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doi:10.1182/blood-2008-04-150342
Prepublished online March 6, 2009;
2009 113: 5176-5185
Claudine Pique
Sébastien Janvier, Nikolaus Heveker, Francis W. Ruscetti, Gérard Perret, Kathryn S. Jones and
Sophie Lambert, Manuella Bouttier, Roger Vassy, Michel Seigneuret, Cari Petrow-Sadowski,
165
VEGF
HTLV-1 uses HSPG and neuropilin-1 for entry by molecular mimicry of
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IMMUNOBIOLOGY
HTLV-1 uses HSPG and neuropilin-1 for entry by molecular mimicry of VEGF165
*Sophie Lambert,1*Manuella Bouttier,1Roger Vassy,2Michel Seigneuret,1Cari Petrow-Sadowski,3Se´bastien Janvier,4
Nikolaus Heveker,4Francis W. Ruscetti,5Ge´rard Perret,2†Kathryn S. Jones,3and †Claudine Pique1
1Centre National de la Recherche Scientifique Unite Mixte de Recherche 8104, Inserm U567, Universite´ Paris-Descartes, Institut Cochin, Paris, France;
2Universite´ Paris 13, Unite Mixte de Recherche 7033, Bobigny, France; 3Basic Science Program, Science Applications International Corporation-Frederick,
National Cancer Institute, Frederick, MD; 4Centre de Recherche 6737, Hoˆ pital Sainte Justine and De´ partement de Biochimie, Universite´ de Montre´ al, Montre´ al,
QC; and 5Laboratory of Experimental Immunology, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, MD
Human T-cell lymphotropic virus type 1
(HTLV-1) entry involves the interaction
between the surface (SU) subunit of the
Env proteins and cellular receptor(s).
Previously, our laboratories demon-
strated that heparan sulfate proteogly-
cans (HSPGs) and neuropilin-1 (NRP-1),
a receptor of VEGF165, are essential for
HTLV-1 entry. Here we investigated
whether, as when binding VEGF165,
HSPGs and NRP-1 work in concert dur-
ing HTLV-1 entry. VEGF165 binds to the
b domain of NRP-1 through both HSPG-
dependent and -independent interac-
tions, the latter involving its exon 8. We
show that VEGF165 is a selective com-
petitor of HTLV-1 entry and that HTLV-1
mimics VEGF165 to recruit HSPGs and
NRP-1: (1) the NRP-1 b domain is re-
quired for HTLV-1 binding; (2) SU bind-
ing to target cells is blocked by the
HSPG-binding domain of VEGF165; (3) the
formation of Env/NRP-1 complexes is
enhanced by HSPGs; and (4) the HTLV
SU contains a motif homologous to
VEGF165 exon 8. This motif directly binds
to NRP-1 and is essential for HTLV-1
binding to, internalization into, and in-
fection of CD4ⴙT cells and dendritic
cells. These findings demonstrate that
HSPGs and NRP-1 function as HTLV-1
receptors in a cooperative manner and
reveal an unexpected mimicry mecha-
nism that may have major implications
in vivo. (Blood. 2009;113:5176-5185)
Introduction
Human T-cell lymphotropic virus type 1 (HTLV-1) is a human
retrovirus responsible for adult T-cell leukemia and inflamma-
tory disorders.1In vivo, HTLV-1 is primarily found in CD4⫹
T cells and can also infect CD8⫹T cells, monocytes/macro-
phages, and dendritic cells (DCs).2-4 In contrast to T cells, which
are thought to be infected via cell-cell contact,5DCs can be
infected by cell-free HTLV-1 and efficiently transmit HTLV-1 to
primary CD4⫹T cells.6
The glucose transporter GLUT1 was the first molecule identified as
a receptor for HTLV-1 and the related virus HTLV-2.7,8 Later studies
showed that HTLV-1 could efficiently enter cells producing minimal
levels of GLUT1 on the cell surface9and that the amount of cell-surface
GLUT1 did not correlate with levels of HTLV-1 binding,10 raising the
hypothesis that other molecules can be used as receptors. Consistent
with this, we reported that heparan sulfate proteoglycans (HSPGs) and
neuropilin-1 (NRP-1) are also critical for HTLV-1 entry.11-13
HSPGs are transmembrane proteins conjugated to negatively charged
sulfated glycan chains. HSPGs have been shown to be essential for the
HTLV-1 surface (SU) subunit binding to target cells11,14 as well as for
HTLV-1 virus binding to and internalization into primary CD4⫹
T cells11,13 and DC-mediated infection of T cells.6NRP-1 is a single-
spanning domain membrane protein notably expressed by activated
T cells12,15 and DCs.15,16 Previously, we demonstrated that NRP-1 binds
to the HTLV-1 Env and regulates target cell infection by HTLV-1–
pseudotyped particles. We also found that HTLV-1 Env can bridge
NRP-1 and GLUT1, suggesting that these 2 molecules function together
to promote HTLV-1 entry.12
NRP-1 is a coreceptor for certain isoforms of vascular endothelial
growth factor (VEGF), primarily VEGF165.17 VEGF165 triggers signal
transduction through VEGF-receptor 2 (VEGF-R2); this signaling
requires VEGF165 to be initially associated with NRP-1, a process
mediated by both HSPG-dependent and HSPG-independent interac-
tions.18-20 HSPGs bridge the heparin-binding domains of VEGF165
(exon 7 domain) and NRP-1 (b domain) and promote dimerization of
the b domain of NRP-1.18,21,22 In addition, the exon 8–encoded domain
of VEGF165 mediates binding to NRP-1 in the absence of heparin.18,23
Both exon 7 and exon 8 peptides interfere with the VEGF165/NRP-1
interaction.24-29 Moreover, in contrast to VEGF165, VEGF121 (which
lacks exon 7) and VEGF165b (which lacks exon 8) bind poorly to NRP-1
and are not capable of bridging NRP-1 and VEGF-R2.23 This suggests
that HSPG-mediated and direct interactions function cooperatively in
forming an extended surface that allows stable binding of VEGF165 to
NRP-1 (Figure 3A).18
In this study, we analyzed the respective roles of HSPGs and
NRP-1 during HTLV-1 entry. We show that HTLV-1 mimics
VEGF165 to recruit HSPGs and NRP-1 as primary entry receptors,
making VEGF165 a potent natural inhibitor of HTLV-1 infection.
