Báo cáo khoa học: Sendai virus N-terminal fusion peptide consists of two similar repeats, both of which contribute to membrane fusion ppt

Sendai virus N-terminal fusion peptide consists of two similar repeats,
both of which contribute to membrane fusion
Sergio G. Peisajovich
1
, Raquel F. Epand
2
, Richard M. Epand
2
and Yechiel Shai
1
1
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel;
2
Department of Biochemistry, McMaster
University Health Sciences Centre, Hamilton, Ontario, Canada
The N-terminal fusion peptide of Sendai virus F
1
envelope
glycoprotein is a stretch of 14 amino acids, most of which are
hydrophobic. Following this region, we detected a segment
of 11 residues that are strikingly similar to the N-terminal
fusion peptide. We found that, when anchored to the mem-
brane by palmitoylation of its N-terminus, this segment
(WT-palm-19–33) induces membrane fusion of large unila-
mellar liposomes to almost the same extent as a segment that
includes the N-terminal fusion peptide. The activity of WT-
palm-19–33 was dependent on its specific sequence, as a
palmitoylated peptide with the same amino-acid composi-
tion but a scrambled sequence was inactive. Interestingly,
two mutations (G7A and G12A) known to increase F
1

-
induced cell-cell fusion, also increased the homology between
the N-terminal fusion peptide and WT-palm-19–33. The role
of the amino-acid sequence on the fusogenicity, secondary
structure, and mechanism of membrane fusion was analyzed
by comparing a peptide comprising both homologous seg-
ments (WT 1–33), a G12A mutant (G12A 1–33), a G7A–
G12A double mutant (G7A–G12A 1–33), and a peptide with
a scrambled sequence (SC 1–33). Based on these experiments,
we postulate that replacement of Gly 7 and Gly12 by Ala
increases the ahelical content of the N-terminal region, with
a concomitant increase in its fusogenic activity. Furthermore,
the dissimilar abilities of the different peptides to induce
membrane negative curvature as well as to promote isotropic
31
P NMR signals, suggest that these mutations might also
alter the extent of membrane penetration of the 33-residue
peptide. Interestingly, our results serve to explain the effect of
the G7A and G12A mutations on the fusogenic activity of
the parent F
1
protein in vivo.
Keywords: viral entry; peptide–lipid interactions; spectro-
scopic studies.
A key step in the infection by enveloped viruses is the fusion
between the viral and the cellular plasma or endosomal
membranes. Most of the specialized viral envelope proteins
directly involved in the fusion process, contain a discrete
region of apolar amino acids, termed the Ôfusion peptideÕ,
which is believed to play an important role in the merging of

the membranes [1]. Although much is known about the 3D
structure of fragments of fusion proteins in the absence of
membranes [2–8], the intimate interplay between fusion
peptides and the membrane is still unknown. Fusion
peptides’ insertion into the cell membrane [9,10], viral
membrane [11,12], or both [13,14] is believed to facilitate
local dehydration [15] and to promote increased negative
curvature strain in the bilayer (reviewed in [16]), factors that
can help to overcome the energetic barriers associated with
the fusion process. In addition, fusion peptides can serve as
membrane anchors that facilitate partition of other regions
of the viral envelope proteins to the membrane, which can
subsequently participate in membrane merging [17].
Viruses from the Paramyxoviridae family are important
respiratory tract pathogens of humans [18]. A salient feature
of Paramyxoviridae infection is the fusion between infected
and noninfected cells [19], a process mediated by the
paramyxovirus envelope glycoprotein F. The F protein is
synthesized as an inactive precursor, which is cleaved by a
host protease, producing two fusion-active subunits, F
1
and
F2 [20]. F
1
remains attached to the membrane by a
transbilayer segment, whereas F2 and F
1
are disulfide
bonded.
Although it was initially thought that viral fusion

glycoproteins contained a single fusogenic region respon-
sible for the actual merging of the membranes, over the last
years a more complex view has emerged. Both the region
consecutive to the N-terminal fusion peptide and the one
immediately before the transmembrane domain of HIV-
1 gp41 were shown to facilitate membrane fusion [17,21].
Furthermore, the F
1
subunit of Sendai and Measles virus
(two distantly related members of the Paramyxovirus
family) were shown to contain, in addition to the N-terminal
fusion domain, an internal fusogenic segment, located
downstream of the N-terminal heptdad repeat [22,23]. The
structural organization of this internal fusogenic region,
postulated based on studies using protein segments [22,24],
was recently confirmed by the X-ray determined structure of
the prefusion conformation of Newcastle disease virus F
protein [25]. Both in the cases of Paramyxovirus and
Correspondence to Y. Shai, Department of Biological Chemistry,
Weizmann Institute of Science, Rehovot 76100, Israel.
Fax: + 972 8 344112, Tel.: + 972 8 342711,
E-mail:
Abbreviations: ATR-FTIR, attenuated total reflection Fourier
transformed infrared spectroscopy; BOC, butyloxycarbonyl;
Cho, cholesterol; DiPoPtdEth, dipalmitoleoylphosphatidylethanol-
amine; DOPtdCho, dioleoylphosphatidylcholine; DOPtdEth, diol-
eoylphosphatidylethanolamine; LUV, large unilamellar vesicles;
NBD-PtdEth, N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidyleth-
anolamine; Rho-PtdEth, N-(lissamine rhodamine B sulfonyl) phos-
phatidylethanolamine; T

