J Biol Chem, Vol. 274, Issue 39, 27875-27884, September 24, 1999
The Anti-HIV Pseudopeptide HB-19 Forms a Complex with the
Cell-surface-expressed Nucleolin Independent of Heparan Sulfate
Proteoglycans*
Sébastien
Nisole
,
Bernard
Krust,
Christian
Callebaut§,
Gilles
Guichard¶,
Sylviane
Muller¶,
Jean-Paul
Briand¶, and
Ara G.
Hovanessian
From the Unité de Virologie et Immunologie
Cellulaire, URA 1930 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
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ABSTRACT |
The HB-19 pseudopeptide
5[K
(CH2N)PR]-TASP, (CH2N) for
reduced peptide bond, is a specific inhibitor of human immunodeficiency virus (HIV) infection in different CD4+ cell lines and in
primary T-lymphocytes and macrophages. Here, by using an experimental
CD4+ cell model to monitor HIV entry and infection, we
demonstrate that HB-19 binds the cell surface and inhibits attachment
of HIV particles to permissive cells. At concentrations that inhibit HIV attachment, HB-19 binds cells irreversibly, becomes complexed with
the cell-surface-expressed nucleolin, and eventually results in its
degradation. Accordingly, by confocal immunofluorescence microscopy, we
demonstrate the drastic reduction of the cell-surface-expressed nucleolin following treatment of cells with HB-19. HIV particles can
prevent the binding of HB-19 to cells and inhibit complex formation
with nucleolin. Such a competition between viral particles and HB-19 is
consistent with the implication of nucleolin in the process of HIV
attachment to target cells. We show that another inhibitor of HIV
infection, the fibroblast growth factor-2 (FGF-2) that uses
cell-surface-expressed heparan sulfate proteoglycans as low affinity
receptors, binds cells and blocks attachment of HIV to permissive
cells. FGF-2 does not prevent the binding of HB-19 to cells and to
nucleolin, and similarly HB-19 has no apparent effect on the binding of
FGF-2 to the cell surface. The lack of competition between these two
anti-HIV agents rules out the potential involvement of heparan sulfate
proteoglycans in the mechanism of anti-HIV effect of HB-19, thus
pointing out that nucleolin is its main target.
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INTRODUCTION |
The external envelope glycoprotein
gp1201 of HIV plays a key
role in the capacity of virus particles to infect CD4+
target cells as a result of the fusion of the viral and cellular membranes. The gp120 has a complex secondary structure in which five
conserved regions (C1 to C5) and five hypervariable regions (V1 to V5)
have been defined. It exists as an oligomer associated noncovalently
with the transmembrane glycoprotein gp41 which at its amino terminus
contains a hydrophobic domain essential for the fusion process (1-3).
Studies on the mechanism of HIV tropism have revealed that chemokine
receptors such as CCR5 and CXCR4 serve as essential cofactors specific
for the entry of macrophage (M)- and T-lymphocyte-tropic HIV-1
isolates, respectively (reviewed in Refs. 3-5). Consequently,
infection of cells by both types of HIV-1 isolates could be inhibited
by chemokines interacting specifically with their respective receptors
(5-7). Besides CCR5, additional 
-chemokine receptors including
CCR2b, CCR3, and CCR8 are used by some HIV-1 variants (5). It should be
noted that chemokines inhibit HIV entry without affecting the
attachment of HIV particles to cells (8, 9). Therefore, chemokines appear to block a step following attachment of HIV particles to cells,
namely fusion between viral and cellular membranes. By incubation of
cells with soluble preparations of gp120, several groups have
demonstrated the formation of a complex between gp120, CD4 and CCR5, or
CXCR4 and have proposed that HIV-1 attachment to CD4+ cells
creates a high affinity interaction site for the coreceptor and that
during this event the V3 loop plays an important role by a mechanism
that remains to be elucidated (3, 9).
The degree of involvement of CD4 in the initial attachment of HIV
particles could be variable according to the cell type (10). In
CD4+ T-cells, HIV attachment is mediated by both
CD4-dependent and -independent interactions (11).
Neutralizing mAbs specific for the V3 loop inhibit both
CD4-dependent and -independent interactions, whereas
neutralizing mAbs against the gp120 binding domain in CD4 or against
the CD4 binding domain in gp120 affect only the CD4-dependent interaction. The observation that anti-V3
loop mAbs also inhibit HIV attachment to CD4
cells (11)
indicates that interaction of the V3 loop with its cell-surface ligands
can occur at a step prior to the interaction of gp120 with CD4. In
accord with this, several studies have suggested that the V3 loop
domain is not necessary for the binding of gp120 to CD4 (12, 13). These
observations and the fact that mAbs against the V3 loop block HIV
attachment to cells without affecting the potential interaction of
gp120 with CD4 (11) indicate that interactions through the V3 loop and
the CD4-binding site of gp120 are two independent events. In accord
with this, spontaneous mutations within the V3 loop have been proposed
to be responsible for CD4-independent entry of the HIV-1 NDK isolate
(14). As the V3 loop is relatively exposed on HIV particles (15) and by
virtue of its positively charged residues, it could interact directly
with negatively charged components of the cell surface independent of
CD4 and chemokine receptors. Potential candidates of such cell-surface
components are on the one hand V3 loop binding proteins on the cell
surface (discussed in Ref. 16) and on the other hand heparan sulfate proteoglycans that are commonly found on the surface of most vertebrate cell types (10, 17-19).
The pseudopeptide 5[K(CH2N)PR]TASP referred to as
HB-19, which presents pentavalently the K(CH2N)PR
tripeptide moiety, is a potent inhibitor of HIV infection of
CD4+ T-cell lines or primary T-lymphocytes and macrophages
(20, 21). HB-19 does not have a significant effect on infection of cells by the simian immunodeficiency virus-mac isolate or by HIV-1 pseudotyped with envelope glycoproteins of other viruses (16, 20).2 We confirm here that
the anti-HIV effect of HB-19 is due to inhibition of the attachment of
HIV-1 particles to target cells leading to inhibition of viral entry.
The biotin-labeled HB-19 but not the control constructs binds
specifically target cells and becomes complexed with the
cell-surface-expressed nucleolin. Interestingly, HIV particles can
compete with HB-19, both in binding to the cell surface and in complex
formation with the cell-surface-expressed nucleolin. On the other hand,
no apparent competition occurs with the fibroblast growth factor-2
(FGF-2), known to use heparan sulfates as low affinity receptors (22,
23). This latter observation rules out the possibility that
cell-surface heparan sulfate proteoglycans serve as additional targets
of HB-19. However, consistent with the implication of heparan sulfates
in the HIV attachment process (10, 18, 19), we show that FGF-2 inhibits
HIV infection by binding to cells and preventing the attachment of HIV
particles. The fact that HIV attachment could be inhibited by two
different agents that do not compete suggests that the HIV attachment
process should be coordinated by two events implicating on the one hand the cell-surface-expressed nucleolin and on the other hand the heparan
sulfate proteoglycans.
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EXPERIMENTAL PROCEDURES |
Materials--
The monoclonal antibody (mAb) specific to human
CD4 and reacting with the gp120 binding domain (11) was kindly provided by Dr. Eugene Bosmans (clone CB-T4-2; Eurogenetics, Tessenderlo, Belgium). mAb CC98 directed against human nucleolin (24) was generously
provided by Dr. Ning-Hsing Yeh, Graduate School of Microbiology and
Immunology, National Yang-Ming Medical College, Shih-Pai, Taiwan,
Republic of China. The mAb D3 specific for human nucleolin and used in
confocal microscopy studies was kindly provided by Dr. Jau-Shyong Deng,
Department of Veterans Affairs, Medical Center, Pittsburgh, PA (25).
