THE JOURNAL
OF
BIOLOGICAL CHEMISTRY
Vol. 272, No. 21, Issue of May 23, pp. 13496 –13505, 1997
Printed in U.S.A.
Fusion Peptides Derived from the HIV Type 1 Glycoprotein
41 Associate within Phospholipid Membranes and Inhibit
Cell-Cell Fusion
STRUCTURE-FUNCTION STUDY*
(Received for publication, January 23, 1997)
Yossef Kliger‡, Amir Aharoni‡, Doron Rapaport‡, Philip Jones§, Robert Blumenthal§,
and Yechiel Shai‡¶
From the ‡Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, 76100 Israel and
the §Section on Membrane Structure and Function, NCI, National Institutes of Health, Bethesda, Maryland 20892
To infect mammalian cells, enveloped viruses have to deposit
their nucleocapsids into the cytoplasm of a host cell. Membrane
fusion represents a key element in this entry mechanism. The
basic unit of most viral fusion proteins is one or two type 1
integral membrane glycoproteins. These often combine into
oligomers that project from the viral envelope (1). A key feature
of most viral fusion proteins is a fusion peptide, a stretch of
highly hydrophobic amino acids that is believed to trigger the
fusion process. It was proposed that fusion is initiated by the
insertion of the fusion peptide into either the target membrane
(2– 6), the viral membrane (7, 8), or both (9 –11).
* This work was supported in part by the Basic Research Foundation
administered by the Israel Academy of Sciences and Humanities and by
the Henri and Françoise Glasberg Foundation. The costs of publication
of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Membrane
Research and Biophysics, Weizmann Institute of Science, Rehovot
76100, Israel. Tel.: 972-8-9342711; Fax: 972-8-9344112; E-mail:
[email protected].
With HIV,1 fusion between the virus and CD41 cells is a
critical step in the viral infection. The fusion peptide located at
the N terminus of the transmembrane viral protein gp41 is
assumed to have a major role in this fusion process (12). A polar
amino acid substitution at position 2 of the fusion peptide of
gp41 (V2E) results in an envelope glycoprotein that dominantly
interferes with both syncytium formation and infection mediated by the wild-type HIV-1 envelope glycoprotein (13, 14).
Although the mechanism of the interference by the V2E mutant is not clear, it does not result from aberrant envelope
glycoprotein synthesis, processing, or transport. This mutant
elicits a dominant interfering effect even in the presence of
excess wild-type glycoprotein, which suggests that a higher
order envelope glycoprotein complex is involved in the membrane fusion (13).
The fusion process is a complex phenomenon that involves an
entire range of biochemical and biophysical interactions (15).
In an attempt to understand the steps involved in the fusion
process, synthetic peptides that resemble or mimic the putative
fusion peptide’s regions of envelope proteins of viruses, or
model peptides, have been synthesized, and their interactions
with liposomes or cells have been examined. Among them there
are peptides corresponding to the fusion sequences of influenza
virus (8, 16 –18), Sendai virus (19), simian immunodeficiency
virus (20), and HIV (21–23).
Despite extensive studies, the mode of action of fusion peptides that promote fusion is still not clear. Interaction of viral
fusion peptides with host-cell membranes was simulated by
computer analysis (24), which led to the conclusion that the
fusogenic helices were obliquely oriented with respect to the
lipid-water interface. This conclusion was experimentally supported by the finding that a mutation that modified the oblique
orientation of the fusion peptide of simian immunodeficiency
virus gp32 reduced the peptide’s fusogenic activity (25). Further support for this oblique orientation comes from a recent
study (19) showing that the fusion peptide of the Sendai virus
is obliquely oriented in its membrane-bound state. Furthermore, the peptide could self-assemble in its membrane-bound
1
The abbreviations used are: HIV, human immunodeficiency virus;
PC, phosphatidylcholine; PS, phosphatidylserine; BOC, butyloxycarbonyl; Rho-PE, N-[lissamine-rhodamine B-sulfonyl]-dioleoylphosphatidylethanolamine; NBD-PE, N-[7-nitrobenz-2-oxa-1,3-diazole-4-yl]dioleoylphosphatidylethanolamine; Rho, 5-(and 6)-carboxytetramethylrhodamine; CMFDA, 5-chloromethylfluorescein diacetate; SUV, small
unilamellar vesicles; LUV, large unilamellar vesicles; aa, amino acid(s);
Chol., cholesterol; DiI(C18:3), 1,19-dioctadecyl-DiI, 3,3,39,39-tetramethylindocarbocyanine perchlorate; PBS, phosphate-buffered saline;
gp, glycoprotein; WT, wild type; Tricine, N-[2-hydroxy-1,1-bis
(hydroxymethyl)ethyl]glycine.
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The fusion domain of human immunodeficiency virus
(HIV-1) envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N terminus of
the transmembrane glycoprotein (gp41). A V2E mutant
has been shown to dominantly interfere with wild-type
envelope-mediated syncytium formation and virus infectivity. To understand this phenomenon, a 33-residue
peptide (wild type, WT) identical to the N-terminal segment of gp41 and its V2E mutant were synthesized, fluorescently labeled, and characterized. Both peptides inhibited HIV-1 envelope-mediated cell-cell fusion and
had similar a-helical content in membrane mimetic environments. Studies with fluorescently labeled peptide
analogues revealed that both peptides have high affinity
for phospholipid membranes, are susceptible to digestion by proteinase-K in their membrane-bound state,
and tend to self- and coassemble in the membranes. In
SDS-polyacrylamide gel electrophoresis the WT peptide
formed dimers as well as higher order oligomers,
whereas the V2E mutant only formed dimers. The WT,
but not the V2E mutant, induced liposome aggregation,
destabilization, and fusion. Moreover, the V2E mutant
inhibited vesicle fusion induced by the WT peptide,
probably by forming inactive heteroaggregates. These
data form the basis for an explanation of the mechanism
by which the gp41 V2E mutant inhibits HIV-1 infectivity
in cells when co-expressed with WT gp41.
