(Received for publication, August 8, 1996, and in revised form, November 14, 1996)
From the The template assembled synthetic peptide
constructs (TASP), pentavalently presenting the tripeptide KPR or RPK,
are potent and specific inhibitors of human immunodeficiency virus
(HIV) infection by preventing viral entry into permissive cells. Here the 5[K HIV1 is an enveloped virus that
infects target cells by the fusion of viral and cellular membranes.
This fusion is initiated by the binding of HIV external and
transmembrane envelope glycoprotein complex to the CD4 receptor and
also is dependent on the presence of species-specific cofactors on the
cell surface (for a review, see Ref. 1). The external envelope
glycoprotein contains the binding site for the CD4 receptor and a
hypervariable region of about 36 amino acids referred to as the V3 loop
(2). The transmembrane glycoprotein contains a potential fusion peptide
at its amino terminus, which is implicated in the membrane fusion
process (3). The external and transmembrane glycoproteins (gp120/gp41
for HIV-1) are associated in a noncovalent manner to generate a
functional complex. The gp120·gp41 complex is essential for binding
of HIV particles to the cell membrane, whereas in HIV-infected cells, this complex expressed on the cell surface is responsible for the
initiation of cell to cell membrane fusion, leading to the formation of
syncytia. The cell surface-expressed gp120·gp41 complex is also
responsible for the initiation of cell death by apoptosis in
HIV-infected cell cultures (4). The V3 loop plays a critical role in
these well-defined functions of the gp120·gp41 complex (1, 2, 5), and
it has been proposed that it might be implicated in post-CD4 binding
events by interacting with specific cell surface proteins (2).
The V3 loop of different HIV-1 and HIV-2 isolates contains a highly
conserved RP dipeptide motif at its NH2-terminal end (6). In view of this, we have designed and synthesized a construction referred to as template assembled synthetic peptide (TASP) in which
templates such as KKKGPKEKGC and KKKKGC were employed to covalently
anchor arrays of tripeptides (RPR, RPK, or KPR) at the CEM cells (clone 13) derived from
human lymphoid cell line CEM and MOLT4-T4 clone 8 cells selected for
high level of CD4 expression (both cell lines were provided by L. Montagnier, Institut Pasteur, France) were cultured in suspension RPMI
1640 medium (BioWhittaker, Verviers, Belgium). Human HeLa cells were
grown in monolayer cultures in Dulbecco's medium. Human peripheral
blood mononuclear cells (PBMC) from a healthy donor were stimulated by
phytohemagglutinin (PHA) and cultured in RPMI 1640 medium containing
10% (v/v) T cell growth factor (Biotest) (7). All cells were cultured
with 10% (v/v) heat-inactivated (56 °C for 30 min) fetal calf
serum.
Infection of CEM cells with the HIV-1 Lai isolate was carried out as
described previously (7). For the assay of the inhibitory effect of the
TASP constructs, CEM cells were first incubated (15 min, at room
temperature) in the presence of different concentrations of each
construct before infection using one synchronous dose of HIV-1 Lai as
described before (8). HIV production was estimated at 4 days
post-infection (p.i.) by monitoring the HIV-1 major core protein p24 in
the culture supernatant (7). The concentration of p24 was measured by
p24 Core Profile ELISA (Du Pont).
Buffer E contained 20 mM Tris-HCl, pH
7.6, 150 mM NaCl, 5 mM MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM
Cells were first washed
extensively in PBS before lysis in buffer E (100 µl/3 × 107 cells), and the nuclei were pelleted by centrifugation
(1,000 × g for 5 min). The nuclei-free supernatant was
then further centrifuged at 12,000 × g for 10 min, and
the supernatant was stored at CEM cells (108
cells) were washed with PBS (2 × 25 ml), and the pellet was
suspended in 20 ml of PBS containing 10 mM
D-glucose and 2 mCi of 125I (100 µCi/ml;
Amersham), 2 units of lactoperoxidase, and 2 units of glucose oxidase
(Calbiochem-Behring). After 10 min of incubation at 22 °C, cells
were washed in PBS, and extracts were prepared as above.
Synthesis of the different
TASP constructs and the measurement of their inhibitory activity on HIV
infection were as described previously (7). The following TASP
constructs were included in this report: 1) control peptides which
manifest no or little activity against HIV infection, such as
5[QPQ]-, 5[KGQ]-, and 5[KER]-TASP; and 2) peptides which are
potent inhibitors of HIV entry and infection, such as 5[KPR]- and
5[K Crude cell extracts were diluted in 2-fold
concentrated electrophoresis sample buffer and analyzed by
polyacrylamide gel electrophoresis (PAGE) in SDS to be
electrophoretically transferred to 0.22 µm polyvinylidene difluoride
sheets (Bio-Rad). The electophoretic blots were saturated with
casein-based blocking buffer (GENOSYS) overnight at 4 °C. In order
to further saturate nonspecific binding sites, the blots were first
incubated at room temperature in blocking buffer containing 5 µM 5[KER]-TASP. After 2 h, the biotin-labeled 5[K Twenty-four
hours after passaging, CEM cells were washed extensively with PBS
before incubation (50 × 106 cells/300 µl of FACS
buffer) at 4 °C for 30 min with different concentrations of the
biotin-labeled TASP molecule. Cells were then washed in FACS buffer
(2 × 15 ml), and nuclear-free cell extracts were prepared using
buffer E (150 µl). Such extracts were first diluted in PBS (600 µl)
before the addition of 100 µl of avidin-agarose (ImmunoPure
immobilized avidin from Pierce) to capture the biotin-labeled TASP
complexed to its cell surface ligand. These suspensions were incubated
at 4 °C for 2 h, and the avidin-agarose bound proteins were
washed batchwise with PBS (5 × 5 ml). Finally, the avidin-agarose
pellet was resuspended in 100 µl of 2-fold concentrated
electrophoresis sample buffer and heated at 95 °C for 5 min. The
eluted proteins were analyzed by SDS-PAGE, and the TASP ligand was
revealed by ligand blotting.
