From the Department of Biological Chemistry, The
Weizmann Institute of Science, Rehovot, 76100 Israel and the
¶ Laboratory of Experimental and Computational Biology, NCI,
National Institutes of Health, Frederick, Maryland 21702-1201
Received for publication, May 15, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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DP178, a synthetic peptide corresponding to a
segment of the transmembrane envelope glycoprotein (gp41) of human
immunodeficiency virus, type 1 (HIV-1), is a potent inhibitor of viral
infection and virus-mediated cell-cell fusion. Nevertheless, DP178 does not contain gp41 coiled-coil cavity binding residues postulated to be
essential for inhibiting HIV-1 entry. We find that DP178 inhibits
phospholipid redistribution mediated by the HIV-1 envelope glycoprotein
at a concentration 8 times greater than that of solute redistribution
(the IC50 values are 43 and 335 nM,
respectively). In contrast, C34, a synthetic peptide which
overlaps with DP178 but contains the cavity binding residues, did not
show this phenomenon (11 and 25 nM, respectively). The
ability of DP178 to inhibit membrane fusion at a post-lipid mixing
stage correlates with its ability to bind and oligomerize on the
surface of membranes. Furthermore, our results are consistent with a
model in which DP178 inhibits the formation of gp41 viral hairpin
structure at low affinity, whereas C34 inhibits its formation at high
affinity: the failure to form the viral hairpin prevents both lipid and
solute from redistributing between cells. However, our data also
suggest an additional membrane-bound inhibitory site for DP178 in the
ectodomain of gp41 within a region immediately adjacent to the
membrane-spanning domain. By binding to this higher affinity site,
DP178 inhibits the recruitment of several gp41-membrane complexes, thus
inhibiting fusion pore formation.
The first step in HIV-11 infection involves the binding of
the viral envelope glycoproteins
gp120-gp41 to CD4 (1-3) and subsequently to a co-receptor (4-8) (for recent review, see Refs. 9-11).
Consequently, gp41 undergoes conformational changes that mediate the
fusion between the viral and the cellular membranes or between infected and healthy cells (12, 13). Gallaher and co-workers (14, 15) postulated
a model of gp41, identifying a fusion peptide followed by a
leucine/isoleucine zipper-like sequence (N-helix) and an amphipathic
helical segment (C-helix) in the viral glycoprotein. The
indispensability of the fusion peptide for viral infection was
confirmed by site-directed mutagenesis (16, 17). Furthermore, gp41 was
found to contain a protease-resistant core consisting of the postulated
N- and C-helices (18). Specifically, peptides corresponding to these
sequences co-crystallized as a six-helix bundle in which the N- and
C-helices are arranged in a three-hairpin structure (19-21). Three N
peptides form a coiled-coil, and the C peptides are packed in an
antiparallel manner into highly conserved, hydrophobic grooves on the
surface of the coiled-coil. Recently, the solution and crystal
structures of the ectodomain of the Simian immunodeficiency virus gp41,
consisting of those two helices as well as the loop connecting them,
confirmed the interplay of the N- and C-helices (22, 23). Remarkably,
the coiled coil is a common motif found in many diverse viral membrane
fusion proteins (24), as well as in proteins involved in vesicular
transport (25-31).
A synthetic peptide overlapping the C-terminal amphipathic helical
segment of gp41 and its tryptophan-rich sequence (32) (DP178, Fig. 1)
was reported to inhibit virus infection at extremely low concentration
(33). Remarkably, DP178 blocks cell fusion and viral entry at a
concentration of less than 2 ng/ml in vitro and was reported
to be a promising drug for treating HIV-1-infected humans (34-37).
