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Journal of Virology, November 2001, p. 10472-10478, Vol. 75, No. 21
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.21.10472-10478.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Synthetic Peptide P-197 Inhibits Human T-Cell Leukemia Virus
Type 1 Envelope-Mediated Syncytium Formation by a Mechanism That Is
Independent of Hsc70
David W.
Brighty* and
Sushma R.
Jassal
Biomedical Research Centre, Ninewells
Hospital and Medical School, University of Dundee, Dundee DD1 9SY,
Scotland
Received 19 January 2001/Accepted 30 July 2001
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ABSTRACT |
Entry of human T-cell leukemia virus type 1 (HTLV-1) into cells is
mediated by the viral envelope glycoproteins gp46 and gp21. The gp46 surface glycoprotein binds to a poorly
characterized cell surface receptor, thereby promoting the
gp21-dependent fusion of the viral and cellular membranes.
Interestingly, a synthetic peptide (P-197) simulating amino acids 197 to 216 of gp46 strongly inhibits envelope-dependent membrane fusion
with Molt-4 target cells. It has been suggested that this peptide acts
by competitively binding to Hsc70, a putative cellular receptor for
HTLV-1. We now demonstrate that P-197 inhibits membrane fusion
among diverse HTLV-1-permissive target cells. Importantly, most of
these cells lack detectable levels of Hsc70, indicating that P-197
inhibits membrane fusion by a mechanism that is Hsc70 independent. We
now suggest that competition for primary receptor
binding is unlikely to account for the inhibitory activity of P-197.
Understanding the mechanism by which P-197 functions may reveal
concepts of general relevance to antiretroviral chemotherapy.
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TEXT |
Efficient entry into, and
infection of, human cells by human T-cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins.
The envelope proteins are expressed as a 68-kDa glycosylated
protein precursor (gp68) that is posttranslationally cleaved by a
cellular protease to yield the mature gp46 surface glycoprotein (SU) and the gp21 transmembrane protein (TM)
(1, 4, 18). The surface glycoprotein remains
associated with gp21 following precursor cleavage, and this SU-TM
complex is anchored to the viral or infected-cell membrane by the
membrane-spanning region within TM. The functionally important domains
required for cellular recognition and receptor binding are contained
within SU, while TM mediates fusion of the viral and target cell
membranes (2, 4, 12, 17-21). By analogy to other
retroviral systems, it is likely that binding of gp46 to one or more
as-yet-uncharacterized cell surface receptors (12, 16, 26, 27,
32) brings the viral and cellular membranes into close proximity and
induces a conformational change within the envelope
glycoprotein complex. This alteration in envelope
conformation activates the fusion domain within gp21 and promotes the
TM-dependent fusion of the closely apposed viral and cellular membranes
(2, 21, 24).
Recently, a peptide scanning approach was used to identify synthetic
peptides derived from envelope that inhibit membrane fusion and
syncytium formation between HTLV-1-infected cells and target Molt-4 T
cells (22). Of the inhibitory peptides identified, one,
P-400, was derived from amino acids 400 to 425 of TM, while another,
P-197, was derived from amino acids 197 to 216 of the gp46 surface
glycoprotein (22). Given that the inhibitory
peptides map to distinct and nonoverlapping regions of envelope, it is likely that these peptides inhibit membrane fusion by functionally distinct mechanisms. In the case of P-197 it has been suggested that
the SU-derived peptide inhibits membrane fusion by competitively binding to a primary cellular receptor for HTLV-1 (22),
which was subsequently identified as heat shock cognate protein 70 (Hsc70) (23). This conclusion was based upon the
observations that P-197 binds to Hsc70 in vitro and that Hsc70 is
efficiently purified from cell lysates by affinity chromatography
against immobilized peptide (23). In support of the view
that P-197 inhibits membrane fusion by competing with SU for Hsc70
binding, it was reported that antibodies directed against Hsc70
antagonize HTLV-induced membrane fusion and block syncytium formation
(23). Here, we have further examined the functional
properties of P-197 and explored the requirement for Hsc70 in
cell-to-cell membrane fusion.