Methods
Cells and transfection
Adherent cells were maintained in Dulbecco modified Eagle medium and
T cells in RPMI 1640, supplemented with 10% fetal calf serum (Invitrogen,
Carlsbad, CA). Blood from healthy donors was collected according to the
Submitted April 11, 2008; accepted February 27, 2009. Prepublished online as
Blood First Edition paper, March 6, 2009; DOI 10.1182/blood-2008-04-150342.
*S.L. and M.B. contributed equally to this work.
†K.S.J. and C.P. contributed equally to this work, and both are last authors.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
5176 BLOOD, 21 MAY 2009 䡠VOLUME 113, NUMBER 21
For personal use only. at INSERM DISC on February 13, 2013. bloodjournal.hematologylibrary.orgFrom
National Institutes of Health–approved Institutional Review Board proto-
cols, and informed consent was obtained in accordance with the Declaration
of Helsinki. Peripheral blood mononuclear cells were isolated by Ficoll-
gradient centrifugation, and the lymphocytes and monocytes were sepa-
rated by countercurrent elutriation as described.13 COS and 293T cells were
transfected using the calcium phosphate precipitation method.
Amino acid sequence analyses
Sequences searches were performed using the UniProt Knowledgebase
Release 12.6 as available at the Expasy server (http://www.expasy.org).
Multiple sequence alignments were generated with the ClustalX program.
The KPxR motif searches were initially performed using the ScanProsite
interface of the Expasy server using specific taxonomy and sequence
description filters.
Plasmids, recombinant proteins, and peptides
The pSG5M,30 CMV-Env-LTR,31 SU-rFc,32 PMT21-HA-NRP-1, and
pMT21-sHA-NRP-1 plasmids12 have been previously described. To gener-
ate HA-NRP-1⌬b, 2 AgeI cleavage sites were introduced in frame in the
HA-NRP-1 plasmid at the junctions of the b domain. The plasmid was then
digested by AgeI to release the b-domain fragment and religated. NRP-1-
Fc, VEGFR2-Fc, and VEGF165 were obtained from R&D Systems (Minne-
apolis, MN) and Tuftsin (TKPR) from Sigma-Aldrich (St Louis, MO). The
VEGF165b control peptide (LTRKD), SU 90-94 (KKPNR), and Tuftsin
analog (TU-A)(TKPPR) were from Eurogentec (Seraing, Belgium). The
15-mer SU peptides were described by Pique et al.33 The 24-mer exon 7
(CSCKNTDSRCKARQLENLERTCRC) and control 24-mer peptide were
from Genscript (Piscataway, NJ). VEGF165 concentration was determined
by enzyme-linked immunosorbent assay (ELISA; R&D Systems).
BIAcore binding analysis
In vitro interactions were performed at 25°C with a BIAcore 2000 using
HBS-EP buffer (10 mM N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic
acid, pH 7.4, containing 150 mM NaCl, 3 mM ethylenediaminetetraacetic
acid, and 0.005% (vol/vol) surfactant P20). Recombinant proteins in
10 mM sodium acetate buffer (pH 5.0) were covalently coupled to a CM5
chip using an amine coupling kit. Regeneration of the sensor chip surface
was achieved by running 5 L HCl, 100 mM, and 5 L NaOH, 100 mM
through the flow cell at 30 L/min. Injected molecules were perfused at a
flow rate of 20 L/min, and the resonance changes were recorded. The
sensorgram of the immobilized surface was subtracted from that of the
control surface, and the data were analyzed with BIAevaluation software
(GE Healthcare, Orsay, France).
Immunoprecipitation, immunoblot, and pull-down
Transfected cells were lysed in lysis buffer12 containing mixed protease
inhibitors (Roche Diagnostics, Meylan, France). Cell lysates were incu-
bated overnight at 4°C with sera from HTLV-1–infected persons before the
addition of Protein-G Sepharose beads for 2 hours at 4°C. Beads were
washed 5 times in lysis buffer, and proteins were eluted by boiling for
5 minutes in Laemmli buffer. Immunoblot analysis were performed using
either the anti-SU 4D4 monoclonal antibody (mAb) provided by Claude
Desgranges (Institut Cochin, Paris, France)34 or the anti-HA antibody
(Roche Diagnostics). For quantification, briefly exposed films were scanned
with an AGFA scanner, and signal densities of Env or HA-sNRP-1 bands
were measured with ImageJ software using the same area. Signal density in
the empty lane was also measured and subtracted from the signal of each
band. The percentage of coprecipitated HA-sNRP-1 (amount of HA-
sNRP-1 in the IP/amount of HA-sNRP-1 in the cell lysate) was calculated
for each condition and was normalized to the level of Env quantified in cell
lysates. The percentage of precipitation found for wt HA-sNRP-1 was set as
100% for comparison.
For heparin pull-down, cell lysates were incubated with 50 L of a 20%
suspension of Sepharose-heparin beads (Sigma-Aldrich) for 2 hours at 4°C.
Beads were washed 5 times in lysis buffer and proteins eluted by boiling for
5 minutes in Laemmli.
Cell-surface expression of HSPGs and NRP-1
Cells were fixed in 4% paraformaldehyde and stained with either the
anti-HSPG F58-10E4 (Seikagaku, Tokyo, Japan) or the anti–NRP-1
BDCA4 (Miltenyi Biotec, Auburn, CA) mAb.11,12 For removal of HSPGs,
cells (106) were resuspended in 200 L heparan sulfate (HS) lyase buffer
and then incubated for 2 hours at 37°C with 10 mU HS lyase (Seikagaku).13
HTLV-1 SU and virion binding assay
The soluble SU protein from HTLV-1 (SU-rFc) or avian retrovirus
(SU-ASLV-rFc) was obtained as described.13 The amount of SU protein was
determined using a rabbit IgG ELISA (ZeptoMetrix, Buffalo, NY), and
200 ng of each SU protein was used. Specific binding of SU-rFc proteins
were performed as previously described.13
HTLV-1 viral stocks were prepared from HTLV-1–producing cells
(MT-2) and the concentration determined by ELISA (ZeptoMetrix) as
previously described.13 Target cells (106) were then incubated at 22°C with
either the concentrated HTLV-1 stock (5 g unless otherwise noted) or
10 L RPMI (negative control) and the amount of virus binding determined
by flow cytometry as previously described.13
HTLV-1 internalization assay
Studies to examine the internalization of HTLV virions were performed as
described.13 For monocyte-derived DCs (MDDCs), cells were incubated
with 25 L of either culture medium (or for Figure 6B, with supernatants
from uninfected T cells) as negative control and with either a 1/1000
dilution of concentrated virus stocks or fresh virus-containing supernatants
from HTLV-1–producing cells (MT-2) at 37°C for 2 hours. For HIV-1
internalization, HIV-1 virions (100 ng/mL) were added on MOLT4 or
MDDC cultures for 2 hours at 37°C and internalization of HIV-1 cores
detected with an anti-HIV core antibody (Beckman Coulter, Fullerton, CA).