5[K Phycoerythrin
(PE)-labeled mAb Ta1 (IgG1; from Coulter, Miami, FL) was used to detect
CD26 (9). Two different FITC-labeled mAbs specific to the CD4 receptor
were used, mAbs OKT4 and OKT4A (both IgG1; Ortho Diagnostics Systems,
Raritan, NJ). In all experiments, PE-labeled mAb B4 (IgG1), specific
for CD19 (Coulter), was used as a control for PE-labeled mAb Ta1, and
FITC-labeled mouse isotype control antibody MCG1 (IgG1; Immuno Quality
Products) was used as a control for FITC-labeled mAbs OKT4 and OKT4A.
Cells were incubated with FITC- or PE-labeled mAbs in the FACS buffer
at 4 °C for 30 min. The cells were then washed twice with PBS and fixed in 1% formaldehyde in PBS and applied to an FACS scan flow cytometer (Beckton Dickinson, Mountain View, CA ). For each sample, 10,000 cells were analyzed with Lysis II Software (Becton
Dickinson).
In order to assay for the binding of FITC- or biotin-labeled TASP
inhibitors to a cell surface antigen, different cells were washed in
PBS, suspended in FACS buffer (5 × 105 cells/100
µl) containing 0.5 µM FITC-labeled or different
concentrations of the biotin-labeled TASP constructs, and incubated at
4 °C for 30 min. The cells were then washed twice with FACS buffer
and fixed in 1% formaldehyde in FACS buffer. The FITC-labeled TASP constructs were analyzed as above, whereas the biotin-labeled TASP
constructs were revealed by using streptavidin·FITC complex (Amersham). The fluorescence intensity was monitored by FACS analysis using Lysis II Software (see Figs. 3 and 4 and Tables 1 and 2) or Cell
QuestTM Software (Becton Dickinson and Macintosh Computer)
(see Fig. 5).
Protease treatment of CEM and
MOLT cells was essentially as described previously (10) with slight
modifications. Briefly, cells were washed in PBS and in RPMI 1640 medium containing 1 mM EDTA before treatment with trypsin
(Sigma; 2.5 mg/ml at 20 °C for 5 min), proteinase K
(Boehringer Mannheim GmbH, Germany; 0.2 mg/ml at 37 °C for 30 min),
or Pronase E (Sigma, 0.1 mg/ml at 37 °C for 45 min). The reactions were stopped by 10-fold dilutions in RPMI 1640 containing 10% fetal calf serum. Cells were then washed in PBS and
FACS buffer and processed for FACS analysis.
A Superose 6 column (1.6 × 50 cm) from Pharmacia Biotech Inc. was equilibrated in buffer GF as
described before (11). The bed volume was 100 ml. The column was
calibrated using extracts (prepared in Buffer E) supplemented with the
molecular mass markers catalase, 202 kDa, and bovine serum albumin, 68 kDa. Elution was performed in buffer GF by collecting 1 ml fractions/2
min, with the void volume (V0) and total column
elution volume (Vc) at 36 and 114 ml, respectively.
Aliquots from each fraction were analyzed by ligand blotting using
biotin-labeled 5[K CEM cells (300 × 106) were washed in PBS before homogenization to prepare
plasma membranes, as described before (11). The presence of TASP ligand
p95 was revealed by ligand blotting using aliquots corresponding to
material from 108 cells.
Recombinant
gp120 (Neosystem) was radioiodinated with the Bolton-Hunter reagent
(DuPont NEN) according to the technique described by the manufacturer.
To study the binding of gp120 to the CD4 receptor, CEM cells (5 × 106), which express high levels of CD4, were incubated in
the culture medium with 125I-labeled gp120 (50 ng, 10 Ci/mg) at 37 °C for 1 h. Cells were then washed twice in PBS (5 ml), and cytoplasmic extracts were prepared by disruption of cell
pellets in buffer E (125 µl). Aliquots (25 µl, corresponding to
material from 106 cells) were diluted in two-fold
concentrated electrophoresis buffer and were analyzed by SDS-PAGE. The
binding of 125I-labeled gp120 to the CD4 receptor was then
revealed by autoradiography (12). The 125I-labeled gp120
band was also quantitated in a PhosphorImager (Molecular Dynamics).
CEM cells (5 × 106) in culture medium (1 ml) were preincubated (at
37 °C for 15 min) in the absence or presence of
5[K We have recently reported that
5[K
In order to further investigate the mechanism of inhibition of HIV
entry, we studied the effect of 5[K
The discrepancy between the binding results of soluble gp120 and viral
particles to CEM cells (Fig. 1) indicates that gp120 complexed to gp41
on the surface of viral particles does not have the same conformational
restrictions as the soluble gp120. Thus, experiments using soluble
gp120 should be interpreted cautiously.
By FACS analysis, we show that the FITC-labeled
5[K In an attempt to determine the proteinaceous nature of the cell surface
component to which peptide-TASP inhibitors bind, cell surface labeling
of MOLT4 cells with FITC-labeled 5[K
The FITC-labeled 5[K Unité de Virologie et Immunologie
Cellulaire,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
(CH2N)PR]-TASP construct,
(CH2N)
for reduced peptide bond, was used in studies to demonstrate its
specific binding to a 95-kDa cell surface protein ligand. Compared to
its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide
5[K
(CH2N)PR]-TASP manifested higher affinity to bind
to its cell surface ligand, increased activity to inhibit HIV
infection, and resistance to degradation when incubated in serum from
an HIV-1 seropositive individual. In ligand blotting experiments, the
biotin-labeled 5[K
(CH2N)PR]-TASP identified a single
95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is
expressed on the cell surface since surface iodination of cells
resulted in its labeling, and moreover, following incubation
of cells with the biotin-labeled 5[K
(CH2N)PR]-TASP, the p95·TASP complex
was recovered by affinity chromatography using avidin-agarose. All
anti-HIV TASP constructs but not their control derivatives affected the
binding of biotin-labeled 5[K
(CH2N)PR]-TASP to p95,
thus emphasizing the specific nature of this binding. Since
5[K
(CH2N)PR]-TASP does not interact with HIV-envelope
glycoproteins, our results suggest that TASP inhibitors mediate
directly or indirectly a block in HIV-mediated membrane fusion process
by binding to the cell surface expressed p95.