Assembly of Fusion Peptides of HIV
EXPERIMENTAL PROCEDURES
Materials—BOC-amino acid phenylacetamidomethyl-resin was purchased from Applied Biosystems (Foster City, CA), and BOC-amino
acids were obtained from Peninsula Laboratories (Belmont, CA). NBDfluoride and other reagents for peptide synthesis were obtained from
Sigma. Egg phosphatidylcholine (PC) and phosphatidylserine (PS) from
bovine spinal cord (sodium salt, grade I) were purchased from Lipid
Products (South Nutfield, UK). Cholesterol (extra pure), purchased
from Merck (Darmstadt, Germany), was recrystallized twice from ethanol. N-[Lissamine-rhodamine B-sulfonyl]-dioleoylphosphatidylethanolamine (Rho-PE), N-[7-nitrobenz-2-oxa-1,3-diazole-4-yl]-dioleoylphosphatidylethanolamine (NBD-PE), 3–39-diethylthiodicarbocyanine iodide,
5-(and 6)-carboxytetramethylrhodamine (Rho), succinimidyl ester,
5-chloromethylfluorescein diacetate (CMFDA), and 1,19-dioctadecyl3,3,39,39-tetramethylindocarbocyanine perchlorate (DiI(C18:3)) were
purchased from Molecular Probes (Junction City, OR). TF228 and
SupT1 cells were grown in RPMI 1640 medium (with 25 mM HEPES,
10% fetal calf serum, 2 mM L-glutamine, penicillin G at 100 units/ml,
and streptomycin sulfate at 100 mg/ml). All other reagents were of
analytical grade. Buffers were prepared using double glass-distilled
water. Phosphate-buffered saline (PBS) was composed of NaCl (8 g/liter), KCl (0.2 g/liter), KH2PO4 (0.2 g/liter), and Na2HPO4 (1.09 g/liter),
pH 7.4. Peptide markers for SDS-PAGE were purchased from Fluka.
Recombinant soluble CD4 was obtained from Intracel (Cambridge, MA).
Peptide Synthesis and Fluorescent Labeling—The peptides were synthesized by a solid phase method on phenylacetamidomethyl-amino
acid resin (0.15 milliequivalents) (27), as described previously (28, 29).
Labeling of the N terminus of the peptides was achieved as described
previously (30, 31); 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. When needed the resin-bound peptides were
reacted with the desired fluorescent probe and finally cleaved from the
resins by hydrofluoric acid, extracted with trifluoroacetic acid, and
precipitated with ether. This procedure yielded peptides selectively
labeled with fluorescent probes at their N-terminal amino acid. The
synthetic peptides were purified (.95% homogenicity) by reverse-phase
high performance liquid chromatography on a C18 column using a
linear gradient of 25– 80% acetonitrile in 0.05% trifluoroacetic acid, for
40 min. The peptides were subjected to amino acid analysis and mass
spectrometry to confirm their composition. Unless stated elsewhere,
stock solutions of concentrated peptides in Me2SO were used to avoid
aggregation of the peptides prior to their use. The final concentration of
the Me2SO in each experiment did not exceed 2%, a concentration that
did not have any effect on the system tested.
Fluorescence Video Imaging Microscopy—The actions of the peptides
upon human cell-cell fusion were investigated using fluorescence video
imaging microscopy. TF228 cells, expressing the HIV type 1 envelope
protein, were labeled with DiI, a lipophilic dye. SupT1 cells, expressing
CD4 receptors, were labeled with CMFDA, an aqueous dye. Transfer of
DiI and CMFDA to another cell indicates membrane mixing and cytosolic mixing, respectively. Both cell lines are derived from lymphocytes.
SupT1 cells were loaded with CMFDA as follows. Cells were pelleted
and incubated in fresh RPMI containing 10 mM CMFDA for 45 min at
37 °C, 5% CO2. Inside the cytosol, acetate groups of CMFDA are cleaved
by esterases, thereby converting a nonfluorescent compound into a
green fluorescent one. Also, the chloromethyl moiety is conjugated to
intracellular thiols, producing cell-impermeant dye-thioether adducts.
The reaction is believed to be mediated by glutathione S-transferase
(Molecular Probes data). The cells were washed twice in RPMI and then
incubated at 37 °C, 5% CO2 for another 45 min to ensure complete
conversion of the dye. The cells were washed twice again and allowed to
settle onto polylysine-coated glass coverslips in serum-free medium for
15 min before addition of DiI-loaded TF228 cells and serum. TF228 cells
were labeled by incubation in 3 mM DiI(C18:3) for 10 min at room
temperature in a 1:1 mixture of diluent C and RPMI medium, followed
by three washes in RPMI.
TF228 and SupT1 cells were incubated for 2 h at 37 °C, 5% CO2 and
video fluorescence images were taken. After 30 min of this incubation
period, different doses of WT, V2E, or N-succinyl-Sendai fusion peptide
were applied. N-Succinyl-Sendai fusion peptide consists of N-succinylated 33 N-terminal amino acids of the Sendai fusion peptide, and it is
not expected to interact specifically with HIV-1. The proportion of
TF228 cells, in contact with SupT1 cells, that had fused to SupT1 cells
was counted. Fused cells are those labeled with both dyes. A control
experiment was performed in which TF228 cells were preincubated at
37 °C for 2 h in the presence or absence of 20 mg/ml soluble CD4
immediately prior to co-culture with the SupT1 cells.
Preparation of Lipid Vesicles—Small unilamellar vesicles (SUV)
were prepared by sonication, from PC/PS/Chol. (4:4:1, w/w). The cholesterol was included to reduce the curvature of the small unilamellar
vesicles (32). Large unilamellar vesicles (LUV) were also prepared from
PC/PS/Chol. (4:4:1, w/w) and, when necessary, with different amounts
of Rho-PE and NBD-PE. The procedure was as follows. Dry lipids were
hydrated in buffer and dispersed by vortexing to produce large multilamellar vesicles. The lipid suspension was freeze-thawed five times
and then extruded eight times through a polycarbonate membranes
with 0.1- or 0.4-mm diameter pores (Nuclepore Corp., Pleasanton, CA).
Peptide-induced Lipid Mixing—Lipid mixing of large unilamellar
vesicles was measured using a fluorescence probe dilution assay, based
on resonance energy transfer measurements (33). Lipid vesicles containing 0.6 mol % each of NBD-PE (energy donor) and Rho-PE (energy
acceptor) were prepared in PBS. A 1:4 mixture of labeled and unlabeled
vesicles was suspended in 400 ml of the buffer at room temperature, and
a small volume of peptide in Me2SO was added. The increase in NBD
fluorescence at 530 nm was monitored with the excitation wavelength
set at 467 nm. The inner filter effect was minimized by using a 0.5-cm
pathlength cuvette. The fluorescence intensity before the addition of
the peptide was referred to as 0% lipid mixing, and the fluorescence
intensity upon the addition of Triton X-100 (0.25% v/v) was referred
to as 100% lipid mixing. All the fluorescence measurements in
the present study were done on a Perkin-Elmer LS-50B
Spectrofluorometer.