Since DP178 is a potent inhibitor of HIV-1-induced membrane fusion,
elucidating its mode of action is of major importance. In essence, this
means finding the stage at which DP178 inhibits the formation of the
fusion-active conformation of gp41. It was suggested that the antiviral
mode of action of DP178 involves interaction with the gp41
leucine/isoleucine zipper motif (38-41). However, DP178 (residues
638-673) does not contain the residues (Trp-628, Trp-631, and Ile-635)
that were shown to be crucial for binding the prominent cavity in the
coiled coil of gp41 (59). Weiss and colleagues (42) demonstrated that
DP178 binds gp41 and inhibits envelope-mediated membrane fusion only
after gp120 interacts with cellular receptors. It is currently accepted
that DP178 binds to the gp41 leucine/isoleucine zipper sequence before the hairpin structure is formed (43-45), thus preventing the HIV envelope glycoprotein from adopting a fusogenic conformation. This
model is supported by the results of Blumenthal and co-workers (6, 46),
suggesting that the pre-hairpin intermediate stage appears to be
induced rapidly upon interaction of gp120 with CD4 and the co-receptor,
but is then relatively stable for several minutes, allowing DP178 to
interact with the exposed leucine/isoleucine zipper sequence.
However, DP178 was reported to inhibit redistribution of lipid and
aqueous dyes as a result of HIV-1 envelope glycoprotein-mediated fusion
with significantly different values of IC50 (46). Binding of DP178 to the leucine/isoleucine zipper sequence in the pre-hairpin intermediate should inhibit both lipid and aqueous redistribution, suggesting the existence of two target sites for inhibition by DP178.
In an attempt to identify an additional target site for inhibition by
DP178, we investigated potential interactions of DP178 with the
membrane, and with the leucine/isoleucine zipper sequence within the
membrane. We find that DP178 binds to the membrane and oligomerizes
within, but does not interact with the leucine/isoleucine zipper
sequence within the membrane. We suggest that by binding to its
corresponding segment in gp41, DP178 inhibits fusion pore formation.
Peptides Preparation and Fluorescent Labeling--
DP178, N36,
and C34 were synthesized by using the Boc chemistry, as described
previously (47, 48). Concentrations were measured by tryptophan and
tyrosine absorbance (at 280 nm) in 8 M urea (49). Labeling
of the N terminus of synthetic peptides was achieved as described
previously (50, 51). Briefly, resin-bound peptides, with their amino
acid side chains fully protected, were treated with trifluoroacetic
acid, to remove the BOC protecting group from their N-terminal
amino groups, while keeping all the other reactive amine groups of the
attached peptides still protected. The resin-bound peptides were then
reacted with the desired fluorescent probe, cleaved from the resins by
hydrogen fluoride, and finally precipitated using ether. This
procedure yielded peptides selectively labeled with fluorescent probes
at their N-terminal amino acids. The synthetic peptides were purified
by reverse-phase high performance liquid chromatography on an
analytical C18 Vydac column 4.6 × 250 mm (pore size
of 300 Å). The column was eluted in 80 min, at a flow rate of 0.6 ml/min, using a linear gradient of 25-80% acetonitrile in water, in
the presence of 0.05% trifluoroacetic acid (v/v).
Dye Transfer Fusion Assay--
Peptide inhibition of cell-cell
fusion was assayed by monitoring the redistribution of fluorescent
probes, both water soluble and lipophilic, between target and effector
cells upon their co-incubation with each other (46). The HIV-1 gp120-41
expressing TF228 cells (52) were labeled with either calcein or a green
fluorescent fatty acid. Cells incubated with 1 µM calcein
for 60 min at 37 °C were then washed and resuspended at 105 cells/Ml
in RPMI. Cells labeled with the fatty acid
(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid
(C1-BODIPY-C12)) were seeded at 105 cells/ml in
RPMI medium containing 10 mg/ml of the
C1-BODIPY-C12 and grown for 3 days prior to
experiment (Molecular Probes, Eugene, OR) (53). The fatty acid was
eventually metabolized into phospholipid, primarily phosphatidylcholine
(data not shown). The cells where then washed and resuspended at 105 cells/ml. Lipid (BODIPY) dye and aqueous (calcein) dye transfer were
measured in separate experiments. The target cells, CD4+ CXCR4+ 3T3
mouse fibroblasts, were plated at 105 cells/ml the night
before the experiment. The next day, they were labeled with 20 µM CMTMR for 1 h at 37 °C, washed several times
and combined with the effector cells (1:3 target-effector cell ratio).