Infection assays
HTLV-1 or HIV-1 virions were obtained from supernatants of concentrated
MT-2 cells or from supernatants or HIV-1–transfected 293T cells, respec-
tively. HTLV-1 or HIV-1 virus concentration was determined by ELISA.
Cells were incubated with virus for 3 hours, washed, and replated in 6-well
plates at 5 ⫻105/well. For experiments in which HTLV-1 infection was
determined by Tax staining, cells were harvested 48 hours after infection
and the amount of intracellular Tax determined by flow cytometry. For
experiments in which infection was determined from Gag levels, the cells
were washed and refed at days 2, 4, and 6, and the culture supernatants were
harvested on day 7 (for DC) or washed at day 2 and collected on day 3 (for
T cells) and the virus concentrations determined by p19 (for HTLV-1) or
p24 (for HIV-1).
Results
VEGF165 blocks HTLV-1 entry into primary DCs
Previously, we reported that the natural NRP-1 ligand VEGF165
blocks binding of the HTLV-1 Env SU to adherent cells.12 Jones et
al recently demonstrated that DCs, including MDDCs, can be
infected with cell-free HTLV-1 virions.6We took advantage of this
new model to examine whether VEGF165 can block HTLV-1 entry
and infection to physiologically relevant target cells. We verified
that MDDC expressed a high level of cell-surface HSPGs and
NRP-1, compared with COS cells (used in Figure 3B for NRP-1
overexpression; Figure 1A). We found that VEGF165 dramatically
reduced the binding of SU to MDDCs (Figure 1B) as well as
HTLV-1 virus internalization into MDDCs (Figure 1C top panel).
In contrast, VEGF165 did not reduce internalization of HIV-1,
which infects MDDCs using different receptors (Figure 1C bottom
INITIATION OF HTLV-1 ENTRY BY HSPG AND NRP-1 5177BLOOD, 21 MAY 2009 䡠VOLUME 113, NUMBER 21
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panels). These data demonstrate that VEGF165 is a selective and
potent competitor of HTLV-1 entry into primary DCs.
Endogenous production of VEGF165 correlates with reduced
HTLV-1 interaction with MDDC
Although MDDC generated by lipopolysaccharide (LPS) stimula-
tion of monocytes do not produce VEGF165, stimulation with both
LPS and prostaglandin E2 (PGE2) leads to VEGF165 secretion.35 We
took advantage of these alternative culture conditions to evaluate
whether endogenous production of VEGF165 would also modulate
HTLV-1 entry. We confirmed that LPS/PGE2-treated MDDCs
produced VEGF165 (1700-2200 pg/mL), whereas the VEGF165
production by LPS-stimulated MDDCs was barely detectable
(⬍30 pg/mL). The levels of cell-surface HSPGs and GLUT1 were
comparable between VEGF165-producing and nonproducing
MDDCs (Figure 2A left panels). The level of NRP-1 was slightly
reduced in VEGF165-producing relative to nonproducing MDDCs,
which was unexpected because VEGF165 has been shown to
up-regulate NRP-1.36 We assumed, therefore, that the lower NRP-1
signal observed in VEGF165-producing MDDCs reflected competi-
tion between the anti–NRP-1 antibody and VEGF165 for NRP-1
binding,rather than reduced production of NRP-1. Culturing
MDDCs in conditions where they produce VEGF165 almost com-
pletely abolished their ability to bind to either the HTLV-1 SU or
virions (Figure 2A right panels) or to internalize HTLV-1 virions
Figure 1. Exogenous VEGF165 inhibits HTLV-1 entry into primary DCs. (A) Cell-
surface levels of HSPGs or NRP-1 on primary MDDCs and COS cells. The gray lines
represent the isotype control; black lines, staining with the anti-HSPG (10E4) or
anti-NRP-1 (BDCA4) mAb, as indicated. (B) Effect of exogenous VEGF165 on the
binding of the HTLV-1 SU to MDDCs. MDDCs were incubated in the presence or
absence of VEGF165 (50 ng/mL) for 30 minutes and then exposed to the HTLV-1
SU-rFc (black lines) or the control SU-ASLV-rFc (gray lines) and the level of binding
determined. (C) Effect of exogenous VEGF165 on the internalization of HTLV-1 or
HIV-1 into MDDCs. MDDCs were incubated in the presence or absence of VEGF165
(50 ng/mL) for 30 minutes and then with culture medium (gray lines) or 100 ng/mL of
either HTLV-1 (top panels) or HIV-1 (bottom panels) virions (black lines). After 2 hours
at 37°C, MDDCs were permeabilized, stained with an antibody directed against the
viral core proteins (HTLV-1 MAp19 or HIV-1 CAp24), and virus internalization
determined.
Figure 2. Endogenous production of VEGF165 by MDDCs correlates with
reduced HTLV-1 entry. (A) Effect of endogenous VEGF165 production by MDDCs on
receptor expression or HTLV-1 binding. (Left panels) Cell-surface expression of
HSPGs, NRP-1, and GLUT-1 on nonproducing (LPS stimulation) and VEGF165-
producing (LPS ⫹PGE2stimulation) MDDCs, determined by flow cytometry. (Right
panels) Nonproducing and VEGF165-producing MDDCs were incubated with either
HTLV-1 SU or HTLV-1 virions (black lines) or control SU-ASLV-rFc or medium (gray
lines) and the amount of binding determined. (B) Effect of endogenous VEGF165
production by MDDCs on the internalization of HTLV-1 or HIV-1 virus. Nonproducing
(top panels) and VEGF165-producing (bottom panels) MDDCs were incubated with
culture medium (gray lines), and 25 ng/mL (low), 50 ng/mL (medium), or 100 ng/mL
(high) HIV-1 (black lines) or with culture medium (gray line) or 100 ng/mL HTLV-1
(black line) and the amount of internalization was determined. (C) Effect of
endogenous VEGF165 production by MDDCs on HTLV-1 or HIV-1 infection. MDDCs
were infected with 100 ng of either HTLV-1 or HIV-1 virions. Seven days later,
supernatants from individual wells were collected (8 wells/condition), and MDDC
infection was measured by quantifying the concentration in supernatants of the viral
core protein, MAp19 for HTLV-1 (left histogram) or CAp24 for HIV-1 (right histogram).