Polyclonal antibodies were raised in rabbits against a synthetic
peptide corresponding to the first 26 amino acid residues of human
nucleolin as we had reported (16). The mAb N11-20 directed against the
V3 loop of HIV-1 Lai isolate was provided by Dr. Jean-Claude Mazie,
Hybridolab, Institut Pasteur, Paris, France. The mAbs specific to human
CXCR4 (12G5) and CCR5 (12D1) were purchased from R & D Systems. The
T-tropic HIV-1 Lai was as described before (20). The M-tropic HIV-1
isolates HIV-1 Ba-L (26) and Ada-M (27) were provided by the AIDS
Research and Reference Reagent Program, AIDS Program, NIAID, National
Institutes of Health. The HIV-1 pseudotyped with VSV envelope
glycoproteins was kindly provided by Dr. Olivier Schwartz (Institut
Pasteur, Paris). FGF-2 (fibroblast growth factor basic) produced in
Escherichia coli was from Sigma. FGF-2 was iodinated (2 × 103 µCi/µmol) using the Bolton-Hunter reagent (NEN
Life Science Products) by a procedure as recommended by the manufacturer.
Cells and Virus Preparations--
Human
HeLa-CD4-LTR-lacZ cells expressing or not CCR5 were referred
to as HeLa P4-C5 and HeLa P4, respectively, and were cultured in
Dulbecco's medium. These HeLa cells were provided by Drs. Olivier Schwartz and Pierre Charneau (Institut Pasteur, Paris, France) and were
cultured with 10% (v/v) heat-inactivated (56 °C, 30 min) fetal calf
serum. The amount of each virus preparation was estimated by the
concentration of the major core protein p24 by p24 Core Profile
enzyme-linked immunosorbent assay (DuPont). Aliquots of virus stocks
were stored at 
130 °C. The virus preparations were filtered
(0.22-µm pore size, Millipore) before infection of cells. For the
competition studies between HIV particles and HB-19, virus particles
were recovered by centrifugation (100,000 × g, for 30 min at 4 °C), and the pellet was suspended in the culture medium and
filtered through 0.22-µm pore size filters (Millipore) in order to
eliminate aggregates and/or cell debris. Such preparations generated
concentrated virus that is highly infectious (11).
Preparation of Nucleus-free Cell Extracts--
HeLa cell
monolayers in 150-cm2 flasks were washed extensively with
PBS before adding 1 ml of the lysis buffer E, 20 mM
Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM
MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM -mercaptoethanol, aprotinin (1000 units/ml), and
0.5% Triton X-100. After 5 min incubation in buffer E (at room
temperature), the flasks were declined and left at a vertical position
to collect cell extracts. By this procedure, nucleus-free extracts of
HeLa cells could be recovered conveniently since nuclei remain attach to the plastic. Such nucleus-free supernatants were then centrifuged at
12,000 × g for 10 min, and the supernatants were
stored at 80 °C.
Peptide Constructs--
The synthesis of
5[K(CH2N)PR]-TASP (herein referred to as HB-19) was as
described previously (20, 28). Control peptides were 5[QPQ]-TASP,
5[R(CH2N)PN]-TASP, and K(CH2N)PR
monomer. For the biotin-labeled compounds, the biotin moiety was
introduced during peptide assembly as an Fmoc
(N-(9-fluorenyl)methoxycarbonyl) Lys-biotin derivative at
the carboxyl terminus of the template in HB-19 and 5[QPQ]-TASP and
the carboxyl terminus of the K(CH2N)PR monomer. The
peptides were obtained at a high purity (>95%), and their integrity
was controlled by matrix-associated laser desorption ionization-time-of-flight analysis (29). The preparation of the
fluorescein isothiocyanate (FITC, Sigma)-labeled HB-19 was as described
before (28). The anti-HIV cyclic peptide TW70 specific for CXCR4 was
synthesized as described (30, 31). The TW70 peptide has the amino acid
sequence RRWCYRKDKPYRKCR; the DK (where DK indicates that the peptide bond is in D
configuration) has been introduced in the sequence of this peptide in
order to stabilize the -turn in the final structure. The disulfide
bridge between the two cysteine residues was generated by air oxidation
at pH 8 in water under vigorous stirring for 2 days at room
temperature. The final cyclic peptide was more than 95% pure.
Detection of Cell-surface Antigens by FACS Analysis--
The
detection of cell-surface antigens was as described before with minor
modifications (28). Briefly, HeLa cells (about 106) in
6-well plates washed in PBS were incubated (30 min at 4 °C) in 500 µl of FACS buffer (PBS containing 1% bovine serum albumin and 0.02%
sodium azide) containing the following antibodies: mAb specific to CD4
(CB-T4, IgG1), CXCR4 (12G5, IgG2a), CCR5 (12D1, IgG2a), or nucleolin
(D3, IgG1). Cells were then washed in FACS buffer and further incubated
(30 min, 4 °C) in the presence of FITC-labeled IgG against mouse
immunoglobulins (Sanofi Diagnostics Pasteur). IgG1/IgG2a were used as
control isotype antibodies (from Becton Dickinson, Mountain View, CA).
After this second incubation, cells were washed and scraped using a
rubber policeman. The cell pellet obtained by centrifugation (350 × g, 8 min) was suspended in PBS, fixed in PBS containing
1% paraformaldehyde, and processed for analysis by FACS scan flow
cytometer (Becton Dickinson). For each sample, 10,000 viable cells were
gated following size (forward scatter, FSC) and granularity (side
scatter, SSC) parameters and analyzed with Cell QuestTM
Software (Becton Dickinson). The capacity of HB-19 to bind specifically different types of cells could be used to monitor labeled cells by FACS
analysis. For this purpose, HeLa cells (as above) were incubated (30 min, either at 4 or at 37 °C) in 500 µl of FACS buffer containing
FITC- or biotin-labeled HB-19. Cells were then washed and processed for
FACS analysis as described before (16, 28).
Purification of Cell-surface-associated Nucleolin--
These
experimental conditions were previously optimized for samples from CEM
cells (16, 28). Briefly, HeLa cell monolayers in 150-cm2
flasks (about 25 × 106 cells/flask) were incubated in
10 ml of culture medium (Dulbecco's medium with 10% fetal calf serum)
containing the biotin-labeled HB-19 (5 µM) for 30 min at
37 °C. After washing extensively in PBS containing 1 mM
EDTA (PBS/EDTA), nucleus-free cell extracts were prepared in lysis
buffer E containing unlabeled HB-19 (50 µM), and the
complex formed between cell-surface-expressed nucleolin and the
biotin-labeled HB-19 was isolated by purification of the extracts using
avidin-agarose (100 µl; ImmunoPure Immobilized Avidin from Pierce) in
PBS/EDTA. After 2 h of incubation at 4 °C, the samples were
washed extensively with PBS/EDTA. The purified proteins were denatured
by heating in the electrophoresis sample buffer containing SDS and
analyzed by SDS-PAGE. The presence of nucleolin was then revealed by
immunoblotting using the monoclonal antibody CC98 as described before
(16).
Assay of HIV Entry in HeLa CD4+ Cells--
HIV entry
was monitored indirectly in HeLa-CD4-LTR-lacZ 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 (32). HeLa-CD4-LTR-lacZ cells expressing recombinant CD4 and
constitutively CXCR4 are permissive to infection by T-tropic HIV-1
isolates (33). When they also express recombinant CCR5, such HeLa cells
become permissive to M-tropic HIV-1 isolates. These two HeLa cell lines expressing or not CCR5 are referred to as HeLa P4-C5 and HeLa P4,
respectively. Cells were plated at 104 cells/well in
96-well plates, and at 24 h later cell monolayers were infected
with the different HIV isolates (a dose corresponding to 20-40 ng/ml
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 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 10-15-min intervals in a microplate reader
using a 570 nm filter. The background value in the -galactosidase
activity in each experiment was measured by including an HIV infection
in the presence of AZT (5 µM) that inhibits the HIV
reverse transcriptase. The background value of -galactosidase
activity was also monitored in cells in which HIV entry was blocked by
the anti-CD4 mAb CB-T4 (5 µg/ml) that reacts with the gp120-binding
site in CD4 (11).