In our study, an inactive envelope-derived control peptide, P-80
(SLYLFPHWTKKPNRNGG; Mw, 2,118), and
the inhibitory peptide P-197 (DHILEPSIPWKSKLLTLVQL;
Mw, 2,331) were chemically
synthesized by standard techniques. HTLV-1 envelope-mediated
cell-to-cell fusion was monitored using a syncytium assay adapted from
published protocols (10, 16). Molt-4, Jurkat, and SupT-1 T
cells were resuspended at 2 × 106 cells/ml,
and 0.5-ml aliquots were seeded into the wells of a 24-well dish.
Subsequently, an approximately equal number of chronically HTLV-1-infected MT2 cells (0.5 ml) were added to each well, and the
cocultures were incubated at 37°C for 18 to 21 h. Syncytia were
scored by gently resuspending the cells, placing 40 µl from each
culture onto a gridded tissue culture dish (Nunc), and counting the
number of syncytia per sample by light microscopy using the high-power
objective. Using this assay we routinely found that cocultures of MT2
cells and target lymphocytic cells contained 800 to 2,000 syncytia/106 target cells (Fig.
1A). We observed no overt differences in
the abilities of Molt-4, Jurkat, or Sup-T1 cell lines to support
syncytium formation, and syncytia were not observed when target
lymphocytes were incubated in the absence of MT2 cells or when MT2
cells were incubated in the absence of target cells.
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FIG. 1.
Peptide P-197 inhibits HTLV-1 envelope-mediated
syncytium formation among diverse target cells. (A) Molt-4, Sup-T1, and
Jurkat target cells were cocultured with HTLV-1-infected MT2 cells in
the presence or absence of the indicated peptides (20 µg/ml). Control
cocultures receiving no peptide were incubated in the presence of
solvent only. The number of syncytia per 106 target cells
was evaluated by high-power light microscopy. Data are means and
standard deviations from six independent assays. (B) HeLa, HOS, or Cos
target cells were cocultured with HTLV-1 envelope-expressing HeLa cells
(transfected with the envelope expression vector pHTE-1) in the
presence or absence of the indicated peptides. The number of syncytia
per low-power field (LPF) was scored by light microscopy. Data are
means and standard deviations from three fields from three independent
assays.
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P-197 inhibits HTLV-1 syncytium formation among diverse target cell
populations.
To examine the inhibitory properties of the
SU-derived peptides, the control peptide P-80, the inhibitory peptide
P-197, or solvent only (dimethyl sulfoxide) was added to target cell
suspensions and allowed to equilibrate at 37°C for 30 min. MT2 cells
were then added to each well, and the cocultures were incubated for 18 to 20 h, whereupon the number of syncytia was scored as described above. Incubation of cocultures with the control peptide P-80 had
little or no effect upon the fusogenic activity of the
envelope-expressing MT2 cells, as cultures pretreated with P-80
contained as many syncytia as controls that received no peptide. In
contrast, coculture of MT2 cells and target cells in the presence of
P-197 resulted in a dramatic reduction in the number of syncytia formed
(Fig. 1A), and the inhibitory activity of the synthetic peptide was observed for all the T-cell lines tested. These results confirm the
findings of Sagara et al. (22) and further demonstrate
that the inhibitory properties of P-197 extend to a range of
lymphocytic cell lines in addition to the T-cell line Molt-4.
Since a variety of non-T-cell lines also support HTLV-1
envelope-mediated syncytium formation and viral entry (
16,
32,
25), we wished to determine if P-197 would interfere with
membrane
fusion in heterologous mammalian cell types. In these assays,
HeLa cells were transfected with the HTLV-1 envelope
glycoprotein
expression vector pHTE-1 (
6),
using Fugene 6 as directed by
the manufacturer (Boehringer Mannheim);
24 h later the cells were
lifted (phosphate-buffered saline
[PBS], 1 mM EDTA) and resuspended
at 1.5 × 10
5 cells/ml. The envelope-expressing HeLa cells
(1 ml) were subsequently
mixed and cocultured with a similar number of
target cells in
the wells of a six-well tissue culture dish. Target
HeLa (human
cervical carcinoma), HOS (human osteosarcoma), or Cos-1
(African
green monkey kidney) cells were incubated in the presence of
test
peptide or in the presence of solvent only for 30 min prior to
addition of the envelope-expressing effector cells. The following
day,
syncytia were scored by counting the number of multinucleate
giant
cells observed per low-power field by light
microscopy.