Electron Microscopy—The effects of the peptides on liposomal suspensions were examined by negative staining electron microscopy. A
drop containing LUV alone or a mixture of LUV and peptide was
deposited onto a carbon-coated grid and negatively stained with 2%
uranyl acetate. The grids were examined using a JEOL JEM 100B
electron microscope (Japan Electron Optics Laboratory Co. Tokyo,
Japan).
Visible Absorbance Measurements—The changes in the vesicles’ size
were measured by visible absorbance measurements. Aliquots of peptide stock solutions were added to 200-ml suspensions of 89 mM PC/PS/
Chol (4:4:1) LUV in PBS. Absorbance was measured using Bio-Tek
Instruments microplate before and after the addition of a peptide.
Membrane Permeability Studies—Membrane destabilization, in the
form of diffusion potential collapse, was detected fluorimetrically as
described previously (28, 34). Briefly, a liposome suspension, prepared
in “K1 buffer,” was added to an isotonic buffer (K1-free buffer), to which
the dye diS-C2-5 was then added. Subsequent addition of a valinomycin
created a negative diffusion potential inside the vesicles by a selective
efflux of K1 ions, resulting in a quenching of the dye’s fluorescence.
Peptide-induced membrane permeability toward all the ions in the
solution caused dissipation of the diffusion potential, as monitored by
an increase of fluorescence. Fluorescence was monitored using excitation at 620 nm and emission at 670 nm. The percentage of fluorescence
recovery (Ft) was defined as shown in Equation 1.
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state, thus suggesting its role in assisting in the assembly of
the envelope protein of the virus. More recently, Martin et al.
(26) showed, using attenuated total reflection fourier transform
infrared spectroscopy, that a short portion of the fusion peptide
of HIV is obliquely oriented in its membrane-bound state. This
oblique orientation was postulated to locally disorganize the
structure of the lipid bilayer and to generate new lipid phases
that are thought to be associated with the initial steps of
membrane fusion.
To understand better the role of fusion peptides in the mediation of cell fusion, a 33-residue peptide that represents the
N terminus of HIV-1 gp41, and its V2E mutant were synthesized and fluorescently labeled. The peptides were then used in
a variety of biophysical and functional studies to characterize
their structure, their abilities to interact with and permeate
phospholipid membranes, to self- and coassemble within membranes, to induce fusion of phospholipid membranes, and to
inhibit cell-cell fusion. The results revealed that the fusogenic
properties of the intact gp41 and its V2E mutant can be modeled using synthetic peptides. Thus, this synthetic peptide
model was used to explore the mechanism in which the V2E
mutant interferes with the fusion peptide activity.
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Assembly of Fusion Peptides of HIV
TABLE I
Amino acid sequences of the peptides and their fluorescently labeled analogues
Peptide no.
Designation
Sequence
1
2
3
4
5
6
7
8
WT
NBD-WT
Rho-WT
V2E
NBD-V2E
Rho-V2E
NBD-A13-pardaxin
Suc-Sendai
H2N-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL-COOH
NBD-HN-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL-COOH
Rho-HN-AVGIGALFLGFLGAAGSTMGARSMTLTVQARQL-COOH
H2N-AEGIGALFLGFLGAAGSTMGARSMTLTVQARQL-COOH
NBD-HN-AEGIGALFLGFLGAAGSTMGARSMTLTVQARQL-COOH
Rho-HN-AEGIGALFLGFLGAAGSTMGARSMTLTVQARQL-COOH
NBD-HN-GFFALIPKIISSALFKTLLSAVGSALSSSGGQE-COOH
Suc-HN-FFGAVIGTIALGVATSAQITAGIALAEAREAKR-COOH
Ft 5 @~It 2 Io!/~If 2 Io!#100%
(Eq. 1)
fh 5
0
!
~@u#222 2 @u#222
100
@u#222
(Eq. 2)
Where u is the experimentally observed mean residue ellipticity at 222
0
100
nm, and values for u222
and u222
, corresponding to 0 and 100% helix
content at 222 nm, estimated at 22,000 and 230,000 degreeszcm2/dmol,
respectively.
NBD Fluorescence Measurements—Changes in the fluorescence of
NBD-labeled peptides were measured upon their binding to vesicles.
NBD-labeled peptide (0.1 mM) was added to 2 ml of PBS, containing
SUV (403 mM). Emission spectra were recorded, with excitation set at
467 nm (10 nm slit), and compared with the emission spectra of the
NBD-labeled peptide in a liposome-free buffer.
Binding Experiments—The degree of peptide association with lipid
vesicles was measured by adding lipid vesicles to 0.1 mM of NBD-labeled
peptides at 28 °C, as has been previously described with tryptophan
containing peptides (37, 38). The fluorescence intensity was measured
as a function of the lipid/peptide molar ratio, with an excitation set at
467 nm (10 nm slit), and with emission set at 530 nm (5 nm slit). The
fluorescence values were 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
amount of vesicles).
Enzymatic Digestion of Membrane-bound Peptides—SUV (500 mM)
composed of PC/PS/Chol. (4:4:1, w/w) were added to 0.1 mM NBD-labeled
peptide, followed by the addition of proteinase K (1 mg/400 ml). Fluorescence intensities as a function of time were obtained before and after
the addition of the enzyme. In these experiments, the peptide/lipid
molar ratio was kept at a level such that more than 90% of the peptides
are assumed to be bound to the vesicles. To estimate the percent of
cleavage, a control experiment was done, in which the enzyme was
added before the addition of the liposomes. The emission at the end of
the control experiment was referred to as 100% cleavage.
Resonance Energy Transfer Measurements—Fluorescence resonance
energy transfer was measured using NBD-labeled peptides serving as
donors and Rho-labeled peptides serving as energy acceptors (39). Fluorescence spectra were obtained at room temperature, with excitation
set at 467 nm using a 10-nm slit width. In a typical experiment, donor
peptide (final concentration 0.04 mM) was added to a dispersion of
PC/PS/Chol. (4:4:1, w/w) SUV (224 mM) in PBS, followed by the addition
of acceptor peptide in several sequential doses. Fluorescence spectra
were obtained before and after addition of 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 acceptor. E was
E 5 ~1 2 Ida/Id! 3 100% .
(Eq. 3)
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 contribution of buffer and vesicles
was subtracted from all measurements.