Assay of HIV Particle Attachment to HeLa CD4+
Cells--
The effect of HB-19 on the amount of HIV-1 Lai particles
attached to cells (i.e. associated with cells) was monitored
after 1 h of incubation at 4 °C. These experiments were carried
out at 4 °C in order to reduce HIV endocytosis (33) and viral entry (34) at its minimum. HeLa P4 and HeLa P4-C5 cells were treated (37 °C, 30 min) with 
-CD4 mAb CB-T4 (at 5 µg/ml), HB-19, FGF-2, and the control peptide constructs before washing cells (with PBS) to
eliminate free inhibitors. Cells were then incubated at 4 °C with
the HIV-1 isolate in the culture medium. One hour after incubation,
cells were washed extensively with culture medium containing 10% fetal
calf serum to eliminate free unbound HIV particles, and the amount of
p24 associated with cells was measured as an estimate for the amount of
HIV binding. In order to demonstrate that most of the HIV associated
with cells represented virus particles bound on the surface of cells,
samples of cells incubated with virus were washed with PBS before
treatment with trypsin to eliminate virus bound on the cell surface as
described before (11). For a control virus, we investigated the effect
of HB-19 on the attachment of HIV-1 Ada, since HB-19 does not affect
entry and infection by this virus isolate. Accordingly, HB-19 resulted
in a significant inhibition of attachment of HIV-1 Lai and Ba-L but not Ada.
Confocal Microscopy--
To confirm the cell-surface expression
of nucleolin, the subcellular localization of nucleolin was
investigated by using a specific monoclonal antibody (mAb D3). Laser
scanning confocal immunofluorescence microscopy (Leica TCS4D) was
carried out by fixing cells either with paraformaldehyde (PFA, 3.7%)
for membrane staining or paraformaldehyde/Triton X-100 solution
(PFA/Triton) for intracellular staining as described previously (35).
Specific cell-surface labeling was also investigated by incubation of
cells directly with the antibody before PFA fixation. HeLa P4 cells were plated 24 h before the experiment in 8-well glass slides (Lab-Tek Brand, Nalge Nunc International, Naperville, IL). The anti-mouse antibodies linked to FITC (Sigma) were used at 1/100 dilution. For a control antibody, mAb LG2-2 specific for histone H2B
(36) was used. The anti-mouse antibodies linked to FITC (Sigma) were
used at 1/100 dilution.
The Binding of the 125I-Labeled FGF-2 to
Cells--
HeLa P4 cells were incubated (30 min, 37 °C) with
125I-labeled FGF-2 (at different concentrations) in culture
medium containing 10% fetal calf serum. Cells were then washed
extensively with PBS before preparation of nuclear-free cell extracts.
Aliquots were then analyzed by SDS-PAGE or counted to evaluate the
amount of FGF-2 binding. The level of the 125I-FGF-2
observed in SDS-PAGE (quantified in a PhosphorImager; Molecular
Dynamics, Sunnyvale, CA) was systematically correlated with the amount
of the radioactivity estimated by counting using an automatic gamma
counter (model 1272 clinigamma, Wallac, Turku, Finland). In order to
remove FGF bound to glycosaminoglycans, cells were washed twice with 2 M NaCl, 10 mM Tris, pH 7.4, following incubation with 125I-FGF-2 as it has been described before
(37). The potential binding of FGF-2 to its high affinity receptor was
also investigated by cross-linking of 125I-FGF-2 using 1 mM 3,3'-bis(sulfosuccinimidyl)suberate (Pierce) (37,
38).
Ligand Blotting and Immunoblotting--
Crude nuclear-free HeLa
cell extracts were diluted in 2-fold concentrated electrophoresis
sample buffer and analyzed by SDS-PAGE to be electrophoretically
transferred to 0.22-µm polyvinylidene difluoride sheets (Bio-Rad).
The electrophoretic blots were saturated with casein-based blocking
buffer (Genosys) and washed extensively before incubation with the
biotin-labeled HB-19 or the 125I-labeled FGF-2. The biotin
was revealed by using streptavidin-horseradish peroxidase complex and
light-based enhanced chemiluminescence reagents as provided by the
manufacturer (Amersham Pharmacia Biotech). Immunoblotting to detect
nucleolin was carried out using either rabbit polyclonal antibodies (10 µg/ml) raised against the amino-terminal peptide of nucleolin or mAb
D3 (0.1 µg/ml). The 125I-labeled protein bands in
SDS-PAGE gels were revealed using a PhosphorImager.
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RESULTS |
HB-19 Inhibits HIV Attachment and Entry in CD4+ HeLa
Permissive Cell Lines--
HeLa P4-C5 and HeLa P4 cells are
recombinant CD4+ cell lines permissive to infection by M-
and T-tropic HIV-1 isolates, respectively. These cells also contain the
bacterial lacZ gene under the control of HIV-1 LTR sequence.
HIV entry and replication in such cells result in the activation of the
HIV LTR, leading to the expression of the lacZ gene.
Consequently, the -galactosidase activity could be measured in order
to monitor HIV entry into cells (see "Experimental Procedures").
One µM of 5[K(CH2N)PR]-TASP (herein
referred to as HB-19) blocked the T-tropic HIV-1 Lai and the M-tropic
HIV-1 Ba-L attachment and entry by more than 90% (Table
I). The inhibitory effect of HB-19 in
these HeLa cells is dose-dependent, as it has been reported
before in other cell lines or in primary T-lymphocytes and macrophages
(16, 20, 21). HB-19 does not affect infection by HIV-1 pseudotyped with
envelope glycoproteins of Moloney murine leukemia or vesicular
stomatitis virus (16, 20),2 thus indicating that its
inhibitory effect is against the HIV envelope glycoprotein-mediated
viral entry process.
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Table I
Inhibition of HIV attachment and entry by HB-19
Two different HeLa cell lines, HeLa P4 (for the T-tropic HIV-1 Lai
isolate) and HeLa P4-C5 (for the M-tropic HIV-1 Ba-L and Ada isolates),
were assayed for the attachment of HIV particles and entry as described
under "Experimental Procedures." For each virus infection (in
triplicate samples), the effect of HB-19
(5[K(CH2N)PR]-TASP) and the control peptides CP-1
(K(CH2N)PR) and CP-51 (5[R(CH2N)PN]-TASP) were
assayed at concentrations as indicated. The percent inhibition of HIV
attachment and entry was calculated considering the value obtained for
each virus isolate in the absence of any inhibitors as 100%. The
background values for virus attachment and virus entry were obtained by
trypsin treatment of cells and by monitoring entry in the presence of 5 µM AZT, respectively.
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The specific inhibitory effect of HB-19 with respect to its structure
was demonstrated by the use of its tripeptide moiety K(CH2N)PR alone (referred to as CP-1) and the control
pseudopeptide analogue in which the lysine and arginine residues were
modified to arginine and asparagine, respectively, to generate the
5[R(CH2N)PN]-TASP construct (referred to as CP-51). No
effect on the attachment and entry of either HIV-1 Lai and HIV-1
Ba-L was observed by these latter constructs (Table I).