-amino
group of the lysine residues in the templates (7). The pentavalent
presentation, 5(RPR)-, 5(RPK)-, or 5[KPR]-TASP molecules, manifested
maximum inhibitory activity on infection of cells by HIV-1 and -2 but
not by simian immunodeficiency virus isolates, thus emphasizing the
specific nature of these TASP inhibitors. In already infected cells,
the TASP inhibitors also blocked syncytium formation and the occurrence
of apoptosis (7). These observations indicated that these TASP
inhibitors block a defined target that should be implicated in the
functioning of the gp120·gp41 complex to initiate viral entry,
syncytium formation, and apoptosis. Structure and inhibitory activity
relationship studies using analogs of 5[KPR]-TASP indicated that the
two basic residues Lys and Arg in the tripeptide are essential and can
be replaced by each other and that their positively charged side chains
play a critical role in the inhibitory structure. Interestingly,
replacement of Leu amino acid residues by Asp amino acids or the
reduction of the peptide bond between the first two amino acids of the
tripeptide generated pseudopeptide-TASP analogs active at
submicromolar concentrations on HIV infection (7). By the use of FITC-
and biotin-labeled 5[K
(CH2N)PR]-TASP constructs, we
demonstrate here that the TASP inhibitors bind specifically to a
95-kDa, cell surface protein (p95). The identity of p95 remains to be
elucidated. However, because the TASP inhibitors block both HIV
envelope-mediated virus to cell and cell to cell membrane fusion
processes and the initiation of apoptosis (7), it can be suggested that
p95 is implicated as a potential cofactor of CD4 in the functioning of
the gp120·gp41 complex to initiate membrane fusion and thus viral
entry. Consistent with this, 5[K
(CH2N)PR]-TASP blocks
the binding of HIV particles to CD4+ permissive cells at
the same efficiency as an anti-CD4 monoclonal antibody.
Cells and HIV Infection
-mercaptoethanol, aprotinin (1000 units/ml), and 0.5% Triton X-100.
Tris-buffered saline contained 25 mM Tris-HCl, pH 7.0, 137 mM NaCl, and 3 mM KCl. Fluorescence-activated
cell sorter (FACS) buffer contained 1% bovine serum albumin and 0.01%
sodium azide in phosphate-buffered saline (PBS). Two-fold concentrated
electrophoresis sample buffer contained 125 mM Tris-HCl, pH
6.8, 2 M urea, 1% SDS, 0.1% bromphenol blue, 150 mM
-mercaptoethanol, and 20% glycerol (v/v). Buffer GF
contained 20 mM Tris-HCl, pH 7.6, 50 mM NaCl,
and 0.1% Triton X-100.
80 °C. Routinely, cell extracts were
prepared 24 h after cell passage.
(CH2N)PR]-TASP. For the preparation of the
biotin-labeled constructs, biotin was incorporated at the beginning of
the synthesis by coupling the Fmoc
(N-(9-fluorenyl)methoxycarbonyl)-Lys(Biotin)-OH derivative (Neosystem, Strasbourg, France) on the resin prior to the
assembly of the template. Thus the biotinylated TASP constructs were
labeled at the COOH terminus of their templates.
(CH2N)PR]-TASP (5 µM) was added to
this solution, and the blots were incubated for another 2 h at
4 °C. The sheets were subsequently washed 3 times (10 min each) in
Tris-buffered saline containing 0.05% (v/v) Tween 20, followed by 2 washes (10 min each) in Tris-buffered saline before revealing biotin by
using streptavidin-horseradish peroxidase complex and light-based
enhanced chemiluminescence reagents as provided by the manufacturer
(Amersham). The enhanced light signal produced was then captured on the
autoradiography film (HyperfilmTM-MP from Amersham
Corp.).
(CH2N)PR]-TASP was labeled with
fluorescein isothiocyanate (FITC; Sigma) by incubating
stoichiometric concentrations (2.5 mM) of each product in
50 mM NaHCO3, pH 9.5, at 20 °C for 2 h (in the dark). This solution (400 µl) was then transferred to a
Microcon Model 3 filter sieve (Amicon, Inc., MA) with a molecular mass
cut-off of 3,000 Da and centrifuged at 12,000 × g for
30 min to filter unbound FITC. The concentrated material was diluted 20-fold in distilled water and purified again using the Microcon filter.
Fig. 3.
The peptide-TASP inhibitor binds to a cell
surface protein resistant to trypsin but sensitive to proteinase K
digestion. MOLT4 cells were treated as described under
"Materials and Methods" with trypsin (TRYP.) (2.5 mg/ml,
5 min at 20 °C) or protease K (PROT. K) (0.2 mg/ml, 30 min at 37 °C) before FACS analysis using the FITC-labeled
5[K(CH2N)PR]-TASP (p19*) and monoclonal
antibodies specific for cell surface proteins, mAb Ta1 against CD26 and
mAbs OKT4 and OKT4A against CD4. The peak C in each section
represents the corresponding control peak obtained by PE-labeled
control mAb B4 (specific to CD19) for mAb Ta1, FITC-labeled MCG1
control antibody for mAbs OKT4 and OKT4A, and 0.5 µM
unlabeled 5[K
(CH2N)PR]-TASP for p19*.
