform 2a (39, 40),
whereas p60 corresponds to a partially degraded product of nucleolin
(Table I). Recently, we reported the
isolation of a 95-kDa cell-surface protein, which binds
5[K
(CH2N)PR]-TASP and forms a stable complex with it
(4). To confirm that this latter protein is nucleolin, the 95-kDa
protein was isolated from the surface of CEM cells and was processed
for NH2-terminal microsequencing (Table I). The 15-amino
acid sequence obtained was found to be 100% identical with that of the
NH2-terminal sequence of human nucleolin (38). Nucleolin is
the major non-histone protein of the nucleolus, which has been
suggested to shuttle between nucleus and cytoplasm (38, 41). Although
primarily localized in the cell nucleoli, nucleolin has been reported
to be also expressed on the cell surface and serve as a binding protein
to different ligands (see Refs. 42-44 and references therein). On the
other hand, although PHAP I and PHAP II have been isolated as putative HLA class II associated proteins, as yet there is no direct evidence to
elucidate their precise function (39). PHAP I is most likely the human
homologue of the rat "leucine-rich acidic nuclear protein" (45),
whereas PHAP II is identical to a phosphoprotein named SET and
localized predominantly in the nucleus (46, 47). Purified preparations
corresponding to PHAP I and PHAP II have been reported to inhibit the
activity of the protein phosphatase 2A (48, 49).
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Table I
Homology of the amino acid sequence of the different peptides from the
V3-BPs to nucleolin, PHAP II, and PHAP I
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The common feature among nucleolin, PHAP II, and PHAP I is their
polyanionic nature due to the presence of extended stretches of amino
acids composed of aspartic and glutamic residues (38, 39). The
comparison of the amino acid sequences of these three proteins revealed
conserved acidic domains overlapping 14-38 residues (data not shown).
These acidic domains in nucleolin, PHAP II, and PHAP I are probably
responsible for the interaction with the V3 loop peptide and the
pseudopeptide 5[K
(CH2N)PR]-TASP. In this respect, it
is worthwhile to mention here that polyanions such as heparin, dextran
sulfate, synthetic double-stranded RNAs, and synthetic
aspartate/glutamate-rich peptides, are potent inhibitors of HIV entry
and infection (50). Interestingly, the mechanism of the inhibitory
effect of polyanions has been proposed to be related to their capacity
to bind the V3 loop domain in gp120 (51).
Nucleolin, PHAP II, and PHAP I Bind the Pseudopeptide
5[K
(CH2N)PR]-TASP and the Synthetic V3 Loop
Peptide--
In order to confirm the identity of the purified
proteins, rabbit antibodies were generated against synthetic peptides
corresponding to the NH2-terminal sequences of nucleolin,
PHAP II, and PHAP I. Such antibodies were shown to be highly specific,
since by immunoblotting each rabbit antiserum reacted only with the
protein corresponding to the peptide that was used for immunization.
Accordingly, in crude CEM cell extracts, the antisera against the
nucleolin, PHAP II, and PHAP I peptides revealed the 95-, 40-, and
30-kDa bands, respectively (Fig. 4,
A, B, and C, lanes
1). The preimmune sera from the different rabbits did not
show any signal (data not shown). In the purified preparations using
the affinity matrix containing either
5[K
(CH2N)PR]-TASP or the V3 loop peptide, the different antisera confirmed that nucleolin, PHAP II, and PHAP I indeed
bind to the V3 loop (Fig. 4, A, B, and
C, lanes 3 and 4). The mAb CC98
specific for human nucleolin, reacted with p95 and p60, thus further
demonstrating that p60 is a partial degradation product of nucleolin
(Fig. 4D). Interestingly, the antibodies directed against
the NH2-terminal peptide of nucleolin did not react with
p60 (Fig. 4A), suggesting that p60 should correspond to the
COOH-terminal portion of nucleolin. The partial degradation of
nucleolin has been well documented in the literature (28). In our
hands, although the major degradation product is p60, other minor bands
could also be observed. None of the V3-BPs were recovered on the
control affinity column containing 5[QPQ]-TASP (Fig. 4, lanes
2). Rabbit antiserum raised against the peptide corresponding to
the NH2-terminal domain of CXCR4 reacted with a 50-kDa
protein in crude cell extracts, but it did not generate a signal in the purified fractions corresponding to the V3-BPs (Fig. 4E).