Different concentrations of peptides, dissolved in phosphate-buffered
saline or Tris buffer, were then added. The cells were co-cultured for
2 h at 37 °C in 12-well plates (Costar, Cambridge, MA). Phase
and fluorescent images were collected using an Olympus IX70 coupled to
a CCD camera (Princeton instruments, Trenton, NJ) with a ×20 objective
lens. An 82,000 optical filter cube (Chroma technology corp.,
Brattleboro, VT) was used for the excitation of calcein (494/517),
BODIPY (500/510), and CMTMR (541/565). Three images per well were
collected and then analyzed using metamorph software (Universal
imaging, West Chester, PA) for dye transfer from the donor to the
acceptor cell. The scoring of fusion events was conducted as previously
(46).
Preparation of Lipid Vesicles--
Dry phospholipids were
hydrated in phosphate-buffered saline and dispersed by vortexing to
produce large multilamellar vesicles. Small unilamellar vesicles (SUV)
were then prepared by sonication.
DNS-tryptophan FRET--
The binding of a peptide to the
membrane was detected by using FRET from the tryptophan residues of the
peptide to DNS chromophores incorporated into lipid vesicles, as
described previously (54). Excitation was set at the tryptophan maximum
absorbance peak (280 nm), and binding was detected by measuring the
increase in the sensitized DNS fluorescence (518 nm), thus diminishing
light scattering.
Membrane Partition of the Peptides--
NBD fluorescence
increases directly with the increase in the environmental
hydrophobicity. The degree of peptide association with lipid membranes
was measured by titration of the NBD-labeled peptide with lipid
vesicles in phosphate-buffered saline. The fluorescence intensity was
measured with excitation set at 467 nm, and emission set at 530 nm. The
fluorescence values were later corrected by taking into account the
dilution factor corresponding to the addition of microliter amounts of
liposomes and by subtracting the corresponding blank (buffer with the
same concentration of vesicles). The binding isotherms were analyzed as
partition equilibria (55, 56), as described in detail previously (50).
The partition coefficient (K) is determined from the slope
of the curve. Free energy ( NBD-Rho FRET--
The interaction of peptides within the
membrane was evaluated using NBD-labeled peptides serving as donors and
rhodamine-labeled peptides as energy acceptors (57, 58). In a typical
experiment, a donor peptide was added to a dispersion of SUV in
phosphate-buffered saline, followed by adding the acceptor peptide in
several sequential doses. Fluorescence spectra were obtained before and
after adding the acceptor. The efficiency of energy transfer
(E) was determined by measuring the decrease in the quantum
yield of the donor as a result of the presence of the acceptor.
E was determined experimentally from the ratio of the
fluorescence intensities of the donor in the presence
(Ida) and absence (Id) of
the acceptor, at the donor's maximum emission wavelength (530 nm and
524 nm for NBD-N36 and NBD-DP178, respectively), E = (1
The correction for the contribution of acceptor emission as a result of
direct excitation was made by subtracting the signal produced by the
acceptor-labeled analogue alone. The contributions of the buffer and
vesicles were also subtracted from all measurements.
Two Overlapping Anti-HIV-1 Peptides Inhibit Cell-Cell Fusion via a
Different Mechanism--
Previously, we reported that DP178 inhibits
redistribution of lipid and aqueous dyes as a result of HIV-1 envelope
glycoprotein-mediated fusion with significantly different values of
IC50 (46). To investigate DP178 mode of action, we compared
its inhibition ability with that of C34, another antiviral peptide
which overlaps with DP178 in 24 out of its 34 residues (59) (Fig.
1). We performed a series of new
experiments to investigate inhibition of HIV-1 envelope
glycoprotein-mediated fusion by DP178 and C34. The experiments with the
two inhibitors were done on the same cells labeled the same way. In our
previous study (46) we used lipophilic carbocyanine membrane probes
which are incorporated into the plasma membrane from the outside and
have the potential of nonspecifically exchanging between cells. To
avoid potential nonspecific dye transfer we used in this study
phospholipid analogs that were produced in the cells by metabolic
incorporation of BODIPY-labeled fatty acids (53).