RESULTS
Peptides representing the 33-amino acid residues N-terminal of gp41 of HIV-1 (LAV1a), as well as its V2E analogue, were
synthesized and fluorescently labeled at their N-terminal
amino acid with either NBD (to serve in the binding experiments and as an energy donor) or rhodamine (to serve as an
energy acceptor). The sequences of the peptides and their designations are given in Table I. The table gives also the sequences of other two membranous a-helical peptides, the Nterminal succinylated analogue of the fusion peptide of Sendai
virus, and a pore forming toxin, A13-pardaxin.
Functional Properties of the Peptides
Inhibition of Cell-Cell Fusion Induced by WT and V2E Peptides—Both WT and V2E peptides inhibited fusion of HIV-1
envelope glycoprotein-expressing cells with a CD41 cell line
(Fig. 1). Statistical analysis using Student’s t test showed that
the data sets of control compared with WT peptides are different to the 97.5% confidence level and that the control compared
with V2E data sets are different to .99% confidence ( p 5
0.0038). The data indicate 50% inhibition of fusion at 0.01 mM
peptide. CEM cells, expressing CD4 receptors but not the envelope protein, were used as negative controls. These cells were
labeled with DiI in the same manner as the TF228 cells and
incubated for 2 h with SupT1 cells. Only 2% of CEM cells
showed dye overlap with SupT1 cells showing that background
overlap is low. Fig. 2 shows representative images of fluorescent cells in the presence of WT, V2E, and control peptides at 4
mg/ml. Images D, H, and L show regions of overlap of CMFDA
and DiI fluorescence (AND function of the “Metamorph” software, Universal Imaging). Little fluorescence overlap is seen in
the case of the WT and V2E peptides, whereas considerable
overlap is observed using the control peptide.
Preincubation for 2 h at 37 °C in the presence or absence of
soluble CD4 (20 mg/ml) prior to co-culture of the TF228 and
SupT1 cells showed that soluble CD4 inhibits cell-cell fusion as
measured by the dye mixing assay. Fusion in the presence of
soluble CD4 was 20.6 6 2.2 and 42.3 6 5.5% in the absence of
soluble CD4 (mean 6 S.E., six experiments). This is the same
sCD4 concentration as that required for half-maximal inhibition of syncytium formation using these cells (40).
Lipid Mixing Induced by Peptides—The induction of intervesicular lipid mixing by the peptides, as a measure of their
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Where It is the fluorescence observed after addition of peptide, at time
t; I0 is the fluorescence after addition of valinomycin; and If is the total
fluorescence prior to addition of valinomycin.
Determining the Oligomeric States of the Peptides by SDS-PAGE—
The experiments were done as described (35), except for a change in the
sample preparation; high performance liquid chromatography-purified
peptide and SDS (1:1, w/w) were dissolved in CHCl3/MeOH (2:1 v/v).
The solvents were evaporated under a stream of nitrogen and then
lyophilized. The peptide and SDS mixtures were resuspended in buffer
composed of 0.065 M Tris-HCl, pH 6.8, and 10% glycerol by sonication.
Fixing, staining, and destaining times were shorted to 1 min, 1 h, and
overnight, respectively, to decrease diffusion effects.
Circular Dichroism (CD) Spectroscopy—CD spectra were obtained
using a Jasco J-500A spectropolarimeter. The spectra were scanned
with a quartz optical cell with pathlength of 2 mm, at room temperature. Each spectrum was the average of eight scans at wavelengths of
250 to 195 nm. Fractional helicities (36) were calculated as shown in
Equation 2.
determined experimentally from the ratio of the fluorescence intensities
of the donor in the presence (Ida) and in the absence (Id) of the acceptor,
at the donor’s maximum emission wavelength (524 nm in the case of the
WT peptide, 522 nm in the case of the V2E mutant, and 531 nm in the
case of the control peptide, A13 Paradaxin). The percentage of transfer
efficiency (E) is shown by Equation 3,
Assembly of Fusion Peptides of HIV
13499
fusogenic activity, was tested with PC/PS/Chol. (4:4:1) LUV
(100-nm diameter) utilizing the probe dilution assay (33). The
dependence of the extent of the lipid mixing process, on the
peptide’s concentration, was examined. In separate experiments, increasing amounts of each peptide were added to a
fixed amount of vesicles. To compare the activity of the two
peptides, the levels of lipid mixing, reached 10 min after the
addition of the peptides, were plotted as a function of the
[peptide]/[lipid] ratio (Fig. 3A). It is evident that only the WT
peptide was able to cause lipid mixing.
The ability of the V2E mutant to inhibit lipid mixing induced
by the WT peptide was tested using the same experimental
system. Addition of premixed WT and V2E using a molar ratio
of 1:2.4, respectively, almost totally abolished the ability of the
WT to induce membrane fusion (Fig. 3B). Furthermore, in a
similar experiment done with 220-nm diameter LUV, 5.3 mM of
the WT peptide induce 28.6% lipid mixing, whereas a mixture
of WT and V2E at a molar ratio of 1:0.75, respectively, did not
cause any fusion (graph not shown). Thus, we conclude that V2E
is able to interfere with the fusion activity of the WT peptide.
Electron Microscopy—To confirm that the intervesicular
lipid mixing was the result of membrane fusion, electron microscopy was used. 100-nm diameter PC/PS/Chol. (4:4:1) LUV
(90 mM), were visualized with an electron microscope before and
after addition of peptides (5 mM). Fig. 4 shows representative
micrographs of the LUV taken at pH 7.3 without peptide (A),
with WT peptide (B), with the V2E peptide (C), and with a
mixture of the WT and V2E (1:3 w/w) (D). The micrographs
demonstrate that the lipid mixing observed with the WT peptide appears concurrently with a size increase of a portion of
the vesicles. Such size increase was not observed with either
the V2E mutant alone or with the mixture of the WT and V2E.
Mechanism for the Loss of the Activity of the V2E Mutant
Induction of Membrane Aggregation by the WT but Not by the
V2E Mutant—Changes in vesicle size distribution resulting
from aggregation and/or fusion can be monitored by following
FIG. 2. Representative images of the actions of WT, V2E, and
control peptides upon TF228 to SupT1 cell fusion. In all cases 4
mg/ml peptide was used. A–D, images of the same field of TF228 and
SupT1 cells after 2 h incubation at 37 °C in the presence of control
peptide (N-succinyl wild-type Sendai). A, bright field; B, green CMFDA
fluorescence; C, red DiI fluorescence; D, overlap between green and red
fluorescence (AND function). E–H, images of the same field of cells after
2 h incubation with wild-type peptide. E, bright field; F, green CMFDA
fluorescence; G, red DiI fluorescence; H, overlap. I–L, images of the
same field of cells with V2E mutant after 2 h incubation at 37 °C. I,
bright field; J, green CMFDA fluorescence; K, red DiI fluorescence; L,
overlap. There is considerably more overlap of red and green fluorescence in the presence of control peptide than in WT or V2E peptides.