Similarly, rabbit antiserum directed against human CD4 reacted with the
55-kDa CD4 protein, but it did not reveal a signal in the purified
preparations of the V3-BPs (Fig. 4F).

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Fig. 4.
Nucleolin, PHAP II, and PHAP I bind the
pseudopeptide 5[K (CH2N)PR]-TASP and the V3 loop
peptide. Aliquots of avidin-agarose (30 µl) in PBS-EDTA were
incubated (18 h, 4 °C) in the presence of either the biotin-labeled
control 5[QPQ]-TASP construct (100 µM; lanes
2), the biotin-labeled 5[K (CH2N)PR]-TASP (20 µM; lanes 3), or the biotin-labeled V3 loop
peptide (100 µM; lanes 4) before washing
extensively in PBS-EDTA. Cell extracts (materials corresponding to
15 × 106 cells) were then added to the affinity
matrix and after 2 h of incubation at 4 °C, the samples were
washed extensively with PBS-EDTA. The purified proteins were eluted by
the addition of 2-fold concentrated electrophoresis sample buffer and
analyzed by SDS-PAGE (4). Such samples (lanes 2-4) along
with cytoplasmic crude extracts (lanes 1) were analyzed by
immunoblotting using the following rabbit antisera (referred to as
): A, -nucleolin; B, -PHAP II;
C, -PHAP I; E, -CXCR4; F, -CD4
(Neosystem); and finally D, mAb CC98 (murine monoclonal
antibody specific for the human nucleolin). The mAb CC98 was used at
5-fold dilution of the hybridoma culture supernatant. The rabbit
antisera against different proteins were used at 100-fold dilution. The
antibodies were revealed with specific immunoglobulins labeled with
horseradish peroxidase (Amersham Pharmacia Biotech). The
numbers on the left give the position of
molecular mass (in kDa) protein markers.
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On the whole, these experiments illustrate that
5[K
(CH2N)PR]-TASP and the V3 loop peptide bind a
similar pattern of proteins, namely p95, p40, and p30. Furthermore,
they provide further confirmation concerning the identity of the V3-BPs
as being nucleolin, PHAP II, and PHAP I, respectively. Moreover, they
point out that CXCR4, the cofactor of CD4 required for the entry of
lymphotropic HIV-1 isolates, does not bind the V3 loop peptide (Fig.
4E).
On the Cell-surface Expression of V3-BPs--
We have shown that
5[K
(CH2N)PR]-TASP forms a stable complex with a
cell-surface-expressed 95-kDa protein (4), which was identified here as
nucleolin (Table I). This binding of 5[K
(CH2N)PR]-TASP to the cell-surface-expressed nucleolin could be detected by using the
biotin-labeled 5[K
(CH2N)PR]-TASP, either by FACS
analysis of cells or by the recovery of the complex formed on the
surface of intact cells (4). This latter property of the pseudopeptide 5[K
(CH2N)PR]-TASP was used as a tool to investigate
the cell-surface expression of PHAP II and PHAP I. For this purpose,
phytohemagglutinin-activated PBMCs from an healthy individual were
incubated with different concentrations of the biotin-labeled
5[K
(CH2N)PR]-TASP at 4 °C in the presence of sodium
azide (to inhibit any potential internalization process). Cells were
then washed extensively to remove unbound 5[K
(CH2N)PR]-TASP before preparation of cytoplasmic
extracts, which were then purified using avidin-agarose to capture the
biotin-labeled 5[K
(CH2N)PR]-TASP complexed to
cell-surface ligands. By this procedure, we could recover
cell-surface-expressed nucleolin, PHAP II, and PHAP I independently
from their cytoplasmic counterparts (Fig.
5).

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Fig. 5.