[View Larger Version of this Image (37K GIF file)]
Fig. 4.
The high affinity of
5[K(CH2N)PR]-TASP to bind its cell surface
ligand. CEM cells were analyzed by FACS analysis using biotin-labeled 5[KPR]-TASP (at 1, 5, and 10 µM),
5[K
(CH2N)PR]-TASP (at 0.25, 0.5, 1, and 5 µM), and control 5[QPQ]- and 5[KGQ]-TASP (at 20 µM) as described under "Materials and Methods." The
peak C gives the fluorescence of cells incubated with the
unlabeled respective TASP constructs (20 µM). The
ordinates give the relative cell number, whereas the
abscissa give the relative fluorescence intensity.
[View Larger Version of this Image (23K GIF file)]
Fig. 5.
The specific binding of
5[K(CH2N)PR]-TASP to a 95-kDa protein. Crude CEM
cell extracts (material corresponding to 106 cells) were
analyzed by ligand blotting using biotin-labeled 5[K
(CH2N)PR]-TASP ("Materials and Methods"). To
show the specificity of binding, the electrophoretic blots, after
saturation with the blocking buffer, were first incubated (4 °C, 30 min) with 50 µM unlabeled 5[KGQ]-TASP (A) or
5[K
(CH2N)PR]-TASP (B) before the addition
of biotin-labeled 5[K
(CH2N)PR]-TASP (5 µM). The numbers in the middle (200, 97, 68, and 43) show the position of molecular mass (in kDa) protein markers.
Note that ligand binding studies shown here and in Figs. 6 and 7 were
performed on reducing gels. It should also be noted, however, that
similar results were obtained on nonreducing gels, i.e. in
the absence of
-mercaptoethanol.
[View Larger Version of this Image (36K GIF file)]
(CH2N)PR]-TASP. Aliquots were also
assayed for dipeptidyl peptidase IV (DPP IV) activity of CD26 and DPP
IV-
by the cleavage of Gly-Pro-para-nitroanilide as
described previously (11). Under these experimental conditions, CD26
and DPP IV-
eluted as monomers of 110 and 82 kDa, respectively, and
these were used as convenient markers to monitor the elution profile of
the TASP ligand p95.
(CH2N)PR]-TASP or mAb against CD4 before addition
of HIV-1 Lai (corresponding to 25 ng of p24). After incubation at
37 °C for 1 h with gentle shaking, cells were diluted 10-fold
in the culture medium and pelleted by centrifugation. Cells at 4 °C
were washed once in RPMI 1640 medium (5 ml) containing 1 mM
EDTA and then washed twice in RPMI 1640 medum (2 × 5 ml). Cell
extracts were prepared in buffer E (50 µl), the nuclei were pelleted
by centrifugation, and the supernatant was assayed for the
concentration of p24. It should be noted that under these experimental
conditions, the values for the bound virus represent particles bound on
the cell surface as well as particles (or cores) entered into
cells.
5[K(CH2PR[-TASP Blocks HIV Entry by Inhibiting the
Membrane Fusion Process
(CH2N)PR]-TASP and related TASP-inhibitors block
HIV entry and thus infection (7). Such inhibition of viral entry could
be demonstrated by different experimental approaches. For example, HIV
entry monitored by the intracellular concentration of p24 (HIV-1 major
core protein), following 1 h incubation of CEM cells with the
virus, is inhibited almost completely in the pesence of 5-10
µM 5[K
(CH2N)PR]-TASP. Similarly, viral entry monitored by the
-galactosidase activity in
HeLa/CD4+ cells expressing the bacterial lacZ
gene placed under the control of the HIV-1 LTR is inhibited by at least
90% at similar concentrations of 5[K
(CH2N)PR]-TASP
(data not shown; as described in Ref. 7). Otherwise, addition of
5[K
(CH2N)PR]-TASP after the viral entry process does
not affect virus infection, monitored by the production of virus at 4 days post-infection (as shown in Fig. 2 in Ref. 7). Here, we further
investigated the timing of the inhibitory effect of
5[K
(CH2N)PR]-TASP during the HIV entry process.
Addition of 5 µM 5[K
(CH2N)PR]-TASP to
cells 1 h before or together with the virus resulted in more than
90% inhibition of virus production. On the other hand, addition of
5[K
(CH2N)PR]-TASP at 2, and 4 h post-infection
reduced its inhibitory effect, and when added at 8 h, there was
almost no effect (data not shown; Ref. 7). The period of 8 h is
necessary for HIV-1 entry in at least 90% of target cells in the CEM
cell culture (8). The effect of 5[K
(CH2N)PR]-TASP on
the viral entry process is most probably the consequence of the
inhibition of the membrane fusion process. In accord with this, we have
previously provided evidence to demonstrate that
5[K
(CH2N)PR]-TASP efficiently blocks the
gp120·gp41-mediated membrane fusion observed in cocultures of
chronically HIV-1-infected cells with uninfected CD4+ cells
(see Fig. 6 in Ref. 7).
Fig. 2.
Specific binding of
5[K(CH2N)PR]-TASP to the cell surface. The
FITC-labeled 5[K
(CH2N)PR]-TASP (referred to here as P19*) at 0.5 µM was added to cultures of
different cell lines, CEM (sections 1, 3, and 4),
MOLT4 (section 2), and HeLa (section 5), or on
the third day of PHA-stimulated PBMC (section 6) in the
absence or presence of 50 µM unlabeled constructs as
indicated: 5[K
(CH2N)PR]-TASP, P19;
5[KGQ]-TASP, P18; and 5[KPR]-TASP, P1. The
fluorescence intensity was monitored by FACS analysis. Peak C gives the autofluorescence of each cell type incubated with unlabeled 0.5 µM 5[K
(CH2N)PR]-TASP. The
ordinates give the relative cell number, whereas the
abscissa give the relative fluorescence intensity. Note that
all the different anti-HIV TASP constructs that manifested inhibitory
activity on HIV infection (described in Ref. 7) could prevent the
binding of FITC-labeled 5[K
(CH2N)PR]-TASP to cells
when added in excess (at 50-100 µM
concentrations).