Fig. 2 shows the difference between
inhibition by the two peptides. The data was plotted and then fit to a
hyperbolic decay function. The concentration at which 50% inhibition
is reached (IC50) can be extracted from each curve. The
IC50 for the DP178 peptide was ~43 ± 10 nM (R2 = 0.96) and 335 ± 125 nM (R2 = 0.89) for solute and lipid
mixing, respectively, while C34 was estimated at 11 ± 3 nM (R2 = 0.97) and 25 ± 4 nM (R2 = 0.98) for solute and lipid
mixing, respectively. The difference in IC50 between solute
and aqueous redistribution of dye for DP178 is 8-fold, whereas that for
C34 is only about 2-fold. In addition, statistical t tests
were conducted on each point in both these curves to compare contents
versus lipid mixing. There is no statistical difference for
any of the concentrations tested for C34, but 100, 200, 400, and 800 nM concentrations of DP178 show a statistical difference
(see Fig. 2).
A reasonable explanation for this difference is that DP178 has two
target sites (46). One is the leucine/isoleucine zipper sequence of
gp41 before it binds the membrane (43-45). We further checked whether
interactions within the membrane may reveal another binding site.
DP178 Binds to the Membrane--
We detected the binding of
peptides to the membrane by using fluorescence resonance energy
transfer (FRET) from the polypeptide tryptophan residues to DNS
chromophores incorporated into the lipid vesicles. Excitation was set
at the tryptophan maximum absorbance (280 nm), and binding was detected
by measuring the changes in the sensitized DNS fluorescence (518 nm).
When DP178 (0.45 µM) was added to 68 µM
PS/PC/cholesterol/DNS-PE (8:8:2:1, w/w) SUV (Fig.
3a), we observed a substantial
increase in the DNS fluorescence, indicating that DP178 binds to the
membrane. Since the DNS groups are attached to the head groups of the
phospholipids, DP178 must be located near the surface of the membrane.
In contrast, when C34 (0.45 µM) was added to the vesicles
(Fig. 3b), there was no change in the DNS fluorescence,
indicating that the tryptophans of C34 do not interact with the DNS
groups, or that the interaction is too weak to be detected by this
assay (see also Fig. 4). Interestingly, while treatment of HIV-1-infected cells with soluble CD4 increases binding of a monoclonal antibody (98-6) to an epitope that spans residues 644-663 (overlaps with C34 and DP178, see Fig. 1), binding of
a monoclonal antibody (2F5) to an epitope that spans residues 662-667
(overlaps with DP178, but not with C34) was reduced (60). This is
consistent with a model in which the conformational change of gp41
results in binding the DP178 region to the membrane, thus hiding the
corresponding epitope.
The biological relevance of this membrane-binding feature is supported
by the location of DP178 just upstream of the transmembrane domain.
Experiments performed with either negatively charged
PS/PC/cholesterol/DNS-PE (8:8:2:1, w/w) vesicles or zwitterionic
PC/cholesterol/DNS-PE (16:2:1, w/w) vesicles qualitatively yielded
similar results (data not shown), indicating that nonelectrostatic
forces are involved in the peptide-membrane interactions.
Determination of the Membrane Binding Energy of the
Peptides--
The high sensitivity of NBD fluorescence to the polarity
of its environment can be used to detect and quantify membrane binding (50, 61). When SUV (PS/PC, 1:1) were added to NBD-labeled DP178 or C34,
an increase concomitant with a blue shift of the NBD fluorescence was
observed, suggesting that both peptides bind to the membrane. To
evaluate the biological relevance of their membrane binding ability, we
measured the energy of peptide-membrane interactions. To this end,
NBD-labeled C34 (62) or DP178 (0.5 µM) were titrated with
SUV. Plotting the resulting increase in the fluorescence intensities of
NBD-labeled peptides as a function of lipid/peptide molar ratios
yielded conventional binding curves (Fig. 4a). The binding
isotherms for the peptides can be obtained by plotting Xb*
(the molar ratio of bound peptide per lipid in the outer leaflets)
versus the equilibrium concentration of free peptide in
solution. The surface partition coefficient (K) is calculated from the slope of the curve. The curve for the binding of
DP178 to phospholipid vesicles has two phases, suggesting positive cooperativity for this binding (Fig. 4b, filled
squares). The initial slope reveals K of 4 × 104 M
The inhibitory activity of DP178 can be partially explained by its
ability to bind to the gp41 leucine/isoleucine zipper sequence before
binding to the membrane (43-45). Here, we examined whether the same
sequence may also be the target for inhibition within the membrane.