The data are the mean ⫾SD of 1 representative experiment of 3 performed in
octuplicate.
5178 LAMBERT et al BLOOD, 21 MAY 2009 䡠VOLUME 113, NUMBER 21
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(Figure 2B HTLV panels). Comparable levels of HIV-1 internaliza-
tion were detected in the 2 MDDC populations, even when a low
dose of HIV-1 was used (Figure 2B HIV panels). The level of
cell-free HTLV-1 infection in MDDC-producing VEGF165 was also
significantly lower relative to the level in the nonproducing
MDDCs (Figure 2C left panel). When the MDDCs were infected in
the presence of exogenous VEGF165, the level of infection was also
reduced, although the effect was less dramatic; this may reflect
lability of added VEGF165 and/or cell-cell spread after the initial
infection. As expected, HIV infection of MDDCs was not inhibited
by either endogenous or exogenous VEGF165 (Figure 2C right
panel). These differences in HTLV-1 binding, internalization, and
infection are probably the result of continuous VEGF165 production
by MDDCs, although the contribution of other secreted molecules
could not be excluded. Along with the findings showing the
inhibitory effect of exogenous VEGF165, these results provide
strong evidence that VEGF165 is a natural regulator of HTLV-1
infection in vivo.
The b domain of NRP-1 is essential for HTLV-1 binding to target
cells
Because the HSPGs and VEGF165 binding sites have been mapped
to the b domain of NRP-1,18,22 we investigated the impact of
b-domain deletion on the binding of HTLV-1 to the cell surface. To
facilitate the study of the effect of exogenously expressed NRP-1,
these experiments were performed in cells that produced low level
of endogenous NRP-1 (COS; Figure 1A). The level of cell-surface
expression similar to that of wild-type NRP-1 was previously
considered as one of the criteria of proper folding for neuropilin
mutants.37,38 We found that an NRP-1 mutant lacking the b domain
(HA-NRP-1⌬b) was synthesized and expressed at the cell surface
at comparable level than WT HA-NRP-1 in COS cells (Figure 3A),
indicating that deletion of the b domain did not induce misfolding
of the mutant. As expected, we also observed that wt HA-NRP-1,
but not HA-NRP-1⌬b, was capable of binding heparin (Figure 3A).
Consistent with their low expression of endogenous NRP-1, a very
low level of virus binding was observed on COS cells transfected
with the control plasmid. Virus binding was increased on cells
overexpressing wt HA-NRP-1, whereas binding was reduced to
almost control level in cells overexpressing HA-NRP-1⌬b (Figure
3B). Statistical analysis indicated a significant difference in binding
between COS cells overexpressing HA-NRP-1 (200% ⫾49%) and
either control cells (normalized to 100%) or cells overexpressing
HA-NRP-1⌬b (94% ⫾32%; Figure 3C). Hence, the b domain of
NRP-1 is essential for binding of HTLV-1 to target cells, account-
ing for the competitive effect of VEGF165 on HTLV-1 entry.
HSPGs enhance the formation of Env/NRP-1 complexes
We next studied whether HSPGs could enhance the binding of the
HTLV-1 SU to NRP-1. Such studies require the quantification of
Figure 3. The HTLV-1 SU/NRP-1 interaction depends on the b domain of
NRP-1 and on HSPGs. (A) Effect of the deletion of the b domain of NRP-1 on
NRP-1 synthesis, binding to heparin, and cell-surface expression. COS cells were
transfected with a control plasmid (Cont.) or a plasmid encoding either wild-type
(wt) HA-NRP-1 or HA-NRP-1⌬b. (Left panels) Total cell proteins and proteins
pulled-down on heparin-coated Sepharose beads were analyzed by immunoblot
using an anti-HA mAb. (Right panel) Transfected COS cells were stained with the
anti-HA mAb and analyzed by flow cytometry. (B) Effect of deletion of the b domain
of NRP-1 on HTLV-1 virus binding. COS cells transfected as in panel A were
incubated with culture medium (gray lines) or concentrated supernatant from
HTLV-1–infected T cells (black lines), and the level of binding of HTLV-1 virions
was determined. (C) Quantification of the level of HTLV-1 virus binding to COS cells
overexpressing wt HA-NRP-1 or HA-NRP-1⌬b. Results correspond to the ratio
between the mean fluorescence intensity (MFI) of cells overexpressing either WT
HA-NRP-1 or HA-NRP-1⌬b and the MFI of control cells (⫻100) and are the mean
⫾SD of 3 independent experiments. *Statistically significant (unpaired ttest
analysis, 2-tailed P⬍.05). ns indicates not significant. (D) Effect of reducing
HSPG synthesis on the formation of complexes between the HTLV-1 Env proteins
and the ectodomain of NRP-1. COS cells were cotransfected with either the
CMV-Env-LTR construct or a control plasmid and a plasmid encoding the soluble
version of HA-NRP-1 (HA-sNRP-1). After 4 hours, cells were either left untreated
(⫺) or treated with 30 mM sodium chlorate for 24 hours (⫹). Total proteins
precipitated with an anti–HTLV-1 serum (IP Env) were examined by immunoblot
using the anti-HA or the anti-SU mAb, as indicated. The levels of HA-NRP-1 and
Env in cell lysates are also shown. The positions of HA-sNRP-1 and of the HTLV-1
Env precursor (gp61) and SU (gp46) are indicated. (Bottom panel) Quantification of
the effect of sodium chlorate on the Env/HA-sNRP-1 coprecipitation. Results
represent the normalized amount of HA-sNRP-1 coprecipitated with Env and are
the mean ⫾SD of 2 independent experiments.