Bright field images were used to confirm that overlap was due to the
presence of both dyes in the same cell rather than just due to one cell
lying over another.
the absorbance of the liposome suspension. The changes in the
absorbance at 405 nm as a function of the peptide to lipid molar
ratio are shown in Fig. 5. The data reveal that in all concentrations tested, the WT peptide caused aggregation and/or fusion of vesicles, whereas the V2E mutant did not. Using subfusion quantities, it was revealed that the WT, but not the V2E
mutant, can cause vesicles’ aggregation.
Induction of Membrane Destabilization by the WT but Not by
the V2E Mutant—To study the potential of the peptides to
destabilize lipid bilayers, their ability to dissipate the diffusion
potential in SUV prepared from PC/PS/Chol. (4:4:1 w/w) was
tested. Peptides, at various concentrations, were mixed with a
solution containing the K1-entrapped vesicles, the fluorescent
dye diS-C2-5, and valinomycin. Addition of valinomycin created
a negative diffusion potential inside the vesicles by a selective
efflux of K1 ions, resulting in a self-quenching of the dye’s
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FIG. 1. Graph of percentage fusion of TF228 cells to SupT1
cells versus log peptide concentration for WT, V2E, and N-succinyl wild-type Sendai (control) peptides. The two cell types were
incubated together for 2 h before video fluorescence microscopy images
were taken. Only TF228 cells in contact with SupT1 cells were counted.
The graphs of both WT and V2E peptides were significantly different
from the control graph indicating that they inhibit fusion. Symbols:
control, circles; WT, squares; V2E, triangles.
13500
Assembly of Fusion Peptides of HIV
be correlated with its lost of activity in induction vesicle fusion,
and the loss of activity of the mutated envelope protein.
Structural Properties of the Peptides
fluorescence. Peptide-induced membrane permeability toward
all the ions in the solution caused dissipation of the diffusion
potential, as monitored by an increase of fluorescence. Recovery of fluorescence was monitored with time using excitation at
620 nm and emission at 670 nm (see inset to Fig. 6). The
maximum level reached as a function of peptide concentration
is shown in Fig. 6. The results demonstrate that the WT peptide is very active, and the V2E mutant is not active, in membrane destabilization.
Oligomerization of the Peptides in SDS-PAGE—The state of
aggregation of several membrane proteins was determined in
SDS, a membrane mimetics environment (41, 42). Here, SDSPAGE revealed that the dominant form of both peptides is a
dimer, but the WT peptide, unlike the V2E mutant, also forms
higher order oligomers, which seems to be tetramers (Fig. 7).
The inability of the mutant to form higher order oligomers, may
Downloaded from http://www.jbc.org/ at BEN GURION UNIVERSITY on June 4, 2017
FIG. 3. A, dose dependence of lipid mixing of PC/PS/Chol. (4:4:1) LUV
induced by the WT or the V2E mutant. Peptide aliquots were added to
mixtures of LUV (22 mM phospholipid concentration) containing 0.6
mol % each of NBD-PE and Rho-PE and unlabeled LUV (88 mM phospholipid concentration) in PBS. The increase of the fluorescence intensity of NBD-PE was measured at 10 min after the addition of the
peptide, and the percentage from maximum is plotted versus the peptide/lipid molar ratio. The fluorescence intensity upon the addition of
Triton X-100 (0.25% v/v) was referred to as 100%. WT, squares; V2E,
triangles. B, V2E inhibition of lipid mixing induced by WT. WT peptide
or WT and V2E pre-mixed solution were added into a mixture of labeled
and unlabeled LUV, as described in A. Continuous line, lipid mixing
induced by WT (4.3 mM) alone. Dashed line, lipid mixing induced by V2E
(10.32 mM) and WT (4.3 mM).
Adoption of Partial a-Helix Conformation in Membrane-mimetic Environments, by Both WT and V2E—The secondary
structure of the peptides was evaluated from their CD spectra
in 40% 2,2,2-trifluoroethanol and in 1% SDS. Both peptides
displayed spectra with minima at 208 and 222 nm in both
solvents (Fig. 8) which is typical of a-helix structure. However,
the a-helical content of both peptides is higher in SDS which is
considered as a better membrane mimetic environment than
40% trifluoroethanol. The a-helical content of V2E in SDS
(40%) is slightly higher than that of the WT peptide (30%). It
should be noted that only ;20 amino acids out of the 33
composed the actual fusion peptide and that 6 of them are
glycines.
Localization of the N Terminals of WT and V2E within the
Hydrophobic Core of the Membrane—The fluorescence emission spectra of the NBD-labeled peptides were monitored in
aqueous solutions and in the presence of vesicles. In aqueous
solution both peptides exhibited emission spectra similar to the
NBD moiety dissolved in water (31, 43), with a maximum at
549 6 1 nm (graph not shown). Upon addition of SUV (403 mM)
composed of PC/PS/Chol. (4:4:1 w/w) to the solution (pH 7.4),
the fluorescence emission intensity increased significantly (3.1
and 5.3 times for the WT and V2E, respectively) concomitant
with blue shifts of the emission maxima of the peptides. The
shift was slightly larger for the V2E peptide (maximum of
522 6 1 nm) than for WT (maximum of 524 6 1 nm) which
agrees with the higher increase of the fluorescence of V2E. Blue
shifts of these magnitudes have been observed when surfaceactive NBD-labeled peptides interacted with lipid membranes
(31, 44, 45) and are consistent with the NBD probe located
within the hydrophobic core of the membrane (43). Note that in
these experiments, the lipid/peptide molar ratio was consistently high (.4000:1) so that spectral contributions of free
peptides could be considered negligible.
Binding of WT and V2E to Phospholipid Membranes—The
increases in the fluorescence intensities of the NBD-labeled
peptides, due to membrane partition, were recorded as a function of the lipid/peptide molar ratios. The fractions of membrane-bound peptides are plotted versus the lipid/peptide molar
ratios in Fig. 9. The shapes of the binding curve of both peptides were very similar, indicating that they have similar surface partition coefficients. Since the peptides aggregate in the
aqueous solution, their binding isotherms were not analyzed
further. The shape of the binding curve which seems to become
saturated at lipid/peptide molar ratios similar to those observed with the NBD-labeled antibacterial peptides DS-b (46)
suggests a partition coefficient on the order of 104 M21 for the
WT and for the V2E mutant. These values are 1 order of
magnitude smaller than the value obtained for the Sendai
virus fusion peptide (19).