Among different HIV-1 isolates, the M-tropic HIV-1 Ada was found to
resist the inhibitory effect of HB-19, and even higher concentrations
consistently resulted in an enhancing effect on the amount of HIV
attachment and entry (Table I). The reason for this latter effect
remains to be investigated. An enhanced HIV-1 Ada replication has also
been reported by others (39) in infected monocytes, upon treatment with
-chemokines which inhibit infection by other M-tropic HIV-1
isolates. The HIV-1 Ada isolate, therefore, appears to behave
differently compared with other known HIV-1 isolates. Whatever is the
case, the HB-19 resistance of the HIV-1 Ada isolate was conveniently
used as a control virus in our experiments described herein.
The Stable Binding of HB-19 to HeLa Cells--
The mechanism of
the anti-HIV effect of HB-19 was investigated in the HeLa cell lines P4
and P4-C5. By FACS analysis using the biotin-labeled HB-19, we first
demonstrated that this inhibitor binds specifically and in a
dose-dependent manner the surface of both cell lines
studied (as in Ref. 16). The binding is stable, since HeLa cells
preincubated with HB-19 could be washed extensively before addition of
HIV, but the inhibitory effect on HIV attachment and entry process
persists for several hours. In contrast, no inhibitory effect is
observed when HB-19 is added a few hours after incubation of cells with
HIV (not shown). Fig. 1 further demonstrates the stable binding of HB-19 to cells by confocal laser
immunofluorescence microscopy. The immunofluorescence signal scanned
toward the middle cross-section of the cell monolayer revealed a
distinct staining at the cell periphery. Staining was also observed on
the cell surface (not shown). However, as cells were scanned downward
the immunofluorescence was observed mainly at the cell periphery thus
indicating that HB-19 is associated with the plasma membrane. It should
be emphasized that under similar experimental conditions, no cell
surface or membrane staining was observed in cells in the presence of
the control pseudopeptide CP-1 (not shown).
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Fig. 1.
The binding of HB-19 to HIV-permissive HeLa
P4 cells revealed by confocal laser microscopy. HeLa P4 cells were
treated (incubation at 37 °C, 20 min) with the biotin-labeled HB-19
(2 µM) in culture medium. Cells were then washed with PBS
and fixed with PFA and processed for confocal microscopy. The biotin
was revealed by streptavidin-FITC complex (Amersham Pharmacia Biotech).
A scan of a cross-section toward the middle of the cell monolayer
showing the FITC labeling and the corresponding phase contrast are
presented.
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HB-19 Forms a Stable Complex with the Cell-surface-expressed
Nucleolin in HeLa Cells--
The specific binding of HB-19 to the
surface of PBMC and CEM cells results in the formation of an
irreversible complex with the cell-surface-expressed nucleolin (16,
28). Similarly, incubation of HeLa cells with the biotin-labeled HB-19
construct resulted in its binding with the cell-surface-expressed
nucleolin and formed an irreversible complex with it (Fig.
2A). For this purpose, cells
were incubated with the biotin-labeled HB-19 under routine experimental
conditions used for the HIV entry assay, before washing extensively and
preparation of nuclear-free extracts with lysis buffer containing
excess unlabeled HB-19. The irreversible complex was then recovered
using avidin-agarose. By this procedure, cell-surface nucleolin was
isolated without its cytoplasmic counterpart (16, 28). Nucleolin
recovered from the cell surface was partially cleaved to give a 60-kDa
product, corresponding to the COOH-terminal part of 95-kDa nucleolin
which could be identified by monoclonal antibodies CC98 and D3 that
react with an internal epitope (16). In some experiments, additional
cleavage products of nucleolin were detectable by the monoclonal
antibodies. Rabbit antibodies directed against the first 26 amino acids
at the amino-terminal of nucleolin (16) did not react with any of the
cleavage products, thus indicating that the cleavage starts at the
amino-terminal end (not shown). As expected, the biotin-labeled
K(CH2N)PR tripeptide did not bind the
cell-surface-expressed nucleolin, even when it is used in excess (Fig.
2A, lane CP-1). Similarly, no binding was observed with the
control biotin-labeled TASP construct 5[QPQ]-TASP (Fig. 2A,
lane CP-40). Therefore, the binding of the biotin-labeled HB-19 to
cell-surface-expressed nucleolin is specific to the structure of the
pentavalently presented tripeptide moiety.
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Fig. 2.
Recovery of the cell-surface-expressed
nucleolin on HeLa cells by the capacity of HB-19 to bind and form a
stable complex with it. A, HeLa P4 cells in the culture
medium were incubated (37 °C, 30 min) at different concentrations of
the biotin-labeled HB-19 (lanes 0, 0.5, 1, and 2.5 µM). As controls, HeLa cells were incubated with
biotin-labeled control peptides: 50 µM
K(CH2N)PR (referred to as CP-1) or 10 µM
5[QPQ]-TASP (referred to as CP-40). Cells were then washed
extensively in PBS containing 1 mM EDTA prior to the
preparation of nucleus-free cell extracts. The extraction of cells
preincubated with the biotin-labeled HB-19 was performed with lysis
buffer E containing 50 µM unlabeled HB-19, in order to
rule out the possibility that the complex formation could occur with
cytoplasmic proteins during preparation of extracts. Extracts were
purified on avidin-agarose to recover complexes formed between
cell-surface proteins and the biotin-labeled peptide constructs (16,
28), and the presence of nucleolin was revealed by immunoblotting using
mAb CC98 (see "Experimental Procedures"). The numbers on
the left show the position of molecular mass (in kDa)
protein markers. On the right is the position of nucleolin
(p95) and its partial cleavage product (p60). Material extracted from
107 cells was analyzed in each lane. B, the
specific binding of HB-19 to the cell-surface-expressed nucleolin. HeLa
P4-C5 cells in the culture medium were incubated (37 °C, 30 min) as
such (lane 1) or with the control tripeptide monomer
K(CH2N)PR (CP-1; 250 µM; lane
2) or unlabeled HB-19 (50 µM; lane 3)
before the addition of the biotin-labeled HB-19 (2.5 µM)
and further incubation at 37 °C for 30 min. The samples were then
processed as in A to recover the biotin-labeled HB-19
coupled to the cell-surface-expressed nucleolin.
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As we demonstrated previously (28) in CEM cells, the binding of the
biotin-labeled HB-19 to the HeLa cell-surface-expressed nucleolin was
specific since it was reduced drastically in the presence of excess
unlabeled HB-19 but not by the control CP-1 pseudopeptide (Fig.
2B, lanes 2 and 3). Nucleolin recovered from the
surface of HeLa cells corresponded to less than 20% that found in the
nuclear-free cytoplasmic fraction. The mechanism of expression of
nucleolin on the cell surface remains to be elucidated, since nucleolin
does not possess a hydrophobic domain as do conventional membrane-associated proteins. The presence of nucleolin at the cell
surface has also been shown by electron
microscopy.3
Studies on the kinetics of the partial cleavage of the cell-surface
nucleolin indicated that cleavage occurs as early as 5 min after the
addition of HB-19 (not shown). Such partial cleavage could still be
observed at 60 min, but at 6 h post-addition of HB-19, only a
trace amount of degraded nucleolin remains detectable (Fig.
3B). The degradation of
nucleolin on the cell surface is specific since the detection of other
cell-surface antigens such as CD4, CXCR4, CCR5 (monitored by FACS
analysis), and the activity of several cell-surface peptidases is not
modified in cells treated with 5-10 µM HB-19 (not
shown). Furthermore, it should be noted that nucleolin in the
cytoplasmic fraction does not appear to be affected (Fig. 3A,
panel Cytoplasm). Therefore, despite the continual presence of CD4
and CXCR4/CCR5 on the cell surface, HIV attachment is inhibited in
cells that bind HB-19. Although the partial cleavage of nucleolin has
been reported before under different experimental conditions (16, 24),
this is the first report for the specific cleavage of the
cell-surface-expressed nucleolin. It is plausible that the binding of
HB-19 to nucleolin changes its conformation making it susceptible to
degradation by a cellular protease.