INITIATION OF HTLV-1 ENTRY BY HSPG AND NRP-1 5179BLOOD, 21 MAY 2009 䡠VOLUME 113, NUMBER 21
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Env/NRP-1 complexes and could therefore not be performed by
flow cytometry, which could not distinguish between SU binding
on HSPG/NRP-1 complexes and SU binding directly to HSPGs or
other molecules. Therefore, the effect of HSPG was examined
using the immunoprecipitation approach we previously used to
demonstrate the interaction of NRP-1 and Env.12 The HTLV-1 Env
proteins are synthesized as a precursor (Pr; gp61) subsequently
cleaved into SU (gp46) and TM (gp21) in the Golgi apparatus.39 We
reasoned that, because heparan chain synthesis also occurs in the
Golgi,40 immunoprecipitation of intracellular proteins could be
used to study the HSPG/SU interaction. Cells were cotransfected
with a plasmid allowing the production of the precursor as well the
cleaved forms of Env and a plasmid encoding the ectodomain of
NRP-1 (HA-sNRP-1) to study the formation of complexes with
Env and the extracellular part of NRP-1. Transfected COS cells
were cultured for 24 hours with or without sodium chlorate
(NaClO3), an inhibitor of heparan chain sulfation.13 NaClO3
treatment reduced the amount of cell-surface HSPGs by approxi-
mately 70% (data not shown) but had little effect on Env or
HA-sNRP-1 synthesis in COS cells (Figure 3D). In contrast, lower
HSPG synthesis was associated with a 40% reduction in the
amount of HA-sNRP-1 coprecipitated with Env, relative to un-
treated cells (Figure 3D). Hence, the formation of intracellular
complexes between the HTLV-1 Env proteins and the ectodomain
of NRP-1 partially depends on HSPGs.
A VEGF165 exon 7 peptide blocks HTLV-1 entry
A peptide corresponding to the exon 7–encoded domain of VEGF165,
which mediates the HSPG-dependent binding to NRP-1 (Figure
4A), was shown to block the VEGF165/NRP-1 interaction.25,29
Using primary CD4⫹T cells, we found that the exon 7 peptide
strongly reduced HTLV-1 SU binding as well as HTLV-1 internal-
ization, compared with binding and internalization in the presence
of the control peptide (Figure 4B). The exon 7 peptide, but not the
control peptide, also inhibited HTLV-1 internalization into DCs,
and this effect was similar to that observed when cell-surface
HSPGs were enzymatically removed by HS lyase (Figure 4C).
Combination of HS lyase and exon 7 further reduced HTLV-1
internalization into DCs. This additive effect could be the conse-
quence of concomitant reduction in HTLV-1 attachment on free
HSPGs (because of the action of HS lyase) and in HTLV binding on
HSPG/NRP-1 complexes (because of addition of the exon 7
peptide). These findings indicate that native HTLV-1 Env binding
to NRP1 involves the same structure recognized by the VEGF165
exon 7 domain, that is, the interface formed between heparan
sulfate chains and the b domain of NRP-1.
The HTLV SUs contain a conserved VEGF165 exon 8–like motif
VEGF165 also contains an HSPG-independent NRP-1 binding site,
which corresponds to the exon 8–encoded domain23,26 (Figure 4A).
Examination of the primary sequence of the HTLV-1 Env revealed
strong homology between the SU 90-94 region (KKPNR) and the
VEGF165 exon 8 domain (CDKPRR). Exon 8–related peptides,
which also bind to NRP-1,28 Tuftsin (TKPR), and Tuftsin analog
(TKPPR), are also highly homologous to the HTLV-1 SU 90-94
region. Strikingly, the KPxR consensus motif that can be deduced
from these 4 sequences contains the 3 residues of VEGF165
previously shown to be critical for direct interaction with NRP-
124,26 (Figure 5A).
The HTLVs, along with their simian counterparts (STLVs),
belong to the primate T-cell lymphotropic virus (PTLV) group of
Figure 4. HTLV-1 interactions with primary CD4ⴙT cells and MDDCs are
blocked by the HSPG-binding domain of VEGF165.(A) Schematic representa-
tion of the interactions between VEGF165, heparan sulfate chains, and NRP-1. (Left
panel) The interactions between VEGF165 exon 7 and heparan sulfate chains (dark
blue arrow), between VEGF165 exon 8 and the b domain of NRP-1 (light blue
arrow), and between the b domain of NRP-1 and heparan sulfate chains (pink
arrow). (Right diagram) The impact of these interactions on the formation of
HSPG/NRP-1/VEGF165 complexes and on NRP-1 dimerization. The subsequent
recruitment of VEGF-R2 by VEGF165 bound to HSPG/NRP-1 complexes is not
depicted. (B) Effect of the VEGF165 exon 7 peptide on HTLV-1entry into CD4⫹
T cells. Primary activated CD4⫹T cells were incubated for 30 minutes with either a
peptide homologous to a portion of VEGF165 exon 7 or a control peptide of the
same length and the levels of HTLV-1 SU binding (left histogram) or HTLV-1 virus
internalization (right histogram) determined. The data are normalized to the control
peptide (100%) and are the mean ⫾SD of 1 representative experiment of 2
performed. (C) Effects of HS lyase and exon 7 peptide on HTLV-1 entry into DCs.
DCs were resuspended in HS lyase buffer and incubated in the presence or
absence of HS lyase. The cells were then washed, incubated for 30 minutes with
either the control peptide or VEGF165 exon 7 peptide, and incubated with either
culture medium (gray lines) or HTLV-1 virions (black lines), and the extent of
HTLV-1 virus internalization was determined.
5180 LAMBERT et al BLOOD, 21 MAY 2009 䡠VOLUME 113, NUMBER 21
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␦retroviruses. Sequence analyses revealed that the KPxR motif
was conserved in the SU proteins of HTLV-2, HTLV-3, and related
STLVs (Figure 5B). Moreover, the KPxR sequence was present as
a single copy and at an identical position in the SU of virtually all
the PTLV sequences available in data banks (Table 1). Interest-
ingly, the motif was not found in the SU sequences of bovine
leukemia virus, another ␦retrovirus that uses a different cell
receptor than HTLV-1.41 The KPxR motif was found in less than
14% of SU sequences from lentiviruses and, among them, in only
0.5% of HIV-1 sequences, consistent with our previous observa-
tions that the HIV-1 Env does not bind to NRP-1.12 Finally, the
KPxR motif was found in less than 6% of the Env proteins from
␥or ␣retroviruses. The motif was found in the single available
sequence from equine foamy virus, but in that case was located in
the extracellular part of the TM, which is not involved in receptor
recognition. This comparison clearly shows that the presence of a
conserved KPxR motif in the SU is a specific feature of the
HTLV/STLV family.