[View Larger Version of this Image (30K GIF file)]
Fig. 6.
Isolation of cell surface p95 complexed to
5[K(CH2N)PR]-TASP. Lanes 2 to 5,
CEM cells were washed and incubated at 4 °C for 30 min in FACS
buffer (which contained sodium azide) with biotin-labeled control
5[KGQ]- or 5[QPQ]-TASP and anti-HIV 5[KPR]- or
5[K
(CH2N)PR]-TASP constructs. Cells were then washed extensively before preparation of cell extracts. The complexes formed
between the biotin-labeled TASP constructs and any cell surface protein
were then recovered by purification using avidin-agarose ("Materials
and Methods"). The presence of p95 in the purified preparations was
then revealed by ligand blotting using biotin-labeled 5[K
(CH2N)PR]-TASP. Lanes 6 to 8,
to show the specificity of complex formation between cell surface p95
and biotin-labeled 5[K
(CH2N)PR]-TASP, cells were first
incubated (22 °C, 10 min) with excess 50 µM unlabeled 5[KPR]-, 5[K
(CH2N)PR]-, and 5[QPQ]-TASP before
addition of 5 µM biotin-labeled
5[K
(CH2N)PR]-TASP and recovery of the complex using
avidin-agarose (as above). Lane 1, Extract
represents the ligand blot analysis of crude CEM cell extracts,
material corresponding to 106 cells. In all the other lanes
representing the recovery of p95 from the cell surface, the material
analyzed corresponded to that from 5 × 106 cells.
Asterisk, the TASP constructs that were biotinylated are referred to as TASP/B.
[View Larger Version of this Image (41K GIF file)]
(CH2N)PR]-TASP on
the binding of gp120 and HIV-1 particles to CD4+ CEM cells.
In the first set of experiments, cells were incubated with different
concentrations of 5[K
(CH2N)PR]-TASP before further incubation with 125I-labeled gp120. Under these
experimental conditions, the binding of 125I-labeled gp120
was specific since it was inhibited by anti-CD4 mAb OKT4A, which is
known to block the binding of gp120 to the CD4 receptor (12, 13). The
binding of gp120 was not affected at 20 µM
5[K
(CH2N)PR]-TASP, whereas there was a slight
inhibition of binding at higher concentrations (Fig.
1A). To investigate the binding of virus to
cells, CEM cells were incubated with HIV-1 particles for 1 h.
Cells were then washed extensively, and the bound virus (including that
which was entered into cells) was estimated by the concentration of p24
in cell lysates. As in the binding of gp120, the binding of virus was
specific since it was inhibited (75%) by mAb OKT4A. Interestingly, at
10 µM 5[K
(CH2N)PR]-TASP, the binding of
HIV particles was also inhibited (78%) at a similar extent as that
exerted by mAb OKT4A alone or when 5[K
(CH2N)PR]-TASP was used combined with mAb OKT4A (Fig. 1B). Thus, the 22%
residual binding in the presence of 5[K
(CH2N)PR]-TASP
should represent unspecific binding. It is plausible, therefore, to
consider that 5[K
(CH2N)PR]-TASP inhibits the
gp120·gp41-mediated membane fusion by affecting the interaction of
this complex with CD4+ cells.
Fig. 1.
The effect of
5[K(CH2N)PR]-TASP on the binding of gp120 or HIV
particles to CD4+ cells. A, the binding of
125I-labeled gp120 to CEM cells. Cells were preincubated
(37 °C, 15 min) in the absence (column C) or presence of
different concentrations of 5[K
(CH2N)PR]-TASP (20, 40, 80 µM; indicated as TASP) or mAb OKT4A (2 µg/ml) before the addition of 125I-labeled gp120 and
further incubation for 1 h. The cells were then washed as
described under "Materials and Methods" and processed for analysis
of the bound gp120. The 100% binding (column C) represents the value obtained in the absence of
5[K
(CH2N)PR]-TASP. B, the binding of HIV
particles to CEM cells. CEM cells were preincubated in the absence
(column C) or presence of 5[K
(CH2N)PR]-TASP
(10 µM; column TASP), mAb OKT4A (10 µg/ml;
column OKT4A), and 5[K
(CH2N)PR]-TASP + mAb
OKT4A (10 µM and 10 µg/ml, respectively) before the
addition of HIV-1. The binding of HIV particles was estimated as
described under "Materials and Methods." The ordinate
gives the concentration of p24 associated with the cells,
i.e. particles bound on the cell surface as well as
particles (or cores) entered into cells. Each value represents the mean
of two identical samples. Similar results were obtained in two other
independent experiments. Note that at these concentrations of
5[K
(CH2N)PR]-TASP (10 µM) and mAb OKT4A
(10 µg/ml), there was a complete inhibition of virus infection.
[View Larger Version of this Image (23K GIF file)]
(CH2N)PR]-TASP to a Cell
Surface Protein
(CH2N)PR]-TASP binds different types of human cells
such as CD4+ T cell lines CEM and MOLT4, PHA-stimulated
PBMC, and the CD4
HeLa cells (Fig. 2). In
all of these cells, the cell surface binding of FITC-labeled
5[K
(CH2N)PR]-TASP was specific since it was prevented
by the unlabeled 5[K
(CH2N)PR]-TASP molecule (Fig. 2,
sections 1, 2, 5, and 6). Interestingly, cell
surface binding of FITC-labeled 5[K
(CH2N)PR]-TASP was
prevented by all TASP constructs active against HIV-infection (not
shown) such as the 5[KPR]-TASP (Fig. 2, section 4), but
not by constructs which were inactive (7) such as 5[KGQ]-TASP (Fig.