The Leucine/Isoleucine Zipper Sequence Is Not a Target for
Inhibition by DP178 within the Membrane--
DP178 interacts with the
leucine/isoleucine zipper sequence (represented by N36, see
Fig. 1) in aqueous solution (45). Contrary, NBD-Rho FRET experiments
that used NBD-DP178 and Rho-N36 (Fig. 5,
open squares) or NBD-N36 and Rho-DP178 (Fig. 5, open
circles) revealed efficiencies of energy transfer that were close
to the calculated values assuming a random distribution of donors and acceptors (Fig. 5, dashed line) (58). These data suggests
that membrane-bound DP178 does not specifically interact with
membrane-bound N36, therefore the leucine/isoleucine zipper sequence is
not the target for inhibition by DP178 within the membrane.
Membrane-bound DP178 Forms Homo-oligomers--
The ability of
DP178 to oligomerize within the membrane was also measured by NBD-Rho
FRET. When Rho-DP178 (a final concentration of 0.035-0.105
µM) was added to a mixture of NBD-DP178 (0.11 µM) and SUV (303 µM), a
dose-dependent quenching of the donor's emission, which is
consistent with energy transfer, was observed (Fig. 5, filled
squares). Note that the acceptor peptide was added only after the
donor peptide was already bound to the membrane, thus preventing any
association in solution. The lipid/peptide ratio in these experiments
was kept high to create low surface density of donors and acceptors to
reduce the energy transfer between unassociated peptide monomers. To
confirm that the observed energy transfer was due to peptide
aggregation, we compared the transfer efficiencies observed in the
experiments with the energy transfer expected for randomly distributed
membrane-bound donors and acceptors (Fig. 5, dashed line).
The results reveal that membrane-bound DP178 forms homo-oligomers.
The striking similarities between structural motifs in various
viral envelope glycoproteins led to the notion that the native conformation of gp41 is metastable and it is stabilized by gp120 (63).
Upon binding of gp120 to its receptors, gp41 is free to form the more
energetically favorable hairpin structure (Fig. 6, a-c). The gp41 ectodomain
core is a six-helix bundle composed of three helical hairpins, each
consisting of an N-helix paired with an antiparallel C helix. The
N-helices form an interior, trimeric coiled coil with three conserved,
hydrophobic grooves; a C-helix packs into each of these grooves. This
structure is believed to correspond to the core of the fusion-active
state of gp41 and shows similarity to the proposed fusogenic structures of envelope fusion proteins from influenza (64), Moloney murine leukemia virus (65, 66), simian parainfluenza virus 5 (67), Ebola virus
(68), and simian immunodeficiency virus (22, 23, 69). Synthetic C
peptides (peptides corresponding to the C-helix), such as DP178 and
C34, potently inhibit membrane fusion by both laboratory adapted
strains and primary isolates of HIV-1 (33). The structural features of
the gp41 core suggest that an intermediate, in which the
leucine/isoleucine zipper sequence is not associated with the C-heptad
repeat, exists before the formation of the hairpin structure, and is
the target for inhibition by C-peptides (43-45). However, no direct
evidence for the existence of this pre-hairpin intermediate exists, and
an alternative for the mechanism of inhibition exerted by the
C-peptides, based on the observed monomer-trimer equilibrium of the
simian immunodeficiency virus gp41 has been postulated (22, 70).