Coculture of envelope expressing HeLa cells with HeLa, HOS, or Cos-1
target cells resulted in rampant syncytium formation
(Fig.
1B). We
routinely found that the Cos-1 cell line produced
slightly (1.5- to
2-fold) more syncytia with envelope-expressing
HeLa cells than the
human cell lines tested. No syncytia were
observed in control
cocultures using mock-transfected HeLa cells.
Importantly, the control
peptide P-80 did not inhibit syncytium
formation in these assays (Fig.
1B). However, treatment of cocultured
cells with the peptide P-197
resulted in a marked (60 to 70%)
reduction in the number of syncytia
formed (Fig.
1B) compared
to control cocultures that had been treated
with solvent only
or with the inactive control peptide P-80. Moreover,
in the presence
of P-197 the sizes of the syncytia were greatly
reduced, as judged
by a decrease in the average number of nuclei within
each syncytium
(data not shown). This inhibitory effect of P197 appears
to be
specific for HTLV-1, since P197 does not inhibit syncytium
formation
induced by human immunodeficiency virus (
22) or
feline immunodeficiency
virus (Fig.
2).
Thus, the accumulating evidence indicates that
inhibition of membrane
fusion by P-197 is specific to HTLV-1 and
is not confined to
lymphocytic cells but is observed with a wide
variety of
HTLV-1-sensitive cell lines.
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FIG. 2.
The peptide P-197 does not block syncytium formation by
an unrelated retrovirus. HeLa cells transfected with the HTLV-1
envelope expression construct HTE-1 (HTLV) or the feline
immunodeficiency virus proviral clone pFIV-34TF10 (FIV) were cocultured
with untransfected target HeLa cells in the presence of the indicated
peptides. Syncytium formation is expressed as a percentage relative to
the number of syncytia obtained with each viral envelope in the absence
of peptide (Control). Data are means and standard deviations from three
low-power fields from three independent assays.
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Most HTLV-1 sensitive cells do not express detectable levels of
surface Hsc70.
Based upon the observation that P-197 binds to the
cellular protein Hsc70, Sagara et al. (23) suggested that
Hsc70 expressed on the cell surface acts as a receptor for HTLV-1 and
that P-197 inhibits syncytium formation by competitively binding to
this putative receptor. Since P-197 inhibits HTLV-1-induced syncytium formation for all cell lines tested, we examined these cells for surface expression of Hsc70. We wished to determine if surface expression of Hsc70 correlates with the ability of cells to support HTLV-1-induced syncytium formation and if inhibition of syncytium formation by P-197 requires surface expression of Hsc70.
Expression of Hsc70 on the surface of HTLV-1 sensitive cell lines was
examined by probing intact cells with the anti-Hsc70
monoclonal
antibody SPA-815 (Stressgen Biotechnologies Corp.)
and detecting the
bound antibody using fluorescein isothiocyanate
(FITC)-conjugated
secondary antibody and flow cytometry. Importantly,
the primary
antibody SPA-815 used in our studies was also used
by Sagara et al.
(
23) to demonstrate cell surface expression
of Hsc70 on
Molt-4 cells. Cells (2.5 × 10
5) were
incubated with monoclonal antibody SPA-815 (1 µg/ml) in
1 ml of RPMI
1640 medium supplemented with 10% fetal bovine serum.
Samples were
incubated at room temperature on a rotary mixer for
1 h. The cells
were spun down, washed in RPMI, and incubated with
secondary antibody,
FITC-conjugated anti-rat immunoglobulin G
(IgG) in RPMI medium at room
temperature for 30 min in the dark.
The cells were washed (PBS, 0.1%
sodium azide), fixed (0.5% paraformaldehyde
in PBS, pH 7.4), and
analyzed for antibody binding by flow cytometry
(FACScan;
Becton
Dickinson).
Surprisingly, we found that the vast majority of cell lines that
support HTLV-1 infection or envelope-mediated syncytium formation
did
not express detectable levels of Hsc70 on the cell surface
(Fig.
3). Moreover, in contrast to previously
reported data (
23),
the Molt-4 cell line used in our
studies (ATCC CRL-1582) did not
express significant amounts of surface
Hsc70. In fact, of all
the cell lines examined, only the T-cell line
SupT-1 expressed
high levels of surface Hsc70, and these cells were no
more prone
to syncytium formation than the Hsc70-negative Jurkat cells
(Fig.