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Fig. 3.
Incubation of cells with HB-19 results in the
cleavage of the cell-surface-expressed nucleolin. A,
the cleavage is specific for the cell-surface-expressed nucleolin. HeLa
P4 cells were incubated at 37 °C for 30 min in culture medium
containing 0, 0.1, and 1 µM of the biotin-labeled HB-19.
Cells were then washed extensively, and nucleus-free extracts were
prepared (as in Fig. 2). The biotin-labeled HB-19 complexed with the
cell-surface nucleolin was then recovered by affinity chromatography
using avidin-agarose (16, 28). The purified proteins (panel Cell
Surface) along with the corresponding nucleus-free crude extracts
(panel Cytoplasm) were assayed by immunoblotting using mAb
CC98 specific for human nucleolin. The numbers on the
left show the position of molecular mass (in kDa) protein
markers. On the right is the position of nucleolin
(p95) and its partial cleavage product (p60).
Material extracted from 1 × 107 and 0.2 × 107 cells in panel Cell Surface and
Cytoplasm, respectively, were analyzed in each lane.
B, kinetics of degradation of cell-surface nucleolin. HeLa
P4 cells, in the culture medium, in the presence of the biotin-labeled
HB-19 (5 µM) were incubated at 37 °C for 1, 6, or
24 h before preparation of nucleus-free extracts and the recovery
of the cell-surface nucleolin complexed with HB-19. Immunoblotting was
as in A. Material extracted from 1 × 107
cells was analyzed in each lane.
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In addition to nucleolin, by using cell extracts we have previously
purified the putative HLA class II-associated proteins I and II (PHAP I
and PHAP II) as two other V3 loop binding proteins (16). However, at
0.5 to 5 µM concentrations of HB-19 that inhibit the
attachment of different HIV isolates to HeLa cells, only nucleolin could be recovered from the cell surface (Fig. 2). Much higher concentrations of HB-19 (20-50 µM) were found to be
required for the isolation of PHAP I and PHAP II from intact HeLa cells
(not shown) and other cell types (16). Therefore, whether PHAP I and
PHAP II are expressed on the cell surface still remains to be
confirmed. The high affinity and the specificity of HB-19 to bind
cell-surface nucleolin point out that nucleolin is the main target of
this pseudopeptide inhibitor of HIV attachment.
Evidence for Cell-surface Expression of Nucleolin Revealed by
Confocal Immunofluorescence Laser Microscopy--
Nucleolin, which is
a major nucleolar protein, has been reported to be also found on the
cell surface (25, 40, 41 and references therein). By FACS analysis
using the mAb D3 specific for nucleolin (25), we could demonstrate the
presence of nucleolin along with CD4 and CXCR4 on the HeLa P4 cells
studied here (Fig. 4). By FACS analysis,
the cell-surface expression of nucleolin has also been shown in primary
macrophages and T-lymphocytes (21).
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Fig. 4.
Cell-surface expression of nucleolin
monitored by FACS analysis using a specific monoclonal antibody.
HeLa P4 cells were processed for FACS analysis with mAb D3, CB-T4, and
12G5 specific for nucleolin, CD4, and CXCR4, respectively (see
"Experimental Procedures"). The ordinate gives the
relative cell number, whereas the abscissa gives the
relative fluorescence intensity.
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The cell-surface expression of nucleolin was further investigated by
using mAb D3 and confocal immunofluorescence microscopy by fixing cells
with PFA. By this procedure, a clear signal was observed on the cell
surface (not shown). As membrane proteins could cluster into patches
when cross-linked by antibodies, HeLa cells were incubated with the mAb
D3 at 37 °C for 1 h before PFA fixing and analysis for confocal
microscopy. Consistently, addition of mAb D3 to unfixed cells resulted
in the redistribution of cell-surface nucleolin into large patches. A
cross-section of such cells is shown in Fig.
5A (panel Control
Cells) demonstrating the clustering of the cell-surface-expressed
nucleolin that was revealed by distinct staining at the periphery of
the HeLa cells (that grow as a monolayer). This strong signal at the
cell membrane was almost completely lost when cells were treated with a
non-ionic detergent used for permeabilization of cells, thus further
confirming that the clustering of nucleolin had occurred on the cell
surface. No cell-surface staining was observed in HeLa cells incubated
with a monoclonal antibody specific to histone H2B, which in
permeabilized cells reacted strongly with histone H2B in the
nucleoplasm (not shown). On the other hand, a similar staining of the
membrane in the HeLa P4 cells was observed by cross-linking CXCR4 with
the specific antibody mAb 12G5 (Fig. 5B, panel Control
Cells).
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Fig. 5.
The cell-surface expression of nucleolin and
its reduction following incubation with HB-19 revealed by confocal
immunofluorescence microscopy. After 24 h of passaging, HeLa
P4 cells were incubated in fresh culture medium in the absence or
presence of 2 µM HB-19 for 5 h. The anti-nucleolin
mAb D3 (A) and anti-CXCR4 mAb 12G5 (B), each at 5 µg/ml, were added in the different cultures during the last hour,
i.e. between 4 and 5 h. Cells were then washed, fixed
with PFA, incubated with FITC-labeled anti-mouse IgG, and processed for
immunofluorescence confocal microscopy. The different panels show an
equivalent layer of a cross-section of HeLa cell monolayers. The
clustering of nucleolin and CXCR4 was consistently observed at the cell
periphery with the anti-nucleolin mAb D3 and the anti-CXCR4 mAb 12G5,
respectively. Note the absence of intracellular nucleolin or CXCR4
staining in intact cells preincubated with the respective monoclonal
antibody. Under similar experimental conditions, no cell-surface
labeling was observed when mAb LG2-2 specific for histone H2B was used
(not shown).
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The prolonged incubation of cells with HB-19 led to degradation of the
cell-surface-expressed nucleolin (Fig. 3). Consistent with this, by
confocal microscopy we show a dramatic reduction of the
cell-surface-expressed nucleolin in cells preincubated with HB-19 for
5 h (Fig. 5A, panel Cells + HB-19). In contrast, no
apparent effect was detectable for the cell-surface-expressed CXCR4 in
HeLa cells preincubated with HB-19 (Fig. 5B), thus
demonstrating that the effect of HB-19 is specific on the
cell-surface-expressed nucleolin.
The clustering of the cell-surface-expressed CXCR4 and nucleolin by
their respective antibodies also occurred at 4 °C (not shown).
Consequently, cell-surface-expressed nucleolin behaves as other well
characterized membrane proteins when it is cross-linked with a specific antibody.
The Binding of HB-19 to the Cell-surface-expressed Nucleolin Is
Inhibited by HIV Particles--
The competition between HIV particles
and HB-19 to bind target cells was demonstrated by the capacity of
HIV-1 Lai particles to inhibit the specific binding of HB-19 to cells
(Fig. 6) and reduce the complex formation
with the cell-surface-expressed nucleolin (Fig.
7). In these experiments, HIV attachment
was carried out at 4 °C in order to block the viral entry process
since fusion between viral and cellular membranes requires incubation
at physiological temperatures. On the other hand, HIV attachment occurs
efficiently at 4 °C,and moreover, the degree of inhibition of this
attachment by HB-19 at 4 °C is comparable to that carried out at
37 °C.
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Fig. 6.
The specific binding of HB-19 to cells is
inhibited by HIV-1 Lai particles. A, the binding of
FITC-labeled HB-19 is inhibited by excess unlabeled HB-19 or by HIV-1
Lai particles. HeLa P4 cells in the culture medium were preincubated
(4 °C, 30 min) in the absence or presence of 50 µM
HB-19 (unlabeled pseudopeptide in excess) or with HIV-1 Lai particles
(corresponding to 200 ng/ml of p24). B, the expression of
CXCR4 is not modified by incubation of cells in excess unlabeled HB-19
or by HIV-1 Lai particles. The experimental procedure used was as in
A. C, the binding of FITC-labeled HB-19 is
inhibited by excess unlabeled HB-19 but not by HIV-1 Ada particles.