2, section 3). These results are consistent with the
suggestion that the different anti-HIV TASP constructs interact with
the same cell surface component since they have the capacity to
competitively block the binding of the FITC-labeled
5[K
(CH2N)PR]-TASP to cells.
(CH2N)PR]-TASP was
investigated after proteolysis with trypsin or proteinase K. As
controls for proteolysis, we investigated the expression of cell
surface CD26 with the mAb Ta1 and CD4 with mAbs OKT4A and OKT4. The
anti-CD4 antibodies recognize different epitopes on the CD4 receptor;
mAb OKT4A is against an epitope in the NH2-terminal extracellular domain, whereas the epitope recognized by Mab OKT4 seems
to be close to the cell membrane because it is resistant to trypsin
treatment (13, 14). As it was expected, trypsin treatment abolished the
OKT4A but not OKT4 epitope to be recognized by their respective
antibody. Under the same experimental conditions, trypsin treatment did
not affect the binding of 5[K
(CH2N)PR]-TASP or mAb
Ta1. However, the binding of the TASP inhibitor was abolished by
proteinase K treatment, which even affected the OKT4 epitope. On the
other hand, the Ta1 epitope remained resistant to proteinase K (Fig.
3). Such results were reproducibly observed in several experiments summarized in Table I; i.e. the
binding of the FITC-labeled 5[K
(CH2N)PR]-TASP was not
affected by trypsin treatment, whereas it was abolished by proteinase K
or Pronase E treatment, which also abolished the OKT4A epitope in CD4.
Once again, however, the Ta1 epitope remained resistant to proteinase K
and to Pronase E (Table I). This latter resistance reveals an
intriguing nature of the cell surface-expressed CD26 to resist
proteolysis, which we have reported recently (11). Taken together, our
results indicate that the 5[K
(CH2N)PR]-TASP-binding
entity on cells is most likely a protein that is resistant to trypsin
but sensitive to proteinase K and Pronase E. Furthermore, such a
potential TASP-binding protein does not seem to be CD4 nor CD26.
(CH2N)PR]-TASP binds to a
cell-surface protein resistant to trypsin but sensitive to
proteinase K and pronase E digestion
(CH2N)PR]-TASP to detect the TASP-ligand, and mAbs
OKT4A and Ta1 specific for CD4 and CD26, respectively. The expression
of CD4 and CD26 in control cells (not treated with different proteases) was considered as 100%. Consequently, the percent positive cells after
protease treatment were estimated by comparison with the untreated
cells.
Protease
Cell surface expression
TASP-ligand
CD4
CD26
% positive
cells
None
100
100
100
Trypsin
92
24
100
Proteinase K
15
6
98
Pronase E
8
9
97
5[K(CH2N)PR]-TASP and its nonreduced
counterpart 5[KPR]-TASP are potent inhibitors of HIV-1 entry and
infection (7), with IC50 values in CEM cells as 0.5 and 5 µM, respectively (Table II). For further
characterization of the TASP-inhibitor binding protein on the cell
surface, biotin-labeled TASP inhibitors along with control TASP
constructs (5[QPQ]- and 5[KGQ]-TASP) that lack activity against HIV
infection, were investigated by FACS analysis (Table II; Fig.
4). Clearly, no cell surface labeling occurred with
control TASP molecules. On the other hand, both biotin-labeled 5[K
(CH2N)PR]- and 5[KPR]-TASP molecules were found
to bind CEM cells with 50% effective binding concentration values of
0.15 and 3.5 µM, respectively. These results illustrate
that 5[K
(CH2N)PR]-TASP manifests, at least, 10-fold
higher activity compared with its nonreduced TASP-counterpart for both
the inhibition of HIV infection and the affinity to bind the cell
surface ligand. This latter favors the hypothesis that inhibition of
HIV infection is a consequence of specific binding of the TASP
inhibitor to its cell surface ligand.
|
5[KPR]- and 5[K(CH2N)PR]-TASP are stable in the FACS
buffer. However, when incubated in serum from fetal calf or from an individual seropositive for HIV-1, 5[KPR]-TASP rapidly loses its activity (probably due to proteolysis) with a half-life of about 1 h. In contrast, 5[K
(CH2N)PR]-TASP retains more than
80% of its activity after 18 h of incubation at 37 °C (Table
II).
Crude extracts from CEM cells
were assayed by ligand blotting using 5 µM of
biotin-labeled 5[K(CH2N)PR]-TASP. A single protein band, migrating just underneath the 97-kDa molecular mass protein marker was revealed; this 5[K
(CH2N)PR]-TASP binding
protein is referred to as p95 (Fig. 5). The binding was
specific since it was abolished in the presence of excess unlabeled 50 µM 5[K
(CH2N)PR]-TASP (Fig.
5B), whereas 50 or 100 µM 5[QPQ]- and
5[KGQ]-TASP had no effect (as in Fig. 5A). Under similar
experimental conditions, biotin-labeled 5[KPR]-TASP construct but not
5[QPQ]- or 5[KGQ]-TASP constructs revealed p95 (data not
shown).
In order to determine the molecular mass of p95 under nondenaturing
conditions, cell extracts were subjected to gel filtration chromatography, and fractions were analyzed by ligand blotting. The
5[K(CH2N)PR]-TASP binding protein eluted as a protein
of molecular mass between 90 and 100 kDa (data not shown; the
experimental conditions were as described under "Materials and
Methods"). A small amount of an 80-kDa protein was detectable in
fractions containing p95. However, since the elution profile of this
80-kDa protein was identical to that of p95, then the 80-kDa protein is
most probably a degradation product of p95, which could have been
generated even during SDS-PAGE. Indeed, if the degradation had occurred
before gel filtration, then the elution of the 80 kDa protein would
have been delayed in relation to that of p95.