1A and
3).
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FIG. 3.
Most cell lines permissive for HTLV-1 syncytium
formation do not express Hsc70 on the cell surface. Binding of the
anti-Hsc70 antibody SPA-815 (1 µg/ml) and the recombinant HTLV-1 SU
derivative sRgp46-Fc (0.6 µg/ml) to target cells was monitored by
flow cytometry. Binding of sRgp46-Fc (0.6 µg/ml) to cells in the
presence of SPA-815 (4 µg/ml) is also shown. In each case the blue
histograms represent basal fluorescence in the presence of irrelevant
control sera and the red histograms represent the fluorescence profile
in the presence of the detected test ligand. The data are summarized in
panel B.
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Importantly, we also examined this panel of cell lines for
receptors recognized by the HTLV-1 surface
glycoprotein gp46. In
these assays, we employed a
functional form of soluble recombinant
gp46 fused to the Fc
region of human IgG (sRgp46-Fc). We have
previously demonstrated that
sRgp46-Fc (referred to as SU-Fc)
(
12) exhibits the
biochemical and immunological properties of
SU and that this
recombinant protein retains the receptor binding
specificity of the
native viral glycoprotein (
12). In particular,
when added to target cells, sRgp46-Fc competitively inhibits
HTLV-1-induced
syncytium formation and viral entry into cells;
demonstrating
that sRgp46-Fc recognizes the primary cell surface
receptor used
by HTLV-1 (
12). As anticipated, all of the
HTLV-1-permissive
syncytium-sensitive cell lines bound sRgp46-Fc,
confirming that
all of these cells express functional receptors for
HTLV-1. Most
importantly, all of the cell lines that are competent for
syncytium
formation and that lack detectable levels of surface Hsc70
bind
sRgp46-Fc. Thus, from our study it would appear that cell surface
expression of Hsc70 is not required for syncytium formation or
for
binding of HTLV-1 SU to cells. To explore these ideas further,
we
examined the ability of the anti-Hsc70 antibody SPA-815 to
block
binding of sRgp46-Fc to cells. We found that pretreatment
of target
cells with monoclonal antibody SPA-815 (4 µg/ml) did
not prevent
binding of sRgp46-Fc to cells (Fig.
3). Significantly,
SPA-815 also
failed to block binding of sRgp46-Fc to Sup-T1, a
cell line which
expresses high levels of surface Hsc70 (Fig.
3).
Taken together, these
results indicate that Hsc70 is unlikely
to be the primary receptor
recognized by HTLV-1
SU.
An anti-Hsc70 monoclonal antibody does not inhibit syncytium
formation for most HTLV-1-sensitive cell lines.
It has been
reported that exogenous addition of the anti-Hsc70
monoclonal antibody SPA-815 inhibits syncytium formation
between cocultured HTLV-1-infected cells and the cell line Molt-4.
However, our results indicate that SPA-815 is unable to block binding
of sRgp46-Fc to cells, suggesting that Hsc70 is not the receptor recognized by gp46. Nevertheless, it is possible, though in our view
unlikely, that the soluble SU fusion protein does not recognize the
HTLV-1 primary receptor in the same way as the native virally expressed
envelope protein. We therefore examined the ability of SPA-815 to
interfere with syncytium formation between HTLV-1 envelope-expressing
cells and diverse target cell populations. In the first assay, the
lymphocytic cell lines Jurkat, Sup-T1, and Molt-4 were cocultured with
the chronically HTLV-1-infected cell line MT2 in the presence or
absence of SPA-815 or antibody directed against the cell adhesion
molecule 
2-integrin.
Anti-2-integrin was employed as a positive
control for these syncytium interference assays, as
2-integrin supports envelope-mediated membrane
fusion and antibodies recognizing this surface antigen block syncytium formation (3, 10). In keeping with previous reports
(3), we found that treatment of cocultured cells with
anti-2-integrin severely disrupted membrane
fusion, as revealed by a 40 to 60% reduction in the number of syncytia
observed in cocultures treated with this antibody (Fig.
4A). In contrast, the anti-Hsc70 antibody SPA-815 had no effect on syncytium formation when either the Jurkat or
Molt-4 cell lines were used as targets. However, when Sup-T1 cells were
cocultured with MT2 cells, we observed a marked inhibitory effect of
SPA-815 on syncytium formation (Fig. 4A), and the inhibitory activity
of SPA-815 was similar to that obtained with the
anti-2-integrin antiserum.