HeLa P4-C5 cells in the culture medium were preincubated (4 °C, 30 min) in the absence or presence of 50 µM HB-19 (unlabeled
pseudopeptide in excess) or with HIV-1 Ada particles (corresponding to
200 ng/ml of p24). After these different preincubations, cells were
further incubated (4 °C, 30 min) in the presence of either the
FITC-labeled HB-19 (1 µM; referred here as
HB-19*) or mAb 12G5 (1 µg/ml) specific to CXCR4
(-CXCR4). Cells were washed and processed for FACS analysis as
described (see "Experimental Procedures"). The ordinate
gives the relative cell number, whereas the abscissa gives
the relative fluorescence intensity. The different samples were as
indicated. The peak control of autofluorescence in A and
C was obtained by preincubation of cells with unlabeled
HB-19 (50 µM). Note, in contrast to unlabeled HB-19,
excess control peptide monomer CP-1 (at 250 µM) did not
exert any significant effect on the binding of the biotin-labeled HB-19
to cells (not shown).
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Fig. 7.
HIV-1 Lai particles inhibit the cross-linking
of the biotin-labeled HB-19 to the cell-surface-expressed
nucleolin. HeLa P4 and P4-C5 cells in the culture medium were
incubated (4 °C, 30 min) with HIV-1 Lai or HIV-1 Ada particles,
respectively, at different dilutions corresponding to 200, 100, 50, and
25 ng/ml p24 (see "Experimental Procedures"). These samples were
further incubated (15 min at 37 °C) with the biotin-labeled HB-19 (5 µM) in the presence of EDTA (5 mM) in order
to block the cleavage of the cell-surface-expressed nucleolin (as shown
in Fig. 3). The sample 0 represents control HeLa cells that have not
been preincubated with HIV. Cells were washed before preparation of
nucleus-free extracts for the recovery of the cell-surface nucleolin
complexed with HB-19 and immunoblotting as described in Fig. 3.
Material extracted from 1 × 107 cells was analyzed in
each lane. A section of the immunoblots at the position of the 95-kDa
nucleolin is presented. The histograms give the quantification of the
intensity of the nucleolin bands in arbitrary units (A.U.)
by quantification using the NIH Image Software.
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The binding of the FITC-labeled HB-19 to the surface of HeLa cells was
shown to be specific, since it was prevented by unlabeled HB-19 (Fig.
6, A and C). HIV-1 Lai particles could also
interfere with the binding of the FITC-labeled pseudopeptide to cells
(Fig. 6A), thus suggesting the HB-19 and HIV-1 Lai interact
with common site(s) on the cell surface. In contrast, the affinity of
mAb 12G5 specific for CXCR4 was not affected by HIV-1 Lai attachment to
cells (Fig. 6B). Because chemokine receptors are implicated in the membrane fusion step, they probably become operational after
attachment of HIV particles to target cells (7, 8, 42, 43). In these
competition experiments, a suitable control was provided by the HIV-1
Ada isolate since this virus is resistant to the inhibitory action of
HB-19, both on virus entry and attachment to cells (Table I).
Accordingly, HIV-1 Ada particles exerted no significant effect on the
binding of HB-19 (Fig. 6C).
The implication of nucleolin in the mechanism of HIV attachment to
CD4+ cells was further demonstrated by the capacity of
HIV-1 Lai but not HIV-1 Ada particles to inhibit in a
dose-dependent manner the complexing of HB-19 to the
cell-surface-expressed nucleolin. Moreover, there was even a slight
increase of HB-19 binding to cell-surface-expressed nucleolin in the
presence of HIV-1 Ada (Fig. 7). It is of interest to note that the
CXCR4-specific anti-HIV peptide TW70 which blocks T-tropic HIV-1 entry
(30, 31) did not affect the attachment of HIV-1 Lai particles to cells
(see below) nor the binding of HB-19 to cell-surface-expressed
nucleolin (not shown).
Inhibition of HIV Entry and Attachment by FGF-2--
Heparan
sulfates present on the cell surface have been shown to be implicated
in the attachment process of HIV particles (10, 18, 19). In order to
define the contribution of cell-surface-expressed nucleolin in respect
to heparan sulfates, we investigated the anti-HIV effect of FGF-2 that
uses heparan sulfates as low affinity receptors (22, 23). The
125I-labeled FGF-2 was shown to bind HeLa P4 cells in a
dose-dependent manner (Fig.
8) but failed to achieve saturation
consistent with the binding of FGF-2 to its low affinity receptor (44).
Indeed, most of the cell-bound 125I-FGF-2 was washed away
by 2 M NaCl treatment (Fig. 8) which disrupts heparan
sulfate but not the high affinity receptor-bound FGF-2 (44). The 2 M NaCl-resistant 125I-FGF-2 binding to cells
did not reach a saturation with the increasing dose of FGF-2, thus
suggesting that it was mainly due to background nonspecific binding
(Fig. 8). HeLa cells therefore, appear to express very low levels, if
any, of the high affinity FGF receptors, and several attempts to
chemically cross-link the 125I-FGF-2 to cell-surface
proteins failed (not shown). Scatchard analysis of the 2 M
NaCl-sensitive binding confirmed that the interaction of FGF-2 with
HeLa cells is of low affinity type with a calculated
Kd of 110 nM. This value is about
1000-fold lower than the Kd reported for the high
affinity binding to the FGF-2 receptor (44). Interestingly, the binding
of 125I-FGF-2 was not affected when cells were preincubated
with HB-19 (Fig. 8), thus pointing out that the cell-surface target of
HB-19 is different from that of FGF-2. Similarly, FGF-2 had no apparent effect on the binding of the FITC-labeled HB-19 revealed by FACS analysis or on the capacity of the biotin-labeled HB-19 to bind the
cell-surface-expressed nucleolin (not shown, similar to the experiments
demonstrated in Figs. 6 and 7). Moreover, in ligand blotting type
experiments, the specific binding of HB-19 to the nucleolin band (16,
28) was not affected by the presence of FGF-2 (not shown).
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Fig. 8.
Association of 125I-labeled FGF-2
with HeLa P4 cells. For the control, cells were incubated in the
culture medium containing different concentrations of the
125I-labeled FGF-2 (abscissa). Cells were then
washed with PBS, and nuclear-free extracts were prepared in order to
measure the amount of the radioactivity associated with cells (in
arbitrary units, A.U., ordinate). For NaCl, as
for the control but after washing with PBS, cells were washed twice
with 2 M NaCl, 10 mM Tris, pH 7.4, to remove
FGF bound to glycosaminoglycans (37). For HB-19, the cells were first
preincubated (37 °C, 20 min) with 25 µM HB-19 before
further incubation with 30, 60, and 120 nM
125I-FGF-2 as the control. Each point represents the mean
of duplicate samples.