CEM cells preincubated with
biotin-labeled control and anti-HIV TASP constructs were washed
extensively, and cell extracts were purified by avidin-agarose, in
order to isolate any potential complexes formed between the
biotin-labeled TASP constructs and cell surface proteins ("Materials
and Methods"). The presence of p95 in such purified preparations was
then revealed by ligand blotting using biotin-labeled
5[K(CH2N)PR]-TASP. Under these experimental
conditions, p95 was recovered when cells were preincubated with
either biotin-labeled 5[KPR]- or
5[K
(CH2N)PR]-TASP (Fig. 6,
lanes 4 and 5), but not with biotin-labeled
control TASP constructs 5[KGQ]- or 5[QPQ]-TASP (Fig. 6, lanes
2 and 3). Consistent with the higher affinity of
5[K
(CH2N)PR]-TASP to bind its cell surface ligand
compared with 5[KPR]-TASP construct (Table II, Fig. 4), an almost
2-fold higher amount of cell surface p95 was recovered by the reduced
compared with the unreduced TASP inhibitor (Fig. 6, lanes 4 and 5). The isolation of cell surface p95 by preincubation of cells with biotin-labeled 5[K
(CH2N)PR]-TASP was
specific since it was completely abolished in the presence of an excess
of unlabeled 5[K
(CH2N)PR]-TASP during the
preincubation period (Fig. 6, compare lanes 5 and
7). Consistent with its lower affinity to bind p95, an
excess of 5[KPR]-TASP abolished about 40-50% (Fig. 6, compare lanes 5 and 6). On the other hand, the control
5[QPQ]-TASP construct had no effect (Fig. 6, lane 8).
These results, therefore, demonstrate that the biotin-labeled TASP
inhibitors bind to the cell surface-expressed p95 and that this complex
is stable since it could be isolated by the strong affinity of biotin
to bind avidin. The 5[K(CH2N)PR]-TASP·p95 complex is
highly stable at physiological salt concentrations but dissociates at
concentrations of NaCl > 200 mM (data not shown). To
confirm that p95 isolated under experimental conditions described in Fig. 6 was indeed from the cell surface, CEM cells were iodinated to
label cell surface proteins, before incubation with biotin-labeled 5[QPQ]- or 5[K
(CH2N)PR]-TASP constructs (Fig.
7). Cells were then washed, extracted, and the
biotin-labeled TASP-protein complexes were isolated by purification
using avidin-agarose. By ligand blotting, we first demonstrated that,
when cells were preincubated with biotin-labeled
5[K
(CH2N)PR]-TASP but not 5(QPQ)TASP, then p95 was
recovered after purification (Fig. 7B, lanes 2 and 3). The hypothetical degradation product of p95,
the 80 kDa protein was once again detected along p95 (Fig.
7B, lane 3). Analysis of the purified preparation
by SDS-PAGE and autoradiography revealed that both p95 and the 80-kDa
by-product were labeled with 125I and were isolated
specifically when cells were preincubated with the biotin-labeled
5[K
(CH2N)PR]-TASP construct (Fig. 7A, lane 3). A highly 125I-labeled 140-kDa protein
was found to bind avidin-agarose independent of the biotin-labeled TASP
constructs (Fig. 7A, lanes 1-3). The identity of
this 140-kDa protein is not known. The isolation of 125I-labeled p95 from labeled cell surface proteins was
also demonstrated by preincubation of cells with biotin-labeled
5[KPR]-TASP (data not shown; similar to those in Fig. 7).
Consequently, the results shown in Fig. 7 provide further
confirmation along those shown in Fig. 6, that a significant proportion
of p95 is expressed on the cell surface and that this protein interacts
specifically with 5[K
(CH2N)PR]-TASP and related
anti-HIV TASP constructs.
Comparison of the estimated amount of the p95 found in crude cell extracts (Fig. 6, lane 1) with that isolated from the cell surface (Fig. 6, lane 5) suggested that cell surface p95 could represent less than 20% of the total cellular p95. This is consistent with the low amount of p95 that we could recover in plasma membrane preparations (see "Materials and Methods") compared with that found in cytoplasmic extracts (data not shown).
The Presence of a p95-like Protein in Different Types of Human and Murine CellsIn ligand blotting-type experiments (data not shown;
experimental procedures as in Figs. 5 and 6) using cell extracts and the biotin-labeled 5[K(CH2N)PR]-TASP, we could
demonstrate the expression of a 95-kDa protein in different types of
human (MOLT4, Jurkat, HeLa, and Daudi) and murine (NIH/3T3, L929, and
hybridoma) cells, similar to p95 in CEM cells.
Several observations indicated that
5[K(CH2N)PR]-TASP does not interact with HIV-1
envelope gp120·gp41 or with other viral proteins (data not shown).
First, by FACS analysis, we demonstrated that FITC- or biotin-labeled
5[K
(CH2N)PR]-TASP does not react with the cell surface
gp120·gp41 complex expressed by chronically HIV-1-infected cells.
Second, HIV-1 particles were not retained on an affinity column
constructed with the biotin-labeled 5[K
(CH2N)PR]-TASP bound to avidin-agarose. Third, 125I-labeled gp120 (as in
Fig. 1A) or metabolically
[35S]methionine-labeled HIV-1 proteins (as described in
Ref. 8) were not retained on the 5[K
(CH2N)PR]-TASP
affinity column. Finally, in ligand blotting-type experiments using
extracts from concentrated HIV-1 Lai particles, we demonstrated that
the biotin-labeled 5[K
(CH2N)PR]-TASP does not interact
with any of the HIV proteins.