Recovery of the cell-surface-expressed V3-BPs
by the capacity of 5[K (CH2N)PR]-TASP to bind and form
a stable complex with them. PBMCs from an healthy donor were
activated by phytohemagglutinin as described previously (23). Four days
after activation, cells were washed extensively with PBS before
incubation (30 min, 4 °C) with different concentration of the
biotin-labeled 5[K (CH2N)PR]-TASP (lanes
0, 2.5, 5, 10, and 20 µM) in PBS containing 1% bovine plasma albumin and
0.01% sodium azide. For a control, cells were incubated with the
biotin-labeled 5[QPQ]-TASP construct (20 µM, lane
C/B*). Cells were then washed extensively in the incubation buffer
prior to the preparation of nucleus-free cell extracts, which were then
purified on avidin-agarose to recover complexes formed between
cell-surface proteins and the biotin-labeled
5[K (CH2N)PR]-TASP construct (4). Material
(corresponding to that from 15 × 106 cells) were
analyzed by immunoblotting using rabbit antisera against nucleolin,
PHAP II, and PHAP I peptides (all NH2-terminal). Lane
E represents crude PBMC cytoplasmic extracts (corresponding to
that from 3 × 106 cells) analyzed in parallel by
immunoblotting.
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The recovery of the cell-surface-expressed PHAP II and PHAP I required
concentrations of 5[K
(CH2N)PR]-TASP
10 µM. Previously, we had reported that p95/nucleolin is the
only cell-surface protein that gets complexed with
5[K
(CH2N)PR]-TASP (4). This latter still holds as long
as the concentration of the peptide is
5 µM (Fig. 5).
The control biotin-labeled 5[QPQ]-TASP construct at 20 µM did not bind to the cell-surface-expressed nucleolin, PHAP II, and PHAP I (Fig. 5, lane C/B*). Similarly, the
monovalent control biotin labeled K
(CH2N)PR construct,
even at concentrations higher than 50 µM, did not bind
any one of the V3-BPs (data not shown).
The capacity of stable complex formation between
5[K
(CH2N)PR]-TASP and the cell-surface-expressed
nucleolin, PHAP II, and PHAP I indicate that a proportion of these
V3-BPs are expressed on the cell surface. On the other hand, the
requirement of higher concentrations of
5[K
(CH2N)PR]-TASP for complexing with PHAP II and PHAP
I points out that the binding of the TASP construct with each one of
these V3-BPs might be at different affinities; the binding with
nucleolin being of the highest affinity. A similar experimental
approach was used to demonstrate the expression of these proteins on
the surface of different cell lines permissive to HIV infection, such
as CEM, PM1, and Jurkat (data not shown). In all of these cells,
5[K
(CH2N)PR]-TASP binding to nucleolin was
consistently observed at lower concentrations of the peptide. It should
also be noted that only a small proportion of nucleolin, PHAP II, and
PHAP I appears to be expressed on the cell surface. For an example, the
proportion of the cell-surface-expressed V3-BPs in PBMCs represents
about 20% of the total amount of these proteins detected in the
cytoplasmic extracts.
The ultrastructural localization of nucleolin, PHAP II, and PHAP I by
electron microscopy revealed the expression of these proteins in both
nuclear and cytoplasmic compartments in addition to their presence at
the plasma membrane.2
High Affinity Binding of gp120 to the V3-BPs--
In an ELISA type
experiment, HIV-1 gp120 but not gp41, corresponding to that of the
HIV-1 Lai isolate, was shown to bind in a dose-dependent
manner the purified V3-BPs (data not shown; described under
"Experimental Procedures"). It could be argued that the binding of
gp120 to the V3-BPs is simply the consequence of a nonspecific
interaction between basic amino acid residues in the gp120 and the
acidic domains in nucleolin, PHAP II, and PHAP I. However, this is most
unlikely, since histone H3, which is rich in basic amino acids, does
not bind the V3-BPs. This latter fact and the observation that binding
does not occur with gp41 confirm that the binding of gp120 to the
V3-BPs is specific. It is also of interest to note that the preparation
of V3-BPs in such ELISA type experiments using specific antibodies, was
shown not to be contaminated with the CD4 receptor or the chemokine
receptor CXCR4 (consistent with the results of immunoblotting analysis
shown in Fig. 4).