According to the alternative model, gp41 exists in equilibrium between
monomer and trimer. In the presence of excess inhibitory peptide, the
equilibrium is driven from homotrimeric gp41 to a heterotrimer of gp41
and C-peptide. Since the peptides are only effective upon gp120
dissociation (42), it follows that the absence of fusogenic activity
displayed by the gp41-peptide heterotrimers is due to the fact that the
heterotrimers can no longer present a sufficient number of fusion
peptides to the target membrane for effective fusion to take place.
Another explanation could be that the homotrimer structure is required
for further oligomerization. The data also suggest that the
homotrimeric state is stabilized by gp120 such that heterotrimers
(which would be expected to bind less tightly to gp120 since they do
not possess a trimeric loop structure) cannot be formed in the presence
of bound gp120 (42). Anyway, the peptides act through a
dominant-negative mechanism, in which exogenous C peptides bind to the
leucine/isoleucine zipper sequence (N-helix) of gp41 and block further
conformational changes needed for fusion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G) was calculated according to
G =
RT
ln([H2O]·K). The universal gas constant was
taken as R = 1.987 cal/(K mol), and H2O
concentration is 55.6 M.
Ida/Id)100%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic view of gp41, showing the location
and sequences of the peptides corresponding to the protease-resistant
core (N36 and C34), and to the antiviral peptide DP178.
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Fig. 2.
Comparison of lipid mixing versus
contents mixing for C34 (a) and DP178
(b). HIV-1 envelope expressing TF228 cells were
labeled with either a water-soluble (calcein) dye or a fluorescent
lipid (C1-BODIPY-C12), and the CD4+ CXCR4+ 3T3 cells were labeled with
CMTMR. After 2 h of co-culture at 37 °C, the cells were
examined for lipid and solute dye transfer. The amount of mixing is
presented relative to the control, which is in absence of peptide.
Three fields are taken of each sample and averaged. The data represent
the average of two to four independent experiments. The error
bars represent the standard error of these experiments. The
filled diamonds represent the calcein mixing while
filled squares represents the lipid mixing. The
concentration at which 50% inhibition is reached (IC50)
can be estimated for each data set: (a) C34 plot yields an
IC50 of 11 ± 3 nM
(R2 = 0.97) and 25 ± 4 nM
(R2 = 0.98) for the solute and lipid mixing,
respectively, (b) DP178 plot yields an IC50 of
43 ± 10 nM (R2 = 0.96) and
335 ± 125 nM (R2 = 0.89) for
the solute and lipid mixing, respectively. Statistical t
tests were conducted on each point in both these curves to compare
contents versus lipid mixing. There is no statistical
difference for any of the concentrations tested for C34, but 100 (p = 0.000003), 200 (p = 0.0049), 400 (p = 0.00019), and 800 (p = 0.002)
nM concentrations of DP178 show a statistical
difference.
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Fig. 3.
Detection of peptide binding to the
membrane. 0.45 µM DP178 (a) or C34
(b) were added to 68 µM lipid vesicles
containing 5% DNS-PE. Excitation, 280 nm; emission, 518 nm.
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Fig. 4.
a, increase in the fluorescence of
NBD-DP178 (filled squares) or NBD-C34 (empty
circles) (0.5 µM each) upon titration with PS/PC
(1:1) phospholipid vesicles. b, binding isotherms derived
from panel a by plotting Xb* (molar ratio
of bound peptide per lipid in outer leaflet) versus
Cf (the equilibrium concentration of free peptide in
solution).
1 (
G =
8.7 Kcal/mol). Remarkably, C34 binds >10-fold weaker and with no
cooperativity (Fig. 4b, open circles). Its surface partition coefficient was 3 × 103 M
1
(
G =
7.1 Kcal/mol).
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Fig. 5.