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FIG. 4.
Antibodies directed against Hsc70 do not block syncytium
formation for most HTLV-1-permissive cells. (A) Molt-4, Sup-T1, and
Jurkat cells were cocultured with HTLV-1-infected MT2 cells in the
presence or absence of the indicated antibodies (2 µg/ml). The
inhibition of syncytium formation relative to untreated control
cocultures is indicated (means and standard deviations from four
independent assays). (B) HeLa, HOS, and Cos target cells were
cocultured with HTLV-1 envelope-expressing HeLa cells in the presence
or absence of the indicated antibodies. The inhibition of syncytium
formation relative to untreated control cocultures is indicated (means
and standard deviations of triplicate determinations from three
independent assays).
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A second syncytium interference assay was performed in which HeLa cells
were transfected with the envelope-expressing plasmid
HTE-1 and the
transfected cells were subsequently cocultured with
HeLa, Cos, or HOS
target cells in the presence or absence of the
test antibodies. Once
again, we found that the anti-Hsc70 monoclonal
antibody had little or
no effect upon syncytium formation, while
the
anti-
2-integrin antibody efficiently
antagonized cell-to-cell
fusion (Fig.
4B). These data correlate well
with the flow cytometry
results (Fig.
3). Thus, the accumulating
evidence indicates that
the vast majority of HTLV-1-permissive cell
lines do not express
detectable levels of Hsc70 on the cell surface,
that Hsc70-negative
cell lines are competent for envelope-mediated
cell-to-cell fusion,
and that antibodies directed against Hsc70 do not
inhibit syncytium
formation for most target cell
populations.
P-197 does not compete with sRgp46-Fc for cell surface
binding.
It has been suggested that P-197 competes with HTLV-1 SU
for binding to the putative primary receptor Hsc70, and that binding of
P-197 to Hsc70 blocks recognition of this receptor by SU. Taken together, the data presented above indicate that Hsc70 is unlikely to
be the primary receptor recognized by HTLV-1 SU. Nevertheless, it is
possible that P-197 binds to some other cell surface antigen used as a
receptor by HTLV-1. To test this hypothesis, we examined the ability of
P-197 to block binding of sRgp46-Fc to HTLV-1-permissive target cells.
Jurkat cells (2.5 × 105) were preincubated
with or without P-197 (40 µg/ml) for 30 min at 25°C, whereupon
sRgp46-Fc (0.6 µg/ml) was added to the cell suspensions and incubated
for a further hour. The cells were washed to remove unbound sRgp46-Fc
and probed for bound sRgp46-Fc using FITC-conjugated anti-Fc antisera;
the cells were then washed and fixed, and the bound antibody was
detected by flow cytometry.
Compared with untreated cells, Jurkat cells incubated with sRgp46-Fc
demonstrated a marked increase in FITC-specific fluorescence,
consistent with the binding of sRgp46-Fc to the cell surface (Fig.
5). Surprisingly, we found that the
synthetic peptide P-197, even
in vast excess, did not block binding of
sRgp46-Fc to Jurkat cells
(Fig.
5). In comparison, binding of the SU
immunoadhesin sRgp46-Fc
to cells was clearly blocked by preincubation
of cells with untagged
sRgp46 (0.6 µg/ml), indicating that these gp46
derivatives compete
for the same primary cell surface receptor.
Moreover, pretreatment
of sRgp46-Fc with a neutralizing antiserum,
SP2-3 (
17), which
is reactive against native viral gp46,
also blocked binding of
SRgp46-Fc to cells. These results indicate to
us that the inhibitory
peptide P-197 does not disrupt primary receptor
binding by the
HTLV-1 surface glycoprotein.
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FIG. 5.
The peptide P-197 does not prevent binding of sRgp46-Fc
to cells. Jurkat cells were treated with P-197 (40 µg/ml) and, for
comparison, sRgp46 (0.6 µg/ml) or neutralizing anti-SU antiserum
SP2-3 (1:1,000). sRgp46-Fc was added and incubated for 1 h with
mixing. Bound sRgp46-Fc was detected using anti-Fc FITC-conjugated sera
and flow cytometry. Basal fluorescence of control samples was
determined from cells treated with irrelevant nonspecific IgG. Means
and standard deviations from three independent assays are shown.