The gp120 from three lymphotropic HIV-1 isolates manifested a high
affinity binding to the V3-BPs, with equilibrium affinity constant
Ka values of 2.1 × 108
M
1, 4.3 × 108
M
1, and 2.3 × 109
M
1 for gp120 of Lai, MN, and SF2,
respectively (Table II). Interestingly, although the unglycosylated form of gp120-SF2 manifested about 10-fold
reduction compared with the glycosylated counterpart, its affinity was
still high with a Ka value of 1.6 × 108 M
1 (Table II). This result
suggests that the polysaccharide side chains of the native gp120
molecule are probably not absolutely necessary for its binding to the
V3-BPs. Under similar experimental conditions, the
Ka values of gp120-Lai, gp120-MN, and gp120-SF2 for
soluble CD4 were 10.8, 7.7, and 2.1 × 108
M
1, respectively (Table II), in accord with
previously published values (52). Therefore, the affinity of gp120 to
bind CD4 and the V3-BPs was of the same order.
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Table II
Kinetic rate and equilibrium affinity constants of
5[K (CH2N)PR]-TASP and gp120 to the V3-BPs and to CD4
Relative affinity of 5[K (CH2N)PR]-TASP and gp120 for the
V3-BPs and CD4 was determined using a biosensor instrument (BIAcore;
see "Experimental Procedures"). The preparation of the V3-BPs
containing nucleolin, PHAP II, and PHAP I was as described in Fig. 3.
The gp120 preparations corresponded to that of lymphotropic HIV-1
isolates Lai, MN, and SF2. The gp120 HIV-1 SF2 2/3 represents an
unglycosylated form of gp120 SF2. The CD4 represented a soluble form of
recombinant CD4.
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The Ka value of 5[K
(CH2N)PR]- and
5[KPR]-TASP to bind the V3-BPs was 9.6 × 109
M
1 and 1.5 × 108
M
1, respectively (Table II). The higher
Ka value observed for
5[K
(CH2N)PR]-TASP is correlated with its higher
activity on HIV entry, compared with the non-reduced counterpart
5[KPR]-TASP (4, 23). Two synthetic V3 loop peptides, corresponding to the amino acid sequence of the T-cell tropic HIV-1 Lai and of the
macrophage-tropic HIV-1 Ba-L isolate (see "Experimental
Procedures"), were investigated for their capacity to interact with
the purified V3-BPs. Both of these V3 loop peptides were found to bind
V3-BPs; however, in contrast to gp120 and to
5[K
(CH2N)PR]-TASP, they manifested somewhat lower
affinity of binding. The Ka value of V3 loop-Lai and
V3 loop-Ba-L to bind the V3-BPs was 5.1 × 106
M
1 and 1.5 × 106
M
1, respectively. By extrapolation,
therefore, the affinity of the V3 loop to bind the V3-BPs might be at
least 3-fold higher for the T-cell tropic compared with the
macrophage-tropic HIV-1 isolates. The presence of a high number of
basic residues in the V3 loop-Lai compared with the V3 loop-Ba-L could
account for this difference (53). Interestingly, the 50% inhibitory
concentration of 5[K
(CH2N)PR]-TASP to inhibit entry of
HIV-1 Lai and Ba-L in HeLa
CD4+/CXCR4+/CCR5+ cells is 0.4 and
0.8 µM, respectively (data not shown).
Competition between gp120 and 5[K
(CH2N)PR]-TASP to
Bind V3-BPs--
In order to demonstrate whether
5[K
(CH2N)PR]-TASP and gp120 interact with similar
domains in the V3-BPs, the binding of
5[K
(CH2N)PR]-TASP and gp120 to the V3-BPs was
investigated in the presence of increasing concentrations of gp120 and
5[K
(CH2N)PR]-TASP, respectively. The results
illustrated that gp120 blocks the binding of
5[K
(CH2N)PR]-TASP to the V3-BPs, and alongwise
5[K
(CH2N)PR]-TASP has the capacity to block the
binding of gp120 to the V3-BPs (Fig. 6,
A and B). The existence of such a competition
between 5[K
(CH2N)PR]-TASP and gp120 to bind the V3-BPs
indicates that the inhibition of HIV entry exerted by
5[K
(CH2N)PR]-TASP is the consequence of inhibition
of gp120 binding to the V3-BPs. In accord with this, there is a close
correlation between the anti-HIV activity in cell cultures and the
capacity of 5[K
(CH2N)PR]-TASP and its non-reduced counterpart 5[KPR]-TASP to block the binding of gp120 to the V3-BPs. Indeed, the IC50 values for the inhibition of HIV infection
and gp120 binding to the V3-BPs are 0.5 µM and 25 nM, respectively, for 5[K
(CH2N)PR]-TASP,
whereas those for 5[KPR]-TASP are 5 µM and 300 nM, respectively (Fig. 6; Ref. 4 and data not shown).