Accessibility of WT and V2E to Proteolytic Digestion in Their
Membrane-bound State—The susceptibility of membranebound NBD-WT and NBD-V2E to proteolytic digestion by proteinase K was investigated by using PC/PS/Chol. (4:4:1 w/w)
SUV (500 mM) as described under “Experimental Procedures.”
The addition of the enzyme to a mixture containing an NBDlabeled peptide and vesicles caused a fast decrease in the NBD
fluorescence, demonstrating its release from the hydrophobic
environment of the vesicles. The level of the final fluorescence
intensity was the same as that obtained for an unbound peptide. The data reveal that both peptides are accessible to proteolytic digestion, with faster kinetics for the V2E mutant
Assembly of Fusion Peptides of HIV
13501
FIG. 4. Electron micrographs of
negatively stained liposomes. A, PC/
PS/Chol. (4:4:1) LUV (90 mM) alone; B,
PC/PS/Chol. (4:4:1) LUV incubated with
WT (5 mM) peptide for 15 min; C, PC/PS/
Chol. (4:4:1) LUV incubated with V2E (5
mM) peptide for 15 min; D, PC/PS/Chol.
(4:4:1) LUV incubated with WT (5 mM)
and V2E (15 mM) for 15 min.
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FIG. 5. Detection of aggregation and/or fusion by observing
absorbance changes at 405 nm of liposomes mixed with peptides. WT or V2E were added to 90 mM PC/PS/cholesterol (4:4:1) LUV in
PBS. The changes in the absorbance at 405 nm are plotted versus the
peptide to lipid molar ratio. WT, squares; V2E, triangles.
(graph not shown). Full digestion of V2E was accomplished
within 6 min, whereas it took more than 10 min to accomplish
full digestion of WT.
Mechanism of Inhibition
Self- and Coassembly of WT and V2E in Their Membranebound State—The aggregation state of the peptides in their
membrane-bound state was monitored by resonance energy
transfer measurements. Peptides that were labeled at their
N-terminal with either NBD, serving as an energy donor, or
with rhodamine, serving as a fluorescence acceptor, were used.
An example of a typical profile of the energy transfer from
NBD-WT to Rho-WT, in the presence of lipid vesicles, is de-
FIG. 6. Dissipation of diffusion potentials in SUV. Peptides were
added to 600 ml of buffer containing a constant concentration of vesicles
(36 mM) pre-equilibrated with the fluorescent dye (diS-C2-5) and valinomycin. The maximum fluorescence recovery is plotted as a function of
the peptide/lipid molar ratio. WT, squares; V2E, triangles. Inset, kinetics of the fluorescence recovery of the WT peptide; peptide/lipid molar
ratios were 0.0076 (top) and 0.0038 (bottom).
picted in Fig. 10A. When Rho-WT (final concentration of 0.04 –
0.12 mM) was added to a mixture of NBD-WT (0.04 mM) and
PC/PS/Chol. (4:4:1) lipid vesicles (224 mM), a dose-dependent
quenching of the donor’s emission, which is consistent with
energy transfer, was observed (Fig. 10A). Dose-dependent
quenching was also observed for the donor/acceptor V2E and
for the donor-V2E with acceptor-WT combination (Fig. 10B).
Note, that the acceptor-peptide was added only after the donorpeptide 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 energy transfer between unassociated peptide monomers. To confirm that the observed energy transfer is due to peptide aggregation, the transfer effi-
13502
Assembly of Fusion Peptides of HIV
FIG. 7. Determining the aggregation states of the peptides, by
Tricine/SDS-PAGE. The molecular masses of the WT and the V2E
mutant are 3264 and 3322 Da, respectively.
membrane-bound values, which were estimated using their
binding curve (Fig. 9). The data reveal that the fusion peptides
are not randomly distributed throughout the membrane but
rather are associated. The finding that the WT and V2E can
coassemble in the membrane may account for the ability of the
V2E analogue to inhibit the fusion activity of the WT peptide.
Self-Association of WT and V2E in Solution—WT and V2E
self-association in solution was tested using rhodamine-labeled
peptides. Since the fluorescence of rhodamine is quenched
when several molecules are in close proximity, an increase in
fluorescence should occur when an aggregated rhodamine-labeled peptide is dissociated, a process that can take place when
the peptide is cleaved by a proteolytic enzyme. When equal
concentrations of Rho-WT or Rho-V2E (0.05 mM each, as determined by UV absorbance at 567 nm in Me2SO) were added to
PBS, the fluorescence of Rho-V2E was 3.7-fold higher than that
of Rho-WT, which suggests that the WT peptide is more aggregated in solution than the V2E mutant. Furthermore, upon the
addition of proteinase K to solutions of the peptides (0.05 mM
each), the fluorescence of Rho-WT increased 6.4-fold and that of
Rho-V2E increased only 1.3-fold (data not shown). To ensure
complete digestion, the labeled peptides were treated for ;30
min with the enzyme, a time that is longer than required for
total cleavage as described above in the section of the accessibility of the peptides to proteolytic digestion in their membrane-bound state.
DISCUSSION
FIG. 8. Circular dichroism spectra of WT and V2E peptides in
40% trifluoroethanol (A) and in 1% SDS (B). Spectra were taken as
described under “Experimental Procedures” at peptides concentrations
of 20 mM. O, WT; - - -, V2E.
ciencies observed in the experiments were compared with the
energy transfer expected for randomly distributed membranebound donors and acceptors (dashed line, Fig. 10B). The levels
of energy transfer between all the three combinations are significantly higher then those expected for randomly distributed
donors and acceptors. The energy transfer between NBD-A13pardaxin and Rho-WT was measured as a control, and the
values obtained were similar to those of random distribution.
The data for the random distribution was calculated assuming
R0 value for the NBD/Rho donor/acceptor pair to be 51 Å (39).
The acceptor concentrations presented in Fig. 10B are the
The interesting observations in this study are the 50% inhibition of HIV-1 envelope glycoprotein-mediated cell fusion at
0.01 mM peptide concentration for both wild-type and the V2E
mutant (Figs. 1 and 2), and the inhibition of WT induced
vesicles fusion by the V2E mutant (Figs. 3B and 4). These
findings are consistent with the trans-dominant effect of the
gp41 mutant protein in inhibiting HIV-1 infectivity in vivo
when co-expressed with WT gp41 (13). We have shown recently
that the trans-dominant mutation also inhibits HIV-1 envelope
glycoprotein-mediated cell fusion when expressed in target
cells (14). Previously, short peptides with sequences corresponding to the N terminus of gp41 were shown to inhibit
HIV-1 envelope glycoprotein-induced syncytium formation.