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The unambiguous identification and characterization of the cellular
receptor recognized by HTLV-1 has remained elusive. Consequently,
there
is considerable interest in defining the molecular events
that promote
envelope-mediated entry of HTLV-1 into cells. Recently,
Sagara et al.
(
22) reported that a synthetic peptide derived
from the
HTLV-1 surface glycoprotein potently inhibits
envelope-mediated
membrane fusion and syncytium formation. It was
suggested that
the inhibitory peptide, P-197, competes with SU for
binding to
a cellular receptor required for HTLV-1 infection.
Subsequently,
a cellular P-197-binding protein, Hsc70, was identified
and proposed
as a candidate receptor for HTLV-1. These
suggestions were intriguing,
since many cell types constitutively
express Hsc70 as a soluble
cytoplasmic protein, and to our knowledge a
signal sequence for
membrane targeting has not been identified within
Hsc70. Moreover,
as a cellular chaperonin Hsc70 is thought to bind to
nascent or
incorrectly folded proteins (
28,
33) and to
peptides produced
in vivo during antigen processing (
15,
27). We have therefore
reexamined the properties of P-197 and
explored the contribution
of Hsc70 to HTLV-1 envelope-induced membrane
fusion.
As reported by Sagara et al. (
22)
, we confirm
that the surface glycoprotein-derived peptide P-197
efficiently interferes
with HTLV-induced membrane fusion and syncytium
formation. Importantly,
we have extended those initial findings by
demonstrating that
the inhibitory properties of P-197 are not confined
solely to
Molt-4 T cell targets but are also observed with other T-cell
and non-T-cell lines. In fact, P-197 inhibits syncytium formation
among
all the HTLV-1-permissive and syncytium-proficient cell
lines tested.
Most surprisingly, we have found that although P-197
potently inhibits
syncytium formation, the peptide was unable
to block direct binding of
a recombinant HTLV-1 envelope protein
to cells. Our results suggest to
us that P-197 does not inhibit
syncytium formation by blocking viral
recognition of a cell surface
receptor.
Based upon the data reported here and for the reasons given below, we
suggest that Hsc70 is unlikely to be a critical receptor
for HTLV-1
entry into cells. First, the majority of HTLV-1-permissive
cells do not
express detectable levels of Hsc70, or express only
exceedingly
low levels of this surface antigen. Second, compared
to Hsc70-negative
cells, cell lines that express high levels of
surface Hsc70 do
not exhibit increased sensitivity to syncytium
formation or greater
resistance to syncytium interference by P-197.
Third, antibodies
directed against Hsc70 do not block syncytium
formation for the
majority of permissive cells, and anti-Hsc70
antibodies do not prevent
binding of recombinant surface glycoprotein
to target
cells. Taken together, these results are inconsistent
with the view
that Hsc70 acts as a critical receptor for HTLV-1
entry. Why should
Hsc70 bind to isolated peptides derived from
envelope or bind to
envelope protein expressed in mammalian cells?
The answer may lie in
the normal physiological role of Hsc70.
As a constitutively expressed
member of the heat shock family
of chaperonins, Hsc70 binds to nascent
proteins facilitating protein
folding and promoting oligomerization of
multiprotein complexes
(
28,
33). Therefore, binding of
Hsc70 to envelope may be a
normal physiological event that promotes
correct processing and
assembly of envelope within cells. A precedent
for this type of
interaction has been observed for membrane proteins of
both cellular
and viral origin and the endoplasmic reticulum-resident
chaperonin
BiP (
9,
13,
14). Alternatively, the
immunoprecipitation
techniques used to demonstrate interaction of Hsc70
with envelope
may partially unfold envelope, thereby promoting
association of
Hsc70 with the partially denatured protein. In addition,
Hsc70
also binds to the synthetic peptide P-197. However, it is well
known that Hsc70 binds to many classes of peptide (
30,
31,
33), and one suggested function for Hsc70 is to bind and deliver
peptides for presentation in the immune response (
15,
27).
Therefore, Hsc70 may possess affinity for particular
classes of
peptide, and binding of P-197 to Hsc70 may merely reflect
the
intrinsic affinity of Hsc70 for peptides. These alternative
explanations
for the association of Hsc70 with P-197 have yet to be
fully
explored.