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Fig. 6.
Characterization of gp120 binding to the
V3-BPs. These experiments were carried out using biosensor
technology as described under "Experimental Procedures." The
purified preparation of the V3-BPs was as described in Fig. 3. The
sample containing the V3-BPs (100 ng/ml) was immobilized on the sensor
chips to characterize the binding with either
5[K (CH2N)PR]-TASP or gp120 (HIV-1 Lai). A,
gp120 prevents binding of 5[K (CH2N)PR]-TASP to the
V3-BPs. The binding of 5[K (CH2N)PR]-TASP (800 nM) to the V3-BPs was carried out in the presence of
increasing concentrations of gp120 (the abscissa). The
ordinate gives the percent inhibition of
5[K (CH2N)PR]-TASP binding to the V3-BPs; the 0%
inhibition value represents the degree of binding in the absence of
gp120. The IC50 value for the inhibition of
5[K (CH2N)PR]-TASP binding to the V3-BPs is around 3 nM gp120. B, 5[K (CH2N)PR]-TASP
prevents the binding of gp120 to the V3-BPs. The binding of gp120 (800 nM) to the V3-BPs was carried out in the presence of
increasing concentrations of 5[K (CH2N)PR]-TASP (the
abscissa). The ordinate gives the percent
inhibition of gp120 binding to the V3-BPs; the 0% inhibition value
represents the degree of binding in the absence of
5[K (CH2N)PR]-TASP. The IC50 value for the
inhibition of gp120 binding to the V3-BPs is around 25 nM
5[K (CH2N)PR]-TASP. C, the binding of gp120
to V3-BPs is prevented by the addition of a mAb against the V3 loop.
The binding of gp120 (800 nM) to the V3-BPs was carried out
in the presence of different concentrations of mAb N11-20 directed
against the V3 loop of the HIV-1 Lai-gp120 (the abscissa).
The ordinate gives the percent inhibition of gp120 binding
to V3-BPs; the 0% inhibition value represents the degree of binding in
the absence of the antibody.
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gp120 Binds the V3-BPs via the V3 Loop Domain--
The binding of
gp120 to the V3-BPs is inhibited in a dose-dependent manner
by mAb N11-20 directed against the V3 loop of gp120 (Fig.
6C). A number of monoclonal antibodies, directed against different domains in the gp120 molecule, were then used to characterize the binding domain in gp120 (Table III).
Three mAbs directed against the V3 loop inhibited the binding of gp120
to the V3-BPs at IC50 values around 100 nM,
whereas mAbs directed against the NH2 and COOH termini of
gp120, including the CD4 binding site, had no effect. The mAb 110-D
directed against an epitope located about 50 amino acid residues
down-stream of the V3 loop had no effect, whereas mAb 110-C directed
against an epitope located about 16 amino acid residues upstream of the
V3 loop resulted in a significant inhibition of binding (Table III).
The epitope of mAb 110-C being close to the NH2 terminus of
the V3 loop, the binding of this mAb might cause conformational changes
in the V3 loop domain, leading to the inhibition of gp120 binding to
the V3-BPs. These results point out that the V3 loop domain is the
binding site in gp120 for the V3-BPs. This conclusion is consistent
with the above results showing that the V3-BPs (p95/nucleolin, p40/PHAP II and p30/PHAP I) bind the synthetic V3 loop peptide (Fig. 4, lanes 4), gp120 binds the V3-BPs with a high affinity (Table
II), and that the pseudopeptide inhibitor of HIV entry that mimics the
V3 loop blocks the binding of gp120 to the V3-BPs (Fig.
6B).
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Table III
Inhibition of gp120 binding to the V3-BPs by monoclonal antibodies
against the V3 loop
The effect of different mAbs on the binding of gp120 Lai to the V3-BPs
was determined using biosensor technology (see "Experimental
Procedures"). The preparation of the V3-BPs containing nucleolin,
PHAP II, and PHAP I was as described in Fig. 3.