However, these short peptides were only effective at concentration of 1 mM (for the 6-aa long peptide (47)) and 0.01 mM (for the
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FIG. 9. Increase in the membrane-bound fraction of NBD-labeled peptides (0.1 mM total concentration) upon titration with
PC/PS/Chol. (4:4:1) SUV vesicles, with excitation at 467 nm and
emission monitored at 530 nm. The experiment was performed at
room temperature in PBS.
Assembly of Fusion Peptides of HIV
13503
TABLE II
Summary of the different properties of WT and V2E analogue
Inhibition of cell-cell fusion
Liposome fusion
Insertion into membranes
Membrane binding
Susceptibility to proteinase K digestion
in membrane bound state
Partial a-helical structure
Self- and coassembly
Membrane destabilization
Induction of vesicle aggregation
Formation of tetramers in SDS gels
a
11-aa long peptide (48)). In another study, Slepushkin et al.
(49) showed that a 22-aa long fusion peptide and its conjugate
with bovine serum albumin inhibited HIV-1 infection at concentrations of 1 mM. The effect of the peptide is certainly enhanced as its length increases. Here, we found that the 33-aa
fusion peptides inhibited fusion at a concentration of 2 orders of
magnitude lower than that reported for the 22-aa peptide (49).
The fusion assay we used is based on the redistribution of
fluorescent dyes about 2 h after incubation of the gp120-gp41expressing cells with target cells and therefore measures initial
events.
Other peptides from the external domain of HIV-1 gp41 also
exhibit antiviral activity. The most potent peptide was derived
from the aa 643– 678 region and was active at 0.001 mM con-
V2E
Figs. where
data are shown
1
1
1
1
1
1
2
1
1
1
1, 2
3, 4
NSa
9
NS
1
1
1
1
1
1
1
2
2
2
8
10
6
5
7
NS, not shown.
centration (50). Peptides derived from other regions (a.a 637–
666 (51) and aa 558 –595 (52)) (90% inhibition at 0.1 mM) are
less potent than those reported herein.
The present study also provides a possible mechanism for
membrane fusion induced by the WT N-terminal gp41 peptide
and its inhibition by the V2E mutant (Figs. 3B and 4). The
observation that, as in the case of viral fusion induced by the
full proteins (13), the parent WT peptide is fusogenic and a
single amino acid substitution results in inactivation of the
peptide suggests that the properties of membrane interaction
discussed below might also play a crucial role in virus-cell
fusion. Fusion of phospholipid membranes is thought to involve
three steps, vesicle aggregation, membrane destabilization,
and merging of membranes (53–55). The WT, but not the V2E
peptide, can induce these three events as discussed below.
First, the WT peptide does not need Ca21 or Mg21 to mediate
fusion, which suggests that the peptide itself is sufficient to
induce vesicle aggregation (Figs. 3A and 4). Indeed, subfusion
quantities of the WT, but not of the V2E mutant, induced
vesicle aggregation (Fig. 5). Contrary to the finding of the
present study, it has previously been reported that a 23-aa
peptide resembling the sequence of HIV could induce phosphatidyl oleoyl palmitoyl glycerol LUV fusion only after their
pre-aggregation by Ca21 or Mg21 ions (21). However, unlike
the parent glycoprotein, a 23-residue synthetic peptide representing the N terminus of the V2E mutant was unable to
inhibit the fusion activity of the wild-type peptide (56). The
33-mer peptides studied herein represent not only the hydrophobic stretch of 21 aa but also the polar border of this hydrophobic region, which consists of a highly conserved series of
polar amino acids, which makes the peptide soluble in aqueous
solution albeit at low concentrations.
Second, the high potency of the WT to dissipate the diffusion
potential demonstrates that this peptide can significantly destabilize lipid membranes (Fig. 6). The potency of the peptide
was higher than that of the fusion peptide of Sendai virus (19),
and the peptide was as potent as pore-forming peptides such as
alamethicin (37) and pardaxin (29). Nevertheless, the kinetics
of membrane permeation by the WT peptide suggest that it
forms irreversible aggregates in the membrane (57). The different membrane-permeating abilities of the WT and the V2E
peptides are not due to a difference in the amount of membrane-bound peptide, since both peptides have similar partition coefficients (Fig. 9). However, it may result from the ability
of the WT to form higher order aggregates, as revealed using
SDS-PAGE (Fig. 7).
Third, the lipid mixing assay revealed that the WT, but not
the V2E peptide, induced membrane fusion (Fig. 3A). Furthermore, V2E inhibited the ability of the WT peptide to induce
membrane fusion (Fig. 3B). The findings that subfusion quantities of the WT, but not of the V2E mutant, induced vesicle
Downloaded from http://www.jbc.org/ at BEN GURION UNIVERSITY on June 4, 2017
FIG. 10. A, fluorescence energy transfer dependence on WT peptide
acceptor concentration. The spectra were obtained for donor peptide
alone or in the presence of various amounts of acceptor peptide. Each
spectrum was recorded in the presence of 224 mM PC/PS/Chol. (4:4:1)
SUV in PBS22. The excitation wavelength was set at 467 nm; emission
was scanned from 500 to 600 nm. Symbols: O, 0.04 mM NBD-WT alone;
z z z, a mixture of 0.04 mM NBD-WT and 0.04 mM Rh-WT; - - -, a mixture
of 0.04 mM NBD-WT and 0.08 mM Rh-WT; -z-, a mixture of 0.04 mM
NBD-WT and 0.12 mM Rh-WT. B, transfer efficiencies between donor
and acceptor-WT (filled squares), donor and acceptor-V2E (filled triangles), donor-V2E and acceptor-WT (open triangles), and donor-pardaxin
A13 and acceptor-WT (open squares) 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 donors and acceptors, and R0 5 51 Å, is given for
comparison (dashed line).
WT
13504
Assembly of Fusion Peptides of HIV
aggregation (Fig. 5), suggest that the inactivity of the mutant
originates at, or before, the stage of the fusion process, where
the bilayers approach each other.
Circular dichroism spectra (Fig. 8) suggest that there is no
significant difference in the secondary structure of the WT and
the V2E peptides. The partial a-helical content in 1% SDS (30
and 40% for the WT and the V2E peptides, respectively) can be
explained by the presence of the polar border of the fusion
peptide. However, the fusion peptide can also contain some
b-sheet conformation, as suggested by others using Fourier
transform infrared spectroscopy (21, 26). However, several reports showed that while helix formation is probably a requirement, it is not sufficient to trigger membrane fusion (8, 16).