Theoretically and experimentally derived
percentage of energy transfer. Transfer efficiencies between donor
and acceptor-DP178 (filled squares), donor-DP178 and
acceptor-N36 (open squares), and donor-N36 and
acceptor-DP178 (open circles) are plotted versus
the bound-acceptor/lipid molar ratio. A theoretical plot showing energy
transfer efficiency as a function of the surface density of the
acceptors, assuming random distribution of donor and acceptor monomers
(57), and Ro = 51 Å (58), is given for comparison
(dashed line). Excitation, 467 nm; emission, 500-600
nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
DP178 inhibits HIV-1 entry at two different
stages. The upper panel is based on the
existence of the pre-hairpin intermediate as suggested by Kim, Wiley,
and colleagues (43, 44): a, native state of HIV-1 envelope
glycoprotein; b, upon binding gp120 (green) to
cellular receptors (not included in the figure), a conformational
change in gp41 is induced, allowing an extension of the N-terminal
fusion peptide (black) toward the cellular membrane.
Subsequently, the leucine/isoleucine zipper (light blue) and
the C-terminal amphipathic sequences (blue) form the
three-hairpin conformation (c). Binding of DP178 to the
leucine/isoleucine zipper sequence can block this step (43-45). The
resulting proximity of the viral and the cellular membranes within a
limited area may allow lipid redistribution. Subsequent steps, not all
of them defined, result in fusion between the viral and the cellular
membranes. Among these steps, several membrane-bound gp41 oligomeric
units interact to form a fusion pore. This step is blocked when DP178
peptide binds to the membrane-bound state of the DP178 region in gp41.
The lower panel is based on the existence of a
monomer-trimer equilibrium of gp41 ectodomain as suggested by Clore and
colleagues (22, 70): I, native state of HIV-1 envelope
glycoprotein. Upon binding of gp120 (green) to cellular
receptors (not included in the figure), shedding of gp120 and a
conformational change in gp41 is induced. It is not known whether the
result is the three-hairpin conformation (IIa), or a
monomeric hairpin conformation (IIb) that will then
trimerize. The resulting proximity of the viral and the cellular
membranes within a limited area may allow lipid redistribution. Binding
of DP178 to the monomeric form shifts this equilibrium toward the
monomer. Subsequent steps, not all of them defined, result in fusion
between the viral and the cellular membranes. Among these steps,
several membrane-bound gp41 oligomeric units interact to form a fusion
pore. This step is blocked when DP178 binds to the membrane-bound state
of the gp41 segment that has a sequence identical to DP178.
Within each coiled-coil interface is a deep cavity, formed by a cluster
of residues in the N-helix coiled coil that has been proposed to be an
attractive target for the development of antiviral compounds. Chan
et al. (59) showed that the inhibitory activity of the
C-peptide C34 depends on its ability to bind to this coiled coil
cavity. Moreover, examining a series of C34 peptide variants with
modified cavity-binding residues, they observed a linear relationship
between the logarithm of the inhibitory potency and the stability of
the corresponding helical-hairpin complexes. A single mutation in the
C34 peptide from Trp-631 Gly increased the IC50 for
cell fusion by about two log units. Reported inhibitory concentrations
of DP178 and C34 appear to be quite similar (we find that the
IC50 for solute mixing is about 4-fold higher for DP178 as
compared with C34). Since DP178 lacks all three residues from the C
helix (Trp-628, Trp-631, and Ile-635) which insert into the cavity, we
would expect the IC50 to be at least 2 orders of magnitude
higher for DP178.
This conundrum has led us to postulate that there are two targets for inhibition by DP178: (i) DP178 inhibits the formation of the heterotrimeric coiled coil by interacting with the leucine/isoleucine zipper sequence in the aqueous solution; (ii) fusion pore formation is inhibited by the interaction of DP178 with the membrane-bound state of its corresponding sequence in gp41 which will enhance melting of the coiled coil soluble form. Accordingly, a mutagenesis study, which revealed that the tryptophan-rich region (overlaps with DP178, see Fig. 1) must function in proximity to the membrane (71, 32) support the notion that this region binds to the surface of the membrane. For clarity, it is worth noting that three segments in the cytoplasmic tail of gp41 may associate with the membrane (48, 72-80), whereas both Rabenstein and Shin (81) and our results suggest that segments in the ectodomain bind to the membrane. These studies suggest that segments both upstream and downstream of the transmembrane domain of HIV-1 gp41 associate with the membrane.