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Purified Preparations of the V3-BPs Inhibit HIV-1
Infection--
The results presented above demonstrated the high
affinity interaction of soluble gp120 with the V3-BPs, thus suggesting
the implication of these V3-BPs as potential receptors of the V3 loop. If this was the case, then soluble V3-BPs, by interacting with the
gp120 expressed on the surface of HIV particles, could block virus
infection as has been reported for the soluble CD4 (54, 55). Indeed,
purified preparations of the V3-BPs were consistently shown to inhibit
HIV infection in a dose-dependent manner. For example,
HIV-1 Lai infection of CEM cells was inhibited by 91 and 67% at 10 and
5 µg/ml, respectively, of the purified preparation of the V3-BPs
(data not shown).
In order to investigate whether this preparation of the V3-BPs block
the HIV entry process, we studied its effect on the HIV infection in
the HeLa-CD4-LTR-
-gal cell model. Upon HIV entry and replication,
the viral transactivator Tat induces the lacZ gene to
express
-galactosidase, which then can be monitored by an enzymatic
assay. Consequently,
-galactosidase activity is directly correlated
to the HIV entry and its replication, since the expression of such
activity does not occur in the presence of anti-CD4 mAb CB-T4, which
blocks HIV entry, or AZT, which blocks viral replication (Fig.
7). In this experimental model, the
V3-BPs had the capacity to block HIV entry in a
dose-dependent manner. Interestingly, the degree of
inhibition by the V3-BPs was comparable to that observed with soluble
CD4 (sCD4) preparation produced in CHO cells (Fig. 7). The inhibitory
effect of the V3-BPs on HIV entry was abolished when the purified
preparation was preheated at 56 or 95 °C, thus confirming the
protein nature of the inhibitory component. It should be noted that
sCD4 produced in the baculovirus expression system had no effect on HIV
entry, and even higher concentrations resulted in an enhancing effect.
The difference between the two sCD4 preparations might be the degree of
glycosylation in the two expression systems used.

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Fig. 7.
The purified preparation of the V3-BPs
inhibits HIV-1 entry. The HIV-1 Lai virus sample was first
incubated with 20, 10, and 5 µg/ml purified preparation of the V3-BPs
(as in Fig. 3) or with the mock preparation (Mock-20 and
Mock-10, corresponding to the volume which was used to
obtain 20 and 10 µg/ml V3-BPs), at 4 °C for 30 min before adding
to HeLa-P4 cells (expressing human CD4 and HIV-1-LTR-lacZ
gene). Similarly, Virus samples were also incubated with V3-BPs
preparations preheated 5 min at 56 °C or 100 °C, or with soluble
CD4 (sCD4) preparations obtained in CHO cells or in the baculovirus
(B. Virus) expression system. As for controls for virus
entry and replication, cells were preincubated (37 °C, 15 min) with
mAb anti-CD4 (CB-T4; 5 µg/ml) or AZT (5 µM)
before addition of HIV. Virus entry was monitored by the activity of
-galactosidase at 48 h after infection. The results are given
as percent -galactosidase activity compared with the control cells
(infected without any additive). The mean ± S.D. of duplicate
samples is shown. The V3-BPs and sCD4 were added only once at the 1-h
incubation period with HIV-1. The mock preparation was generated by
purifying extracts on an affinity matrix constructed with the
biotin-labeled 5[QPQ]-TASP.
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The capacity of the purified V3-BPs to inhibit HIV infection suggests
their potential interaction with HIV particles, and provides a
mechanism by which the purified proteins could block what might be
happening under normal conditions, i.e. the interaction of
HIV particles with the cell-surface-expressed V3-BPs.