H
, bilayer to hexagonal phase transition
temperature; DTGS, deuterated triglyceride sulfate.
(Received 1 April 2002, revised 14 July 2002, accepted 24 July 2002)
Eur. J. Biochem. 269, 4342–4350 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03132.x
Retrovirus internal fusogenic regions, the mechanism by
which these segments destabilize membranes remains
unknown.
In an attempt to further our understanding of the
process of viral infection and to determine whether
the presence of a fusogenic region consecutive to the
N-terminal fusion peptide is a characteristic common to
unrelated viral families, here we analyzed the role in
membrane merging of the N-terminal domain of Sendai
virus F
1
protein. The first 14 amino acids of this region
are termed Ôthe N-terminal fusion peptideÕ. Following this
apolar segment, we detected a stretch of about 11
residues strikingly similar to the N-terminal fusion
peptide (Fig. 1). We found that, when anchored to the
membrane by palmitoylation of its N-terminus, this
segment (WT-palm-19–33) induces membrane fusion of
large unilamellar liposomes to almost the same extent as
a longer fragment that also includes the N-terminal
fusion peptide (WT 1–33); whereas a palmitoylated
peptide with the same amino-acid composition of WT-
palm-19–33, but a scrambled sequence (SC-palm-19–33),
was inactive. In addition, we analyzed the role of the
amino-acid sequence on the fusogenicity, secondary

structure, and mechanism of membrane fusion exerted
by the F
1
N-terminal region.
EXPERIMENTAL PROCEDURES
Materials
BOC-amino acids were purchased from Novabiochem AG
(La
¨
ufelfingen, Switzerland), and BOC-amino acid phenyl-
acetamidomethyl (PAM)-resin was obtained from Applied
Biosystems (Foster City, CA, USA). Reagents for peptide
synthesis were obtained from Sigma. Dioleoylphosphatidyl-
choline (DOPtdCho), dioleoylphosphatidylethanolamine
(DOPtdEth), and dipalmitoleoylphosphatidylethanolamine
(DiPoPtdEth) were purchased from Avanti Polar Lipids
(Alabaster, AL, USA); cholesterol (Cho) was purchased
from Lipid Products (South Nutfield, UK. NBD-PtdEth
and Rho-PtdEth were purchased from Molecular Probes
(Eugene, OR). All other reagents were of analytical grade.
Buffers were prepared using double glass-distilled water.
NaCl/KCl/P
i
is composed of NaCl (8 gÆL
)1
), KCl (0.2
gÆL
)1
), KH
2

PO
4
(0.2 gÆL
)1
), and Na
2
HPO
4
(1.09 gÆL
)1
),
pH 7.3.
Peptide synthesis
The peptides (derived from Sendai virus F
1
protein Swiss-
prot entry P04856) were synthesized by a standard solid
phase method using a Boc-strategy on PAM-resin as
described [26]. The peptides were cleaved from the resin
by HF treatment and purified by RP-HPLC. Purity
( 99%) was confirmed by analytical HPLC. The peptide
compositions were determined by amino-acid analysis and
mass spectrometry.
Preparation of lipid vesicles
Large unilamellar vesicles (LUV) were prepared from
DOPtdCho, DOPtdEth, and Cho (1 : 1 : 1) and when
necessary with different amounts of Rho-PtdEth and NBD-
PtdEth, as follows: dry mixed lipid films were suspended in
NaCl/KCl/P
i