Some peptides failed to induce efficient membrane fusion, even
though they could bind to lipid bilayers and exhibited the
appropriate secondary structures. Therefore, it was proposed
that in addition to the other requirements for fusogenic activity, such as membrane binding, destabilization of the bilayer,
and helical conformation, self-aggregation of the peptide monomers within the membrane and their appropriate orientation
are also essential.
Attempts to define the oligomeric state of the HIV-1 envelope
glycoprotein have yielded conflicting results. Several reports
indicated that the envelope glycoprotein of HIV-1 is a tetramer
in its membrane-bound state (58 – 60). On the other hand, other
reports revealed that it forms trimers (61– 63). In the gp41
non-fusogenic form the fusion peptide is buried; thus it is not
responsible for the oligomerization of the protein. However,
once the envelope glycoproteins are recruited to form a fusion
complex the fusion peptides will self-assemble. Here, fluorescently labeled WT and V2E peptides were used to show that
both peptides tend to self-aggregate efficiently within PC/PS/
Chol. vesicles. Furthermore, the two peptides can coassemble
in their membrane-bound state (Fig. 10B). The size of the
aggregates was determined by using SDS as a membrane mimetic environment, as has been done with several other mem-
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FIG. 11. Cartoon illustrating the membrane fusion process induced by the fusion peptide of HIV-1. Homotetramers of the fusion
peptide cause the membranes to approach each other (top), followed by
membrane mixing (bottom).
brane proteins (41, 42). SDS-PAGE revealed that the dominant
form of both peptides is a dimer but that the WT peptide also
forms higher order oligomers, which seem to be tetramers (Fig.
7). The inability of the V2E mutant to form higher order oligomers (Fig. 7) may be correlated with its loss of activity.
Furthermore, the coassembly of the two peptides in their membrane-bound state (Fig. 10B) suggests that the inhibition of
membrane fusion exhibited by the V2E mutant occurs via its
association with the WT. This inhibition may occur either because the V2E mutant decreases the portion of higher order
oligomers of WT or because the V2E mutant forms nonfunctional higher order oligomers with the WT peptide. Since the
V2E mutant gp41 elicits a dominant interfering effect even in
the presence of excess wild-type glycoprotein (13), the second
possibility is a more likely mechanism. Furthermore, when the
V2E peptide was pre-mixed with the WT peptide, prior to
SDS-PAGE, there was no significant difference in the proportion of higher order aggregates (data not shown).
Whether the peptides also self-associate in solution as well
was assessed using rhodamine-labeled peptides and exposure
to a proteolytic enzyme. Quenching experiments with rhodamine-labeled peptides in an aqueous solution revealed that
both WT and V2E peptides self-associate in aqueous solutions. The self-quenching of the rhodamine-labeled WT peptide was 6.4-fold and that of the rhodamine-labeled V2E mutant was only 1.3-fold, which suggests that the WT peptide
forms higher order aggregates than the V2E mutant also in
aqueous solutions.
Another characteristic of a peptide’s interaction with membranes is the extent of a peptide’s penetration into the lipid
bilayers and its orientation. The studies with the NBD-labeled
WT and V2E peptides revealed that the N terminus of both
peptides was inserted into the membrane, with that of the
non-fusogenic V2E mutant being inserted a little deeper than
that of the WT. This might be due to the repulsion between the
negative charge of the carboxylate at the N terminus of the V2E
peptide and the acidic head groups of the lipids. These findings
are similar to those obtained with the Sendai virus fusion
peptides, in which the N terminus of the more fusogenic G12A
mutant was exposed to the surface of the membrane more than
the N terminus of the WT (19). Even though the N termini of
both peptides are inserted into the hydrophobic core of the
membrane, both peptides were cleaved efficiently by proteinase
K when bound to membranes (data not shown), which is in
contrast to other membrane-inserted helices, which were totally protected from enzymatic cleavage in their membranebound state (64). These findings suggest an oblique orientation
for the fusion peptides, rather than a transmembrane orientation, as has been suggested by others (26). Such an oblique
orientation is consistent with recent data (65, 66) that indicate
no merger of the inner with the outer leaflets of viral or liposomal membranes during fusion. Therefore, the peptides do not
need to penetrate deeply into the inner leaflet to induce fusion.
The more rapid enzymatic digestion of membrane-bound V2E,
as compared with WT, is consistent with the higher order
aggregates formed by WT, which may better protect the peptide
from enzymatic digestion.
Synthetic peptides can only partly mimic the complex fusogenic properties of a viral protein (67). However, many of the
properties of the HIV gp41 fusion peptide demonstrated in the
present study, such as strong binding to membranes and assembly therein, orientation in the membrane-bound state, ability to destabilize membrane-packing, and a-helix secondary
structure, were proposed to be requirements of viral proteininduced fusion (68). The similarities and distinctions between
the physicochemical properties of the WT and V2E shown in
Assembly of Fusion Peptides of HIV
Table II may shed some light on the mode of action of the fusion
peptide and the mechanisms of inhibition of this fusion by V2E.
The WT and the V2E mutant are similar in their partial a-helical structure, ability to bind to membranes, and to self-assemble therein, and susceptibility to proteinase K digestion in the
membrane-bound state. However, the WT and mutant differ in
their ability to form higher order peptide aggregates in membranes, to induce vesicle aggregation, to destabilize lipid packing, and to cause membrane fusion. A model for the peptideinduced membrane fusion is shown in Fig. 11. Homotetramers
of the fusion peptide cause the membranes to approach each
other (top), which is followed by membrane mixing (bottom).
Acknowledgments—We thank Dr. Zdenka Jonak for the TF228.1.16
cell line and Drs. A. Puri, S. Durell, D. Dimitrov, and R. Garry for
helpful suggestions. We also thank Dr. Y. Marikovsky for help in
visualization of the phospholipid vesicles using electron microscopy and
A. Bren and G. Jona for their help in SDS gels.
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13505
Fusion Peptides Derived from the HIV Type 1 Glycoprotein 41 Associate within
Phospholipid Membranes and Inhibit Cell-Cell Fusion: STRUCTURE-FUNCTION
STUDY
Yossef Kliger, Amir Aharoni, Doron Rapaport, Philip Jones, Robert Blumenthal and
Yechiel Shai
J. Biol. Chem. 1997, 272:13496-13505.
doi: 10.1074/jbc.272.21.13496
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