The V3-BPs (Nucleolin, PHAP II, and PHAP I) as Cofactors in the
Mechanism of HIV Binding--
The pseudopeptide
5[K
(CH2N)PR]-TASP, designed to mimic the conserved RP
dipeptide motif and basic lysine and arginine residues in the V3 loop
of HIV isolates, is a potent and specific inhibitor of HIV infection
(23). Here, we demonstrate that an identical pattern of proteins
composed of nucleolin, PHAP II, and PHAP I can be purified from cells
using either the pseudopeptide 5[K
(CH2N)PR]-TASP or a synthetic V3 loop peptide (Fig. 4, lanes 3 and
4), suggesting that 5[K
(CH2N)PR]-TASP can
indeed mimic the V3 loop. This observation, together with the fact that
5[K
(CH2N)PR]-TASP is a potent inhibitor of HIV entry
by binding to cell-surface components of protein in nature (4),
suggests that the V3-BPs described here are targets of this
pseudopeptide inhibitor. The interaction of
5[K
(CH2N)PR]-TASP with the V3-BPs is of high affinity
(Table II). Otherwise, the purification of these V3-BPs by just a
single step would not have been possible. The control peptide
5[QPQ]-TASP construct does not bind the V3-BPs, whereas the
tetravalent 4[KPR]-TASP construct, which has very little activity
against HIV, binds poorly the V3-BPs but along with many other
proteins. These observations therefore emphasize the unique specific
nature of the pentavalent 5[K
(CH2N)PR]-TASP construct.
In addition, the affinity to bind the V3-BPs and the anti-HIV activity
of the different TASP constructs (23) are tightly correlated. These
V3-BPs therefore appear to be implicated as cofactors in the mechanism
of HIV binding to permissive CD4+ cells. Such a cofactor
role is enforced by several observations: 1) specific binding of
5[K
(CH2N)PR]-TASP to cell-surface proteins (4); 2)
inhibition of HIV particle binding to CD4+ cells in the
presence of 5[K
(CH2N)PR]-TASP (4, 23); 3) binding of
5[K
(CH2N)PR]-TASP and the V3 loop peptide to each one
of the V3-BPs, nucleolin, PHAP II and PHAP I (Fig. 3, panels 3 and 4); 4) formation of a stable complex between
5[K
(CH2N)PR]-TASP and cell-surface-expressed
nucleolin, PHAP II, and PHAP I (Fig. 5); 5) demonstration that gp120
binds the V3-BPs via its V3 loop (Tables II and III); 6) competition
between gp120 and 5[K
(CH2N)PR]-TASP to bind the V3-BPs
(Fig. 6); 7) inhibition of HIV infection and entry using purified
preparations of the V3-BPs (Fig. 7). By virtue to bind the V3 loop
domain, the V3-BPs could interact with the gp120 on the surface of HIV
particles and thus become implicated in the HIV binding process.
Consequently, agents that can interfere in the interaction of the V3
loop domain of gp120 with the cell-surface-expressed V3-BPs block HIV
binding and thus entry (results herein; Refs. 4 and 22). For examples,
HIV binding to CD4+ cells could be blocked either by
5[K
(CH2N)PR]-TASP, which binds the
cell-surface-expressed V3-BPs, or by anti-V3 loop mAbs, which bind the
V3 loop in gp120 exposed on HIV particles. We found out that all human
and murine cells of lymphoid or non-lymphoid origin which were
investigated express nucleolin, PHAP II, and PHAP I (data not shown).
In view of this, and the fact that the expression of human CD4 and
CXCR4 or CCR5 is sufficient for efficient entry of different HIV-1
isolates into heterologous cells (7-13), the V3-BPs should therefore
represent a complementary coreceptor for HIV that is not
species-specific. Previously, by biochemical approaches different
peptidases such as tryptase TL2, cathepsin, and dipeptidyl peptidase IV
(CD26), or unidentified proteins have been reported to interact with
the V3 loop of gp120 (56-59); however, the relationship between such
interactions and their putative role in HIV infection has not been
determined. Consequently, this is the first report in which an
inhibitor of HIV binding was used to isolate its potential target(s).
In the case of CD26, several reports have now demonstrated that it can
regulate the rate of HIV envelope-mediated viral entry, cell-fusion
process, and apoptosis (53, 60-63). Furthermore, evidence has been
presented for the specific interaction of CD26 with gp120. However,
this latter interaction appears to be located outside the V3 loop, at
the C3 region between the V3 and V4 loop (64).