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The effect of FGF-2 on the entry of T-tropic HIV-1 Lai and M-tropic
HIV-1 Ba-L was monitored by the -galactosidase activity in HeLa P4
and P4-C5 cells (see "Experimental Procedures"). The value of
-galactosidase activity obtained in the presence of AZT was referred
to as the background value in each experiment. In the presence of
inhibitors of HIV entry, such as the mAb CB-T4 that reacts with the
gp120-binding site in CD4 (11) and HB-19 which binds
cell-surface-expressed nucleolin (Figs. 2-7), the -galactosidase activity values were comparable to the background value observed in the
presence of AZT in both of HIV-1 Lai and Ba-L samples (Fig. 9). On the other hand, the CXCR4-specific
peptide TW70 (30, 31) inhibited entry of HIV-1 Lai but not that of
HIV-1 Ba-L (Fig. 9). This latter result is in accord with the action of
TW70 on the CXCR4- but not CCR5-mediated membrane fusion during the HIV
entry process (31). In this well defined experimental model, FGF-2
blocked both the HIV-1 Lai and Ba-L entry in a
dose-dependent manner, with a 50% inhibitory concentration
(IC50) value of 20-25 nM (Fig. 9). At 120 nM FGF-2, the inhibitory effect was comparable to that
observed in the presence of HB-19. Interestingly, the degree of
inhibition of HIV-1 Lai entry in the two cell types (HeLa P4 and P4-C5)
was similar at the different concentrations of FGF-2, thus indicating
that expression of CCR5 does not modify its inhibitory efficacy.
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Fig. 9.
The inhibition of T- and M-tropic HIV-1 entry
into HeLa cells by FGF-2. Two different cell clones, HeLa P4 and
HeLa P4-C5 cells (as indicated), were treated (incubation at 37 °C,
30 min) with AZT (5 µM), anti-CD4 mAb CB-T4 (-CD4; 5 µg/ml), the nucleolin-binding HB-19 (0.5 µM), the
control peptide CP-1 (500 µM), CXCR4-binding peptide TW70
(0.2 µM), and different concentrations of FGF-2 (30, 60, and 120 nM) before infection with the T-tropic HIV-1 Lai
and the M-tropic HIV-1 Ba-L isolate or the HIV-1 pseudotyped with the
VSV envelope glycoproteins (VSV/HIV-1). At 48 h post-infection,
the -galactosidase activity was measured in cell extracts directly
in order to monitor HIV entry. The mean ± S.D. of triplicate
samples is shown.
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In order to study the specificity of FGF-2 with respect to the HIV
envelope glycoprotein-mediated viral-entry process, we investigated its
inhibitory effect on infection by an HIV-1 pseudotyped with envelope
glycoproteins of vesicular stomatitis virus (VSV, Fig. 9).
Infection of HeLa cells by this pseudotyped HIV was inhibited by AZT
but not by the anti-CD4 mAb CB-T4, as the viral binding and entry in
this case is mediated by the VSV envelope proteins. Consistent with our
previous reports (16) and Footnote 2, HB-19 exerted no significant
effect on the infection with HIV-VSV pseudotyped virus, thus confirming
that its inhibitory effect is specific and concerns only virus
particles presenting the HIV envelope glycoproteins. In contrast to the
specific effect of HB-19, FGF-2 inhibited entry of the HIV-VSV
pseudotyped virus in a dose-dependent manner, thus
indicating that its effect on HIV is not specific. Indeed, FGF-2 has
been reported to inhibit infection by herpes simplex virus (45),
whereas cell-surface heparan sulfates have been implicated in the
infection of different types of viruses, such as herpes,
adeno-associated, vaccinia, dengue, and Sindbis virus (38, 46-50).
We next investigated the effect of FGF-2 on the amount of HIV-1 Lai
particles attached to cells (i.e. associated with cells) after 1 h of incubation at 4 °C (Fig.
10). A significant amount of HIV
particles was associated with cells, and most of it was found to be on
the cell surface as demonstrated by its trypsin sensitivity. Indeed,
treatment of HIV-incubated cells with trypsin resulted in more than
90% reduction of the amount of HIV particles associated with cells.
Although entry of HIV-1 Lai was inhibited by the neutralizing mAb
against CD4, virus attachment was only slightly affected in accord with
a recent report indicating that attachment of HIV to HeLa cells is
mostly CD4-independent (10). The neutralizing mAb against the V3 loop
of HIV-1 Lai and HB-19 inhibited significantly the attachment of this
virus in accord with our previous results (11, 28). The CXCR4-specific
TW70 peptide had no significant effect on HIV attachment (Fig. 10), although HIV entry was inhibited (Fig. 9). On the other hand, FGF-2
resulted in a dose-dependent inhibition of HIV particle attachment to cells (Fig. 10), and the degree of inhibition at the
different concentrations of FGF-2 was correlated to that of the
inhibition of HIV entry.
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Fig. 10.
FGF-2 inhibits the attachment of HIV-1 to
permissive cells. HeLa P4 cells were treated (37 °C, 30 min)
with -CD4 mAb CB-T4 (at 5 µg/ml), HB-19 (2 µM), the
control tripeptide monomer CP-1 (50 µM), the anti-CXCR4
peptide TW70 (0.2 µM), and different concentrations of
FGF-2 (30, 60, and 120 nM) before washing cells (with PBS)
to eliminate unbound inhibitors. To demonstrate the effect of the
neutralizing anti-V3 loop antibody, the HIV-1 Lai preparation was
incubated (37 °C, 30 min) with mAb N11/20 (-V3 mAb at 5 µg/ml),
before addition to cells at 4 °C as the other samples (11). Cells
were incubated at 4 °C with HIV-1 Lai for 1 h. Cells were then
washed extensively with culture medium containing 10% fetal calf serum
to eliminate free unbound HIV particles, and the amount of p24
associated with cells was measured as an estimate for the amount of HIV
binding ("Experimental Procedures"). In order to demonstrate that
most of the HIV associated with cells represented virus particles bound
on the surface of cells, a sample of cells incubated with virus were
washed with PBS before treatment with trypsin to eliminate virus bound
on the cell surface (sample trypsin). The mean ± S.D. of
triplicate samples is shown.
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DISCUSSION |
The results presented here further demonstrate that the HB-19
pseudopeptide is a potent and a specific inhibitor of HIV infection. At
concentrations that block HIV attachment to cells, HB-19 binds cells
and forms an irreversible complex with the cell-surface-expressed nucleolin (Fig. 2). Consistent with this, HIV particles can prevent the
capacity of HB-19 to bind cells and form a stable complex with the
cell-surface-expressed nucleolin (Figs. 6 and 7), thus pointing out
that nucleolin is implicated in the HIV attachment process. Nucleolin
is one of the major RNA-binding proteins of the nucleolus which has
been suggested to shuttle between the nucleus and cytoplasm (51, 52).
Although its localization has been emphasized to the nucleoli,
nucleolin has been reported to be also expressed on the cell surface
and serve as a binding protein to different ligands (Refs. 25, 40, and
41 and references therein). Here we have confirmed that nucleolin is
expressed on the cell surface, by demonstrating that incubation of
intact cells with a specific antibody results in the clustering of the
cell-surface-expressed nucleolin (Figs. 4). The role of nucleolin in
the HIV attachment and entry processes is under investigation. As
nucleolin is characterized as a shuttle protein (51, 52), it might be
plausible that interaction of HIV particles with the
cell-surface-expressed nucleolin might be functional for chaperoning
the viral entry process.
Other groups by enzymatic digestion of cell-surface heparan sulfates
have suggested the implication of proteoglycans in the HIV attachment
process (10, 18, 19). In order to confirm this under physiological
conditions, we used the growth factor FGF-2 which is a physiological
ligand of heparan sulfates. Indeed, fibroblast growth factors have been
shown to exert their effects on target cells by using a dual receptor
system, heparan sulfate proteoglycans as low affinity receptors and a
family of FGF receptors as the signal-transducing high affinity
receptors (22, 23). The binding of FGF with heparan sulfates appears to
induce a conformational change and/or formation of the FGF dimer
required for interaction with FGF receptors (44). FGF bound to its high
affinity receptor is stable and persists after washing cells with 2 M NaCl. On the other hand, the binding of FGF to heparan
sulfates is disrupted by 2 M NaCl. Consequently, the amount
of cell-associated FGF, sensitive and resistant to 2 M
NaCl, is considered as binding to heparan sulfate and the FGF
receptors, respectively (37). By such a procedure, we demonstrated here
that association of FGF-2 with HeLa P4 cells is mainly due to binding
to the low affinity receptors, i.e. the
cell-surface-expressed heparan sulfates (Fig. 8). Consistent with
previous data on the implication of surface proteoglycans in the HIV
attachment process (10, 18, 19), FGF-2 was demonstrated to be a potent
inhibitor of HIV attachment and entry in the HeLa cell model studied
here (Figs. 9 and 10). These results and the fact that FGF-2 does not
compete with HB-19 indicate that FGF-2 and HB-19 have a distinct mode
of action in the process of HIV particle attachment to permissive
cells. Furthermore, these observations rule out the potential
interaction of HB-19 with cell-surface heparan sulfate proteoglycans.