buffer by vortexing to produce large multi-
lamellar vesicles. The lipid suspension was freeze-thawed six
times and then extruded 20 times through polycarbonate
membranes with 0.1 lm-diameter pores (Nuclepore Corp.,
Pleasanton, CA, USA).
Peptide-Induced lipid mixing
Lipid mixing of large unilamellar vesicles was measured
using a fluorescence probe dilution assay [27]. Lipid vesicles
containing 0.6 mol% each of NBD-PtdEth (energy donor)
and Rho-PtdEth (energy acceptor) were prepared in NaCl/
KCl/P
i
as described above. A 1 : 4 mixture of labeled and
unlabeled vesicles (110 l
M
total phospholipid concentra-
tion) was suspended in 400 lL of NaCl/KCl/P
i
,andasmall
volume of peptide in dimethylsulfoxide was added. The
increase in NBD fluorescence at 530 nm was monitored
Fig. 1. Sendai virus F
1
N-terminal fusion domain is composed of two
repeats. Panel A, the segment ranging from residue 7 to residue 17 is
homologous to the segment 21–31. The mutations G7A and G12A,
known to increase cell-cell fusion between cells expressing the F
1
protein and normal cells [30], increase also the homology between the
two segments: G7 matches A21 and G12 matches A26. Panel B,

sequence alignment of the segment 7–31 of Sendai virus with homol-
ogous regions in other paramyxoviruses (HPIV 1, Human Parainflu-
enza virus 1; Measles virus; Rinderpest virus; CDV, Canine Distemper
virus;Mumpsvirus;SV5,SimianParainfluenzavirus5;NDV,New-
castle Disease virus). The consensus sequence consists of those amino
acids that are present in at least 50% of the aligned sequences. Panel C,
alignment between the segments 7–17 and 21–31 of the consensus
sequence.
Ó FEBS 2002 Sendai virus-mediated membrane fusion (Eur. J. Biochem. 269) 4343
with the excitation set at 467 nm. The fluorescence intensity
before the addition of the peptide was referred to as zero
percent lipid mixing, and the fluorescence intensity upon the
addition of Triton X-100 (0.05% v/v) was referred to as
100% lipid mixing.
Electron microscopy
The effects of the peptides on liposomal suspensions were
examined by negative-staining electron microscopy. A drop
containing DOPtdCho/DOPtdEth/Cho (1 : 1 : 1) 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).
Differential scanning calorimetry
A Nanocal instrument from Calorimetry Sciences Corpora-
tion (Spanish Fork, UT, USA) was used for all scans. Films
composed of DiPoPtdEth and increasing mole fractions of
peptide were prepared by dissolving the lipid in chloroform:
methanol (2 : 1) and adding appropriate amounts of a
dilute methanolic solution of peptide. The lipid DiPoPtdEth

was used for determining the effects of the peptides on
curvature because this lipid has a sharp bilayer to hexagonal
phase transition at a moderate temperature of 43 °Csothat
small shifts in the temperature of the transition of this lipid
can easily be measured. This is not the case for the
DOPtdCho/DOPtdEth/Cholesterol mixture used for other
purposes in this manuscript. The films were dried in a test
tube under a stream of nitrogen and then kept for 2–3 h in a
vacuum dessicator. They were hydrated with Pipes buffer
pH 7.40 (20 m
M
Pipes, 0.15
M
NaCl, 1 m
M
EDTA and
20 mgÆL
)1
NaN
3
) to give a final lipid concentration of
7mgÆmL
)1
, vortexed extensively and loaded into the
calorimeter sample cell. The same buffer was placed in the
reference cell. Heating scan rates of 0.75 °Cmin
)1
were
used. The bilayer to hexagonal phase transition was fitted
using parameters to describe an equilibrium with a single

van’t Hoff enthalpy and the transition temperature reported
as that for the fitted curve. Data was analyzed with the
program
ORIGIN
5.0.
31
P NMR Spectroscopy
The
31
P NMR spectra were measured using suspensions
of about 10 mg of a lipid mixture containing equimolar
amounts of DOPtdCho, DOPtdEth and cholesterol, with
or without the addition of peptide at a lipid to peptide
molar ratio of 200 : 1. The lipids and peptide were mixed
in organic solvent and dried, as described for the DSC.
The lipid film was hydrated with 200 lLof20m
M
Pipes,
1m
M
EDTA, 150 m
M
NaCl with 20 mgÆL
)1
NaN
3
,
pH 7.40. Spectra were obtained using a Bruker AV-500
spectrometer operating at 202.456 MHz in a 5-mm
broadband inverse probe with triple axis gradient capa-