The SU 90-94 region is a direct binding site to NRP-1
Given the homology between the SU 90-94 and exon 8 sequences,
we examined using the BIAcore system whether a SU 90-94
peptide also directly binds to NRP-1, as previously demonstrated
for VEGF165 exon 8 and Tuftsin.18,23 Recombinant NRP-1 or, as a
control, VEGF-R2, was immobilized on the sensor chip and the
peptides were injected (Figure 5C). Because the amplitude of the
signal obtained in the BIAcore system depends on both the affinity
for the ligand and on the mass of the injected molecule, we used
control peptides of the same size as the SU 90-94 peptide: as a
positive control, the 5-mer peptide Tuftsin analog (TU-A), and as
negative control, a 5-mer peptide corresponding to the C-terminal
sequence of VEGF165b, which does not bind to NRP-1.42 Binding to
NRP-1 (Figure 5C left panel), but not VEGF-R2 (right panel), was
observed for both SU 90-94 and TU-A (curves 1 and 2), whereas
the control VEGF165b peptide did not bind (curves 3). Because of
the small size of the exon 8–like peptides (5-mer), the binding
signals did not permit determination of the affinities for NRP-1.
However, this analysis clearly showed that the SU 90-94 and
Tuftsin analog directly bind to recombinant NRP-1 in vitro.
We next investigated whether longer SU peptides can also bind
to NRP-1. Efficient binding to NRP-1 was observed for both
peptide containing the KPxR motif: the SU 81-95 peptide (Figure
5D left panel, curve 1) and, to a lesser extent, the SU 86-100
peptide (curve 2). In contrast, no binding was found for the SU
Figure 5. The HTLV-1 SU contains a KPxR motif homolo-
gous to the VEGF165 exon 8 domain. (A) Sequence align-
ment between the VEGF165 exon 8 domain, the exon 8–like
peptides Tuftsin and Tuftsin analog, and the aa 90-94 region
of the HTLV-1 SU. The conserved consensus KPxR motif
deduced from the alignment is also shown. (B) Local se-
quence alignment of VEGF165 exon 8 sequence with the
region of the HTLV/STLV SUs encompassing the KPxR motif.
Sequence positions corresponding to conserved and chemi-
cally homologous residues between viral sequences are
highlighted in dark and light gray, respectively, and the KPxR
motif is highlighted in black. The UniProt sequences used
for the alignment are, respectively, VEGFA_HUMAN,
ENV_HTL1A, ENV_HTLV2, ENV_HTL3P, O41897_9STL1,
O09243_9DELA, and Q6XQ01_9DELA. The numbering re-
fers to the complete unprocessed sequences. (C) In vitro
binding of exon 8–like peptides to NRP-1 in the BIAcore
system. The SU 90-94 peptide, Tuftsin analog (TU-A), or the
control peptide (Cont.) was injected (15 M each) on either
recombinant NRP-1 (left panel) or recombinant VEGF-R2
(right panel) previously immobilized on the sensor chip. The
sensorgram displays the kinetics of peptide binding after
injection (time 0) and is 1 representative experiment of
2 performed. RU indicates resonance unit. (D) In vitro binding
of longer SU peptides to NRP-1 in the BIAcore system.
Fifteen-mer SU peptides either containing or lacking the
KPxR motif (shown in bold) were injected (10 M each) on
recombinant NRP-1 immobilized on the sensor chip. (Left
panel) The kinetics of peptide binding to NRP-1 after injection
(time 0), which is 1 representative experiment of 2 performed.
(Right panel) The binding of the SU 81-95 peptide on NRP-1,
corresponding to the maximal RU value of the binding curve,
and the absence of binding on BSA or VEGF-R2. (E) The SU
90-94 and exon 8–like peptides compete with VEGF165 for
NRP-1 binding. (Left panel) Recombinant NRP-1 was in-
jected on immobilized VEGF165 in the presence of buffer or of
the SU 90-94, Tuftsin, Tuftsin analog (TU-A), or control
peptide (100 M each). The maximal RU value of each curve
was used to quantify NRP-1 binding to VEGF165, and binding
was normalized to condition without peptide (100%). Data are
the mean ⫾SD of 2 independent experiments. (Right panel)
Dose-dependent inhibition of the NRP-1/VEGF165 interaction
by the SU 90-94 and exon 8–like peptides.
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61-75 peptide, which lacks the motif (curve 3). The binding of the
SU 81-95 peptide to NRP-1 was dose-dependent, and no binding of
this peptide to either BSA or VEGF-R2 was observed (Figure 5D
right panel).
The effect of the peptides on the VEGF165/NRP-1 interaction
was also characterized. NRP-1 was injected with or without the
peptides on immobilized VEGF165 (Figure 5E). These settings also
allowed us to compare the SU 90-94 peptide to the 4-mer peptide
Tuftsin because the binding of NRP-1 was measured in these
experiments. The association of NRP-1 to VEGF165 was signifi-
cantly reduced in the presence of SU 90-94, Tuftsin, or TU-A at
100 M (Figure 5E left panel). A dose-dependent inhibition of the
VEGF165/NRP-1 interaction was found for all 3 inhibitory peptides
(Figure 5E right panel). The IC50 for Tuftsin and TU-A was similar
(20 and 30 M, respectively) and was only slightly higher for SU
90-94 (50 M), showing that the SU 90-94 is also a potent inhibitor
of VEGF165 binding to NRP-1.
The SU 90-94 or exon 8–like peptides inhibit HTLV-1 entry
To further investigate the property of the exon 8–like motif of the
HTLV SU, we examined whether the KPxR-containing peptides
block HTLV-1 binding to and infection of target cells. Initially, we
preincubated CD4⫹T cells with peptides and determined the effect
on SU binding. Whereas the control peptide had minimal impact,
both SU 90-94 and TU-A reduced SU binding in a dose-dependent
manner (Figure 6A). The SU 90-94 and TU-A, but not the control
peptide, also strongly reduced HTLV-1 virion binding to and
internalization into CD4⫹T cells (Figure 6B left panel). In
contrast, exon 8–like peptides had minimal effect on HIV-1 infec-
tion of CD4⫹T cells, even when a low dose of HIV-1 was used
(Figure 6C).
We also evaluated the effect of the peptides on HTLV-1
interactions with DCs. The SU 90-94 and TU-A peptides dramati-
cally reduced the binding of the HTLV-1 SU to DCs as well as
HTLV-1 infection of DCs, whereas the control peptide did not
(Figure 6D). The amount of the viral protein Tax, which can be
detected in cells only after proviral de novo production of viral
proteins, was reduced to below the level of detection for SU 90-94
and at almost control level for TU-A (Figure 6D bottom panel).
These analyses clearly demonstrate that the KPxR motif of the
HTLV-1 SU is a receptor binding domain and extend the impor-
tance of NRP-1 to cell-free HTLV-1 infection of primary DCs.
Table 1. Occurrence of the KPxR motif in the Env sequences of infectious retroviruses
Retrovirus
Sequences*
Occurrence, percentage
Motif type
(no.)