Our results are consistent with the notion that C34 inhibits HIV-1-induced membrane fusion by blocking heterotrimeric coiled coil formation only. The relative low affinity of C34 to the membrane (Figs. 3 and 4) does not allow it to interfere with steps that occur within the membrane. DP178 interacts with this leucine/isoleucine zipper sequence when in aqueous environment (38, 45), but this interaction cannot take place in the membrane environment (Fig. 5, open squares and open circles). On the other hand, DP178, which was found to be monomeric in aqueous solution when its concentration was less than 10 µM (38), forms oligomers even at 100 times lower concentration in the membrane (Fig. 5, filled squares). However, we cannot rule out the possibility that an increase in its membrane local concentration might shift gp41 closed heterotrimeric (soluble)-gp41 open bundle (membrane-bound) equilibrium toward the closed bundle soluble form. In other words, DP178 can prevent melting of the heterotrimeric hairpin complex. The fact that DP107 (a shifted version of N36) inhibited (although at higher concentrations) HIV-1-mediated cell fusion with no differences between lipid and aqueous dye redistribution (46) may be correlated with its inability to interact with the membrane-bound state of the segment corresponding to DP178.
In the case of influenza hemagglutinin-mediated fusion, Chernomordik and co-workers (82) have shown that at reduced envelope glycoprotein surface density there is more hemi-fusion than full fusion. In the presence of different concentrations of peptide, the effective surface density of envelope glycoprotein is reduced, giving rise to different extents of hemi-fusion as compared with full fusion. This explains the 2-fold difference between inhibition of lipid and aqueous redistribution by C34 (Fig. 2a). However, the striking difference between inhibition of lipid versus content mixing in the case of DP178 (Fig. 2b) leads us to propose the following mode of inhibition by DP178 (see Fig. 6).
The binding of DP178 to the leucine/isoleucine zipper sequence
(light blue) at an intermediate stage (Fig. 6, upper
panel, b; lower panel, IIb) inhibits the formation of
the heterotrimeric coiled coil. How the binding of DP178 to the
membrane-bound state of its corresponding segment in gp41 actually
inhibits pore formation remains an open question. We can speculate that
by binding to its corresponding segment in the membrane, DP178
prevents, by a dominant negative manner, further oligomerization of
gp41. This possibility implicates the presence of another intermediate
in the fusion reaction that takes place after the formation of the heterotrimeric coiled coil, but before the formation of the fusion pore. Indeed, it is believed that the recruitment of several oligomeric units of the fusion proteins is needed for complete fusion to occur
(83-88). Accordingly, by binding to its corresponding segment in the
membrane, DP178 inhibits the formation of fusion pores (Fig. 6, the
transition from c to d). Further studies are
needed to understand the mechanism by which DP178 inhibits this process.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Zdenka Jonak for a gift of the TF228 cells, and Drs. D. Littman and V. Kewal Ramani for the NIH3T3/CD4/CXCR4 cells obtained through the National Institutes of Health AIDS Reagent program. We thank Drs S. Ausborn for suggesting the BODIPY protocol, and Dr. A. Puri for assistance.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health intramural AIDS targeted antiviral program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Contributed equally to the results of this work.
Supported by a Harold S. and Harriet B. Brady Professorial
Chair in Cancer Research. To whom correspondence should be addressed: Dept. of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel. Tel.: 972-8-9342711; Fax: 972-8-9344112; E-mail:
Yechiel.Shai@weizmann.ac.il.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M004113200
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; gp, glycoprotein; CMTMR, 5-(and-6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine; DNS-PE, N- (5-dimethylaminonaphthalene-1-sulfonyl)-sn-glycero-3-phosphoethanolamine; FRET, fluorescence resonance energy transfer; NBD, 7-nitrobenz-2-oxa-1,3-diazole-4-yl; PC, egg phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; Rho, tetramethyl- rhodamine; SUV, small unilamellar vesicles.
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