The potential role of each one of the V3-BPs and the mechanism by which
they are implicated in the process of HIV-particle binding to cells
requires further investigation. However, the fact that
5[K
(CH2N)PR]-TASP or anti-CD4 mAb CB-T4 block the
binding of HIV to CD4+ cells by two independent mechanisms
is consistent with the suggestion that the V3-BPs are as important as
CD4 in the HIV binding process (4, 22). The degree of inhibition of HIV
binding by 5[K
(CH2N)PR]-TASP is not enhanced when it
is used combined with an anti-CD4 antibody, indicating that the
pseudopeptide-mediated inhibition affects specific (i.e.
functional) binding of HIV particles to CD4+ cells (4).
Consequently, these observations suggest the existence of two specific
binding events occurring between gp120 molecules on HIV particles and
distinct cell-surface components, namely the CD4 molecule on one hand,
and V3-BPs on the other hand. There should be a cooperativity between
these two interactions in order to achieve functional binding, since
antibodies against CD4 or the pseudopeptide
5[K
(CH2N)PR]-TASP disrupt the functional binding process. Once HIV is stably bound to permissive cells, the chemokine receptors CXCR4/CCR5 could then become implicated in the fusion process
to allow viral entry.
Taken together, our recent works (4, 22) and the results presented here
suggest that there should be two distinct domains in the gp120 molecule
on an HIV particle, responsible for direct binding events with the cell
membrane; the first domain is the well described site of binding to CD4
(20), whereas the second domain is the V3 loop, which binds the V3-BPs.
This secondary binding event appears to be as functional as binding to
CD4, since 5[K
(CH2N)PR]-TASP or antibodies directed
against the V3 loop inhibit efficiently viral binding and thus the
entry process (4, 22), as is the case with anti-CD4 antibodies against
the gp120 binding site (22). It should be noted that
cell-surface-expressed heparan sulfates have been implicated in the
interaction of the V3 loop with the cell surface, but this latter
interaction is restricted to the T lymphocyte-tropic HIV-1 isolates (5,
6, 15). In contrast, 5[K
(CH2N)PR]-TASP inhibits
binding of both T lymphocyte- and macrophage-tropic HIV-1
isolates,2 thus pointing out the wider significance of
V3-BPs in the mechanism of viral binding. Accordingly, the V3-BPs
represent novel targets for the development of potential anti-HIV
reagents.
 |
ACKNOWLEDGEMENTS |
We thank Nadine Robert for excellent
technical assistance, and Olivier Schwartz for advice on the
preparation of the HIV-1 pseudotyped virus. Rabbit antiserum raised
against a purified preparation of human nucleolin was generously
provided by Drs. M. Erard and C. Faucher, Center de Recherche de
Biochimie et de Génétique Cellulaire du CNRS, Toulouse,
France.
 |
FOOTNOTES |
*
This work was supported by grants from Institut Pasteur,
Paris, CNRS, Agence Nationale de la Recherche sur le SIDA, and
SIDACTION; by a grant from Agence Nationale de la Recherche sur le SIDA
(to C. C.); by scholarships from SIDACTION (to N. B. and
N. S.); by postdoctoral fellowships from Ministerio de Educacion y
Ciencia (Spain) and Ministère des Affaires Etrangères
(France) (to J. B.); and by a grant from Fondation Mondiale pour
la Recherche et Prevention SIDA (France) (to E. 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.
¶
To whom correspondence should be addressed. Tel.:
33-1-4568-8776; Fax: 33-1-4061-3012; E-mail:
arahovan{at}pasteur.fr.
1
The abbreviations used are:
HIV, human immunodeficiency virus; TASP, template-assembled
synthetic peptide; PHAP I and PHAP II, putative HLA class II-associated
proteins I and II; gp120, the external envelope glycoprotein of HIV-1;
V3 loop, the hypervariable region of about 36 amino acids in gp120;
V3-BPs, V3 loop-binding proteins; PBMC, peripheral blood mononuclear
cell; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis; FACS, fluorescence-activated cell sorting; AZT,
azidothymidine; MMLV, Moloney murine leukemia virus; CHO, Chinese
hamster ovary; ELISA, enzyme-linked immunosorbent assay; PBS,
phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; LTR,
long terminal repeat; Fmoc,
N-(9-fluorenyl)methoxycarbonyl.
2
A. G. Hovanessian and F. Puvion-Dutilleul,
manuscript in preparation.
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