Motif
position†Total no. No. containing KPxR
␦retrovirus
HTLV-1 255 254 99.6 KPnR (246)
KPyR (7)
KPsR (1)
91-94 (SU)
91-94 (SU)
91-94 (SU)
HTLV-2 15 14 93.3 KPnR (14) 87-90 (SU)
HTLV-3 2 2 100 KPnR (1)
KPdR (1)
91-94 (SU)
91-94 (SU)
STLV-1 33 32 97 KPnR (32) 91-94 (SU)
STLV-2 2 2 100 KPnR (2) 87-90 (SU)
STLV-3 4 4 100 KPnR (4) 89-92 (SU)
Baboon TLV 1 1 100 KPnR (1) 91-94 (SU)
BLV 113 0 0
Lentivirus
HIV-1 71364 345 0.5 KPvR
KPiR
KPrR
KPkR
KPyR
KPgR
286-289 (SU)
286-289 (SU)
498-501 (SU)
498-501 (SU)
185-188 (SU)
729-732 (TM)
HIV-2 968 22 2.3 KPrR
KPkR
KPgR
177-180 (SU)
177-180 (SU)
374-377 (SU)
SIV 3695 12 0.3 KPkR
KPdR
KPiR
420-423 (SU)
751-754 (TM)
15-18 (signal)
FIV 455 61 13.4 KPrR 606-609 (SU)
Visna-Maedi virus 163 20 12.3 KPsR 7-10 (signal)
EIAV 795 2 0.3 KPvR 11-14 (signal)
␥or ␣retrovirus
MLV 134 0 0
RSV 18 1 5.6 KPgR 191-194 (SU)
Spumavirus
EFV 1 1 100 KPiR 834-837 (TM)
PFV/HFV 1 0 0
STLV indicates simian T-lymphotropic virus; HTLV, human T-lymphotropic virus; BLV, bovine leukemia virus; SIV, simian immunodeficiency virus; FIV, feline
immunodeficiency virus; EIAV, equine infectious anemia virus; MLV,murine leukemia virus; RSV, Rous sarcoma virus; EFV,equine foamy virus; PFV, primate foamy virus; HFV,
human foamy virus; SU, surface Env subunit; and TM, transmembrane Env subunit.
*For T-lymphotropic viruses, only complete Env sequences and Env fragments encompassing the 90-94 region of the SU were considered. For other retroviruses, all
complete and fragment Env sequences were considered.
†Whether the consensus KPxR motif is found within the signal peptide, the SU or TM subunit of Env is indicated in parentheses.
5182 LAMBERT et al BLOOD, 21 MAY 2009 䡠VOLUME 113, NUMBER 21
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Discussion
Although recent studies have independently identified GLUT1,
HSPGs, and NRP-1 as key players in HTLV-1 entry, the specific
roles of each of these molecules in this process have not been well
characterized. The work presented here was aimed at clarifying the
respective contribution of HSPGs and NRP-1 during HTLV-1
binding, entry, and infection.
Among other physiologic roles, NRP-1 functions as a receptor
for VEGF165. Using the new model of HTLV-1 infection involving
primary DCs and cell-free virus, we first demonstrated that
VEGF165 is a specific and potent inhibitor of HTLV-1 infection.
It was previously reported that, like VEGF165, the HTLV-1 SU
binds to HSPGs14 as well as to NRP-1.12 However, it was unclear
whether HSPG and NRP-1 function together, as during VEGF165
signaling, or individually. Here we report that (1) binding of
HTLV-1 to the cell surface requires the b domain of NRP-1; (2) HSPGs
enhance the formation of complexes between HTLV-1 Env and the
NRP-1 ectodomain; and (3) binding of the HTLV-1 SU to target
cells is partially blocked by the domain of VEGF165, which
mediates the HSPG-dependent interaction with NRP-1. These
findings reveal that HSPGs and NRP-1 also cooperate during
HTLV-1 SU binding, providing the functional explanation for the
previously demonstrated critical importance of these 2 molecules
during HTLV-1 entry.
VEGF165 also contains residues located in the exon 8 domain
that mediate interaction with NRP-1 in a heparin-independent
manner.23 Using sequence comparisons and in vitro binding
experiments, we identified a VEGF165 exon 8–like motif in the
HTLV-1 SU (amino acids [aa] 90-94: KPxR) and demonstrated that
this sequence mediates direct binding to NRP-1 and competes with
VEGF165 for NRP-1 binding. Because of technical limitations, we
were unable to produce sufficient quantities of the HTLV-1 SU to
study the direct SU/NRP-1 interaction. However, we demonstrated
that 15-mer SU peptides containing the KPxR motif could also bind
to NRP-1. The aa 90-94 motif is in an internal position in the SU,
whereas the exon 8 sequence constitutes the C-terminus of VEGF165.
This could explain why the SU 81-95 peptide, which contains only
a single residue after the motif, has higher affinity for NRP-1 than
the SU 86-100 peptide, in which the motif is in an internal position.
We further demonstrated that both exon 8 peptides and the SU
90-94 peptides block HTLV-1 binding, entry, and infection in
assays using the primary target cells of this virus (CD4⫹T cells and
DCs). No inhibition was found when HIV-1 was used as a control
virus, showing that the peptides selectively impact HTLV-1 entry.
These findings confirm both the importance of the exon 8 domain
in NRP-1 binding and the key role of NRP-1 in HTLV-1 entry.
Because the SU KPxR motif is involved in HTLV-1 entry, one
would expect that it would correspond to an exposed and neutraliz-
ing region. Indeed, antibodies raised against the aa 89-107 of the
HTLV-1 SU recognize the native form of the SU, suggesting that
this region is indeed exposed on the surface of the protein.43
Furthermore, the SU 89-107 region, containing the KPxR motif,
has been characterized as neutralizing epitope,43,44 and the corre-
sponding antibodies were shown to preferentially recognize the
(KKPNRN) sequence.43 In addition, we previously demonstrated
that the residue R94 in SU, the arginine residue in the KPxR motif,
Figure 6. The SU 90-94 and exon 8 peptides block HTLV-1 entry into CD4ⴙ
T cells or DCs. (A) Effect of the peptides on HTLV-1 SU binding on CD4⫹T cells.