By structure and inhibitory activity relationship studies using analogs
of HB-19, previously we had demonstrated that the positively charged
side chains of the two basic residues in the tripeptide moiety of HB-19
are essential for the inhibitory structure (20). In addition to this
positive charge, the pentavalent presentation of the tripeptide moiety
is a complementary determining factor for the anti-HIV activity of
HB-19, since the tripeptide moiety alone is not active (Table I),
whereas the tetravalent presentation of the tripeptide moiety generates
a product with reduced inhibitory activity (20). HB-19 has several
biochemical properties that are also manifested by a synthetic V3 loop
peptide (16, 20, 28). Indeed, both HB-19 and the V3 loop peptide
compete together to bind cells and form a complex with the
cell-surface-expressed nucleolin. This interaction is direct as
demonstrated by the capacity of HB-19 and the V3 loop peptide to bind
nucleolin in ligand blot type experiments and also to purify nucleolin
by affinity chromatography using cell extracts. It should be noted that
in our direct binding assays, HB-19 and the synthetic V3 loop peptide
do not bind CD4 or CXCR4. With partially purified preparations of
nucleolin, we could demonstrate that different preparations of
recombinant gp120 bind nucleolin with a high affinity comparable to the
binding of gp120 to soluble CD4. Such binding is inhibited by HB-19 or by monoclonal antibodies against the V3 loop but not against the CD4-binding domain in gp120. In view of these observations, we suggested that gp120 could bind nucleolin via its V3 loop domain (16).
The equilibrium affinity constant Ka value for the
binding of a synthetic V3 loop peptide to the nucleolin preparation is
5.1 × 106 M1, whereas that
of soluble gp120 is 1.1 × 109
M1 (16). Interestingly, the
Ka value for the binding of HB-19 to the nucleolin
preparation is 9.6 × 109 M1
(16). This high affinity binding of HB-19 to nucleolin is most probably
due to the presence of several stretches of amino acids composed of
aspartate and glutamate residues at the amino terminus of nucleolin
(53). Such polyanionic domains in nucleolin should provide a well
defined structure to account for its specific binding to the HB-19
construct but not to the K(CH2N)PR tripeptide moiety (Fig. 2A). Consequently, the polyanionic domains in
nucleolin might be responsible for the interaction of gp120, especially through the basic residues in the V3 loop, and thus provide a potential
receptor for the V3 loop to mediate virus attachment. Our hypothesis is
that by virtue of binding the V3 loop domain, nucleolin could interact
with gp120 on the surface of HIV particles and thus become implicated
in the mechanism of HIV attachment to CD4+ cells.
Therefore, agents such as mAb anti-V3 loop or HB-19 which interfere in
the interaction of the V3 with the cell-surface-expressed nucleolin
block HIV attachment and thus entry.
The V3 loop is relatively exposed on HIV particles (15, 54). It
contains several conserved positively charged lysine and arginine
residues that account for its net positive charge (55). Consequently,
polyanionic molecules that interact with the V3 loop inhibit attachment
of HIV particles to cells (56, 57) as is the case with neutralizing
monoclonal antibodies specific to the V3 loop (11, 58). In view of this
and the ability of chemokines (3, 8, 9) or compounds that interact
specifically with chemokine receptors (42, 43) (Figs. 9 and 10) to
block HIV entry without affecting HIV attachment, it might be plausible to suggest that the V3 loop by virtue of its positively charged residues could interact directly with negatively charged components of
the cell surface independent of CD4 and chemokine receptors. Potential
candidates of such cell-surface components are on the one hand heparan
sulfate proteoglycans that are commonly found on the surface of most
vertebrate cell types (10, 17-19) and on the other hand V3 loop
binding proteins such as nucleolin (16, 53). Specificity in the case of
HIV attachment could be mediated in part by nucleolin and CD4, since
heparan sulfates have been shown to serve as low affinity receptors for
different viruses and ligands such as the fibroblast growth factors and
chemokines (22, 23, 38, 46-50, 59). Heparan sulfates by providing negatively charged molecules could interact nonspecifically with the V3
loop (18, 19, 56, 57), whereas nucleolin by virtue of the defined
structure of its acidic amino acid domains could interact specifically
with the V3 loop (16, 53). In accord with this, we show that FGF-2
blocks entry of HIV-1 pseudotyped with the envelope glycoproteins of
VSV, whereas HB-19 has no effect on such a pseudotyped virus (Fig. 9).
However, it should be noted that there should be cooperativity between
heparan sulfates and nucleolin, since HIV attachment could be inhibited
by acting independently either on heparan sulfates using FGF-2 or on
nucleolin using HB-19. The heparan sulfates may be necessary for the
concentration of HIV particles on permissive cells to allow efficient
subsequent interaction with nucleolin and CD4. Indeed, interaction of
ligands with cell-surface heparan sulfates have often been shown to
facilitate a secondary interaction with specific high affinity
receptors (22, 23, 59, 60).
The wide spectrum of the specific inhibitory action of HB-19 on
different types of HIV isolates, along with its distinct mode of action
and stability in serum (16, 20, 21, 28) (results herein),2
make this pseudopeptide inhibitor a potential drug candidate for
HIV-infected individuals.
| |
ACKNOWLEDGEMENTS |
We thank Josette Svab, Nadine Robert,
Elizabeth Dam, and Solen Loaec for excellent technical assistance.
| |
FOOTNOTES |
*
This work was supported in part by grants from Institut
Pasteur, Paris, CNRS, and Ensemble Contre le Sida, SIDACTION.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.
§
Supported by a postdoctoral fellowship grant from Agence Nationale
de la Recherche sur le SIDA.
To whom correspondence should be addressed: Unité de
Virologie et Immunologie Cellulaire, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-4568-8776; Fax: 33-1-4061-3012; E-mail: arahovan@pasteur.fr.
2
S. Nisole, B. Krust, E. Dam, N. Seddiki, C. Callebaut, G. Guichard, S. Muller, J. P. Briand, and A. G. Hovanessian,
submitted for publication.
3
F. Puvion-Dutilleul, unpublished results.
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ABBREVIATIONS |
The abbreviations used are:
gp120, the external
envelope glycoprotein of HIV-1;
HIV, human immunodeficiency virus;
M-tropic, macrophage tropic;
T-tropic, T-lymphocyte tropic;
TASP, template-assembled synthetic peptide;
HB-19, 5[K(CH2N)PR]-TASP;
CP-1, K(CH2N)PR;
CP-40, 5[QPQ]-TASP;
CP-51, 5[R(CH2N)PN]-TASP;
TW70, CXCR4 specific anti-HIV peptide;
FGF-2, fibroblast growth factor 2;
V3
loop, the hypervariable region of about 36 amino acids in gp120;
mAb, monoclonal antibody;
PAGE, polyacrylamide gel electrophoresis;
FACS, fluorescence-activated cell sorting;
AZT, azidothymidine;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
LTR, long
terminal repeat;
PFA, paraformaldehyde;
VSV, vesicular stomatitis
virus.
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ABSTRACT
INTRODUCTION