HIV-1 gp120·gp41 complex expressed on the surface of viral particles and infected cells initiates the fusion processes between virus and cell membranes, to allow HIV entry, and between cell to cell membranes, to generate the formation of multinucleated cells or syncytia (1). Independent of syncytium formation, cell membrane-expressed gp120·gp41 complex is responsible for the occurrence of apoptosis in HIV-infected cell cultures (4). In all of these functions of the gp120·gp41 complex, the cell surface CD4 molecule is essential but not sufficient. Accordingly, throughout the years, several potential cofactors of CD4 have been proposed (1). By biochemical approaches, different cell surface proteins have been reported to interact with the V3 loop of gp120 (15-20) and gp41 (21-24); however, in most cases the relationship between the interaction and a putative role in HIV infection has not been determined. We have previously reported that CD26, through a potential interaction with the V3 loop, may serve as a cofactor of HIV entry (25-27). Using other experimental approaches, several groups initially were not able to produce similar results; their criticisms with our response have been published (28-32). Recently, more evidence has been provided by another group to show the implication of CD26 in HIV entry and its cytopathic effect, and this has been correlated with the structure of the V3 loop (33). Along with this, we have further demonstrated that the level of CD26 may determine the rate of HIV envelope-mediated cell to cell membrane fusion (26). Furthermore, we have shown that in a given cell line, the gp120·gp41-induced cell killing by apoptosis is enhanced with increased expression of CD26 (34). The work of other groups has suggested the participation of a cell surface protease, similar to trypsin, referred to as tryptase TL2 (16, 35), the Fc receptor (36), adhesion molecules LFA-1 (37-39) and ICAM-3 (40), major histocompatibility complex class I and class II molecules (41-42), cell surface antigens CD7 (43) and CD44S (44), and finally cell surface membrane-associated components such as heparan sulfates (45), lectins (46), and glycolipids (47). In CD4 negative cells, galactosyl-ceramide has been shown to be the responsible factor for binding of HIV particles (48-50). In view of all these observations, it is conceivable that several cell surface antigens may coordinate the complex machinery of the membrane fusion process in which the HIV gp120·gp41 envelope complex plays a key role. The requirement for some of the individual components might depend on the cell line studied and also might vary between the virus-cell or cell-cell fusion processes (1, 27, 32, 39). Whatever is the case, the number of cofactors implicated and their precise role in the HIV-mediated membrane fusion process still remains to be determined.
More recently, convincing evidence has been provided by several laboratories to show that the G protein-coupled receptors belonging to the large family of seven-transmembrane-spanning (7tm) cell surface proteins, such as HUMSTSR/LESTR/Fusin and CC-CKR-5, serve as species-specific cofactors for the entry of T cell and macrophage-tropic HIV-1 isolates, respectively (51-55). Moreover, the cofactor role of CC-CKR-5 was shown to be influenced by the structure of the V3 loop (54). However, as yet, it is not known whether the latter is due to a direct interaction with the V3 loop.
The results presented here demonstrate that
5[K(CH2N)PR]-TASP binds specifically to a cell
surface-expressed 95-kDa protein. The close relationship between the
anti-HIV activity and the affinity to bind such a cell surface protein
indicates that the inhibition of HIV entry might be a direct
consequence of a complex formation between
5[K
(CH2N)PR]-TASP and p95. Thus, p95 might be acting as a potential cofactor of CD4 during the functioning of the
gp120·gp41 complex. Consistent with this,
5[K
(CH2N)PR]-TASP and related anti-HIV TASP constructs
block major functions of the gp120·gp41 complex to initiate membrane
fusion, viral entry, and apoptosis (Ref. 7; see "Results and
Discussion"). Preliminary results obtained in our laboratory have
indicated that a synthetic V3 loop peptide corresponding to the HIV-1
Lai gp120 sequence has the capacity to reveal p95 in ligand blotting
type experiments and also form a stable complex with the cell
surface-expressed p95 in CEM cells (similar to the results described in
Figs. 5 and 6). As the TASP inhibitors were originally designed to
mimic the conserved RP dipeptide motif in the V3 loop of HIV-1 and
HIV-2 isolates (7), it is plausible then to suggest that such
inhibitors manifest their anti-HIV effect by blocking the potential
interaction of the V3 loop with p95. It is unlikely that p95 is either
Fusin or CC-CKR-5 since these recently described cofactors have
molecular masses less than 50 kDa (56, 57). Nevertheless, it will be essential to determine the implication of p95 in relation to these cofactors in the HIV envelope-mediated membrane fusion mechanism. The
interaction of 5[K
(CH2N)PR]-TASP with a p95-like
protein in different types of human and murine cells is consistent with the hypothesis that p95 is a cofactor that might not be
species-specific. This was somewhat expected since the expression of
human CD4 along with fusin or CC-CKR-5 has been demonstrated to be
sufficient in rendering heterologous cells permissive to HIV-1
infection (51-55). The fact that 5[K
(CH2N)PR]-TASP
blocks the association of HIV-1 particles to permissive cells with a
similar efficiency as an anti-CD4 mAb (Fig. 1B) points out
the important contribution of p95 in the gp120·gp41-mediated binding
of HIV to CD4+ cells. Whatever is the case, our results
suggest that the binding of HIV particles to permissive cells is
complex and should involve other cell surface components besides the
CD4 receptor.
The demonstration that biotin-labeled
5[K(CH2N)PR]-TASP binds with a high affinity to p95,
under native and denatured conditions, provides a powerful experimental
approach essential for the purification of p95 in sufficient quantities
necessary for its further characterization, identification, and
determination of its role in HIV infection. In the meantime, the
capacity of 5[K
(CH2N)PR]-TASP to interact with a
specific cell surface ligand, its potent anti-HIV activity (7), and its
stability in human serum, indicate that this TASP construct represents
a potential candidate for the development of an efficient antiviral
drug for treatment in AIDS patients.
We thank Denis Cointe for helpful discussion.