Although anti-Hsc70 antibodies do not interfere with membrane fusion
for most target cells, inhibition of syncytium formation
was observed
with Sup-T1 cells. However, Sup-T1 cells express
high levels of surface
Hsc70. Consequently, inhibition of syncytium
formation by anti-Hsc70
antibodies may be due to steric effects
that occlude the HTLV-1
receptor and block receptor engagement
by envelope. Such inhibitory
mechanisms need not necessarily involve
direct binding of antibody to
the receptor, as similar inhibitory
effects on HTLV-1 syncytium
formation have been observed for antisera
directed against molecules of
the major histocompatibility complex
(
11). Thus, the
proximity of a bulky surface-bound antibody
molecule to a small and
easily masked receptor epitope may be
all that is required to block
access of the virally expressed
envelope protein to the receptor,
thereby disrupting membrane
fusion. In contrast, the sRgp46-Fc fusion
protein used in this
study may enjoy unrestricted access to the
surface-bound receptor,
even in the presence of anti-Hsc70 antibodies,
since the recombinant
protein is freely diffusible and unrestrained by
presentation
on a cellular
membrane.
An important finding of this study is that, even at high
concentrations, the peptide P-197 does not inhibit binding of
recombinant
SU to cells. These data indicate to us that P-197 does not
inhibit
membrane fusion by competitively binding to a primary cellular
receptor that is recognized by HTLV-1. Although the mechanism
by which
P-197 disrupts membrane fusion is not yet clear, several
models can be
proposed for the observed inhibitory activity: One
possibility is that
the peptide disrupts an interaction with a
secondary receptor required
for HTLV-1 entry. If this is the case,
it is unlikely that Hsc70 is the
coreceptor, since most HTLV-1-sensitive
cells lack detectable levels of
this protein on the cell surface.
Alternatively, P-197 may disrupt
critical interactions between
envelope subunits. Retroviral
envelope glycoproteins form the
characteristic "knobs"
or "spikes" that have been observed on
virions or infected
cells. These spikes likely consist of a trimeric
association of SU-TM
complexes, held together by intersubunit
interactions between TM-TM,
SU-TM, and perhaps SU-SU subunits.
We suspect that perturbation of the
envelope glycoprotein complex
by synthetic peptides would
have a profound inhibitory effect
on envelope-mediated membrane fusion.
Finally, a growing body
of evidence suggests that retroviral envelope
glycoproteins undergo
dramatic alterations in conformation
upon receptor engagement
(
2,
8,
24,
34). These structural
transitions represent
critical events in envelope-catalyzed membrane
fusion, and elegant
studies have demonstrated that interfering with
these molecular
transitions can block membrane fusion
(
24). Thus, a particularly
attractive model to account for
the inhibitory activity of P-197
is that P-197 binds directly to HTLV-1
SU and interferes with
a conformational change in SU that is a
prerequisite for membrane
fusion. We are now designing experimental
strategies to explore
the validity of these alternative
models.
In conclusion, our studies indicate that the cellular factor
Hsc70 is not the direct target of the fusion-inhibitory
peptide
P-197 and that Hsc70 is not the receptor recognized by
HTLV-1.
Importantly, we now suggest that the synthetic peptide P-197
inhibits
envelope-mediated membrane fusion by a mechanism that does not
involve competition for a primary cellular receptor. Understanding
the
mechanism by which synthetic peptides, such as P-197, inhibit
membrane
fusion may provide new opportunities for the rational
development of
clinically useful antagonists of HTLV-1
infection.
| |
ACKNOWLEDGMENTS |
We thank the members of the HTLV-1 European Research Network (HERN)
for reagents and helpful discussions and Richard Pöhler for
review of the manuscript.
This work was funded in part by grants from the Leukemia Research Fund,
Tenovus-Scotland, and the Misses Barrie Charitable Trust to D.W.B.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biomedical
Research Centre, Ninewells Hospital and Medical School, Level 5, University of Dundee, Dundee DD1 9SY, Scotland. Phone: 44 (0)1382
660111, ext. 33513. Fax: 44 (0)1382 669993. E-mail:
brighty{at}icrf.icnet.uk.
| |
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Journal of Virology, November 2001, p. 10472-10478, Vol. 75, No. 21
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.21.10472-10478.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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