MOLT4 were incubated in medium alone or in medium containing 2, 20, or
50 g/mL peptides before incubation with 200 ng/mL HTLV-1 or ASLV SU-rFc. The
data show the specific binding (MFISU-rFc–MFISU-ASLV-rFc) normalized on cells
incubated with no peptide (100%) and are from 1 representative experiment of 2
performed. (B) Effect of the peptides on HTLV-1 virus binding to or internalization
into CD4⫹T cells. (Top panel) CD4⫹T cells were preincubated with 20 g/mL
peptides, then HTLV-1 virions were added 30 minutes later, and the amount of
virus binding was determined as described in “BIAcore binding analysis,” except
that 1 g rather than 5 g virus was used. (Bottom panel) CD4⫹T cells were
incubated with 50 g/mL peptides for 30 minutes. The cells were diluted and
incubated with HTLV-1 virions for 3 hours (final concentration, 10 g/mL peptide),
and the amount of virus internalization was determined. HTLV-1 binding and
internalization are normalized to the control peptide (100%), and the data are mean
⫾SD of at least 2 independent experiments. (C) Effect of the peptides on HIV-1
infection of CD4⫹T cells. CD4⫹T cells were preincubated with 50 ng/mL peptides
for 30 minutes, and cells were infected with either a low dose (30 ng/mL) or high
dose (80 ng/mL) of HIV-1 virions for 2 hours at 37°C. HIV-1 production was
assessed 3 days after infection by measuring the amount of released particles
using the anti-CAp24 ELISA. (D) Effect of the peptides on HTLV-1 binding to and
infection of MDDCs. (Top panel) Cells were incubated with 20 g/mL peptides for
30 minutes, exposed to the HTLV-1 SU-rFc (black lines) or the control SU-ASLV-
rFc (gray lines), and the level of binding determined. (Bottom panel) Cells were
incubated with 20 g/mL peptides for 30 minutes and infected by cell-free HTLV-1.
The extent of infection was determined by flow cytometry at 48 hours after
exposure to virus by intracellular staining for the HTLV-1 Tax protein. The black
lines represent the staining with Tax-specific antibody; gray lines, the staining with
an isotype control.
INITIATION OF HTLV-1 ENTRY BY HSPG AND NRP-1 5183BLOOD, 21 MAY 2009 䡠VOLUME 113, NUMBER 21
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was essential for HTLV-1 infectivity.31 Our data thus clarify the
previous data showing the importance of the SU 89-107, by
revealing that this region is indeed a receptor-binding domain.
For VEGF165, blocking either the exon 7– or exon 8–mediated
interactions is sufficient to prevent binding to NRP-1.23,25 We show
here that both the exon 7 and exon 8 peptides can block HTLV-1
entry. These observations indicate that HTLV-1 entry is also
governed by both HSPG-mediated and direct interactions between
the SU and NRP-1, in a cooperative manner. The initial interactions
of VEGF165 with HSPG/NRP-1 complexes permit the subsequent
presentation of VEGF165 to VEGF-R2, thereby forming the stable
complex competent for signal transduction. This is reflected by the
ability of VEGF165 to bridge NRP-1 and VEGF-R2.45 Strikingly,
we previously found that the HTLV-1 SU is able to bridge NRP-1
and GLUT1.12 These observations strongly suggest that the forma-
tion of the HTLV-1 entry receptor complex mirrors the formation of
the HSPG/NRP-1/VEGF-R2 complex. We therefore envision a
model in which NRP-1 functions as an intermediate that recruits
HTLV-1 particles clustered on HSPGs and present them to GLUT1,
forming an HSPG/NRP-1/GLUT1 binding structure that renders
Env competent for fusion. Alternatively, the stable binding of the
SU to NRP-1 could trigger conformational changes in the SU,
favoring its subsequent interaction with GLUT1, a receptor/
coreceptor model reminiscent of what occurs during HIV entry. In
both models, GLUT1 functions as a postbinding receptor, in
agreement with observations that GLUT1 overexpression increases
HTLV-1 Env-mediated fusion or infection but not HTLV-1 virus
binding.7,10
In conclusion, our study provides a model for HTLV-1 entry that
reconciles and extends the previous observations concerning
HSPGs, NRP-1, and GLUT1. Moreover, the molecular mimicry we
demonstrated opens new perspectives for the understanding of
HTLV-1 propagation in vivo. In contrast to T cells, which spontane-
ously produce VEGF165,46 VEGF165 production by DCs is restricted
to certain stimulation conditions.35 It could therefore be speculated
that this lack of VEGF165 production accounts, at least in part, for
the particular susceptibility of DCs to cell-free HTLV-1 infection.
More generally, we propose that HTLV-1 tropism is governed by a
subtle balance between the levels of HSPGs and NRP-1 at the cell
surface and the amount of extracellular NRP-1 ligands, especially
VEGF165. Future elucidation of these complex interactions would
greatly improve the understanding of HTLV-1 transmission and
pave the way for the design of novel therapeutic strategies against
HTLV-1 infection.
Acknowledgments
The authors thank A. Strazec, C. Vander Kooi, and M. Cre´pin
for helpful discussions and D. C. Bertolette and M. Chazal for
technical assistance.
This work was supported in part by the Intramural Research
Program of the National Institutes of Health (NIH), National
Cancer Institute (NCI), Center for Cancer Research; Association de
Recherche contre le Cancer (no. 3123) or Sang pour Cent la Vie;
the Canadian Institutes for Health Research; the Immune and Viral
Diseases Program of the Sainte Justine Research Center (Montreal,
QC); and, in whole or in part, by the NCI, NIH (contract no.
N01-CO-12400). S.L. was the recipient of grants from the French
Ministry of Research and Association de Recherche contre le Cancer.
The content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human Services,
nor does mention of trade names, commercial products, or organi-
zations imply endorsement by the US government.
Authorship
Contribution: S.L., M.B., R.V., C.P.-S., and S.J. performed research
experiments and analyzed the data; M.S. performed sequence
comparisons; G.P. and F.W.R. contributed to the experimental
design and provided vital new reagents; N.H. contributed vital new
reagents and wrote the paper; K.S.J. contributed to the experimen-
tal design, analyzed the data, and contributed to the writing of the
paper; and C.P. designed the research project, analyzed the data,
and wrote the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Claudine Pique, Institut Cochin, 22 rue
Mechain, 75014 Paris, France; e-mail: claudine.pique@inserm.fr;
or Kathryn S. Jones, NCI-Frederick, SAIC-Frederick, Bldg 567,
Rm 253, Frederick, MD 21702; e-mail: joneska@mail.nih.gov.
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INITIATION OF HTLV-1 ENTRY BY HSPG AND NRP-1 5185BLOOD, 21 MAY 2009 䡠VOLUME 113, NUMBER 21
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