bility. The spectra were acquired over a 48.544-kHz sweep
width in 32K data points (0.338 s acquisition time). A 90°
pulse width of 9.9 ls(90° flip angle) and a relaxation
delay of 3.0 s were used. Composite pulse decoupling was
used to remove any proton coupling. Generally, 700 free
induction decays were processed using an exponential line
broadening of 100 Hz and were zero-filled to 64K prior to
Fourier transformation. Probe temperature was main-
tained at 25 °C by a Bruker B-VT 3000 variable tem-
perature unit. Temperatures were monitored with a
calibrated thermocouple probe placed in the cavity of
the NMR magnet.
ATR-FTIR Measurements
Spectra were obtained with a Bruker equinox 55 FTIR
spectrometer equipped with a deuterated triglyceride sulfate
(DTGS) detector and coupled with an ATR device. For
each spectrum, 150 scans were collected, with resolution of
4cm
)1
. Samples were prepared as previously described [28].
Briefly, DOPtdCho/DOPtdEth/Cho (1 : 1 : 1) (0.78 mg)
alone or with peptide (23 lg) were deposited on a ZnSe
horizontal ATR prism (80 · 7 mm). Prior to sample
preparation the trifluoroacetate (CF
3
COO
–
) counterions,
which strongly associate with the peptide, were replaced
with chloride ions through several washings of the peptides

in 0.1
M
HCl and lyophilizations. This allowed the elimin-
ation of the strong C¼O stretching absorption band near
1673 cm
)1
[29]. Peptides were dissolved in methanol, and
lipids in a 1 : 2 methanol/CHCl
3
mixture. Lipid-peptide
mixtures or lipids with the corresponding volume of
methanol were spread with a Teflon bar on the ZnSe prism.
Drying under vacuum for 30 min eliminated the solvents.
Polarized spectra were recorded and the respective spectra
corresponding to pure phospholipids in each polarization
were subtracted from the sample spectra to yield the
difference spectra. The background for each spectrum was a
clean ZnSe prism. Hydration of the sample was achieved by
introduction of excess of deuterium oxide (
2
H
2
O) into a
chamber placed on top the ZnSe prism in the ATR casting
and incubation for 30 min prior to acquisition of spectra.
Any contribution of
2
H
2
O vapor to the absorbance spectra

near the amide I peak region was eliminated by subtraction
of the spectra of pure lipids equilibrated with
2
H
2
O under
the same conditions.
ATR-FTIR Data analysis
Prior to curve fitting, a straight base line passing through the
ordinates at 1700 cm
)1
and 1600 cm
)1
was subtracted. To
resolve overlapping bands, the spectra were processed using
PEAKFIT
TM
(Jandel Scientific, San Rafael, CA, USA)
software. Second-derivative spectra were calculated to
identify the positions of the component bands in the
spectra. These wavenumbers were used as initial parameters
for curve fitting with Gaussian component peaks. Positions,
bandwidths, and amplitudes of the peaks were varied until
good agreement between the calculated sum of all compo-
nents and the experimental spectra were achieved
(r
2
> 0.995), under the following constraints: (a) the
resulting bands shifted by no more than 2 cm
)1

from the
initial parameters, and (b) all the peaks had reasonable half-
widths (< 20–25 cm
)1
). The relative contents of different
secondary structure elements were estimated by dividing the
areas of individual peaks, assigned to particular secondary
structure, by the whole area of the resulting amide I band.
The experiments were repeated twice and were found to be
in good agreement.
4344 S. G. Peisajovich et al. (Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
Sendai virus F
1
N-terminal fusion domain is composed
of two repeats
The N-terminal fusion peptide of Sendai virus F
1
envelope
glycoprotein is formed by the 14 most N-terminal amino
acids. Following this apolar region, we detected a segment
of 11 residues strikingly similar to the N-terminal fusion
peptide. As shown in Fig. 1A, the segment ranging from
residue 7 to residue 17 is similar to the segment 21–31.
Interestingly, the mutations G7A and G12A, known to
increase cell–cell fusion between cells expressing the F
1
protein and normal cells [30], increase also the identity
between the two segments: G7 matches A21 and G12
matches A26. Furthermore, as shown in Fig. 1B,C, homo-

logous regions exist in other paramyxoviruses. This intrigu-
ing finding prompted us to investigate the role played by the
region consecutive to the Sendai virus N-terminal fusion
peptide in membrane fusion. To this end, we synthesized a
peptide corresponding to amino acids 19–33 from Sendai F
1
protein (WT-palm-19–33, see Table 1) and compared its
ability to induce lipid mixing of large unilamellar liposomes
with that of a longer segment (WT 1–33) that includes the
N-terminal fusion peptide. In order to facilitate partition of
the short WT-palm-19–33 to the membrane, its N-terminus
was palmitoylated. To ensure that palmitoylation did not
cause lipid mixing per se, we used as a control a palmitoy-
lated peptide with the same amino-acid composition of
WT-palm-19–33, but with a scrambled sequence (SC-palm-
19–33).
WT-palm-19–33 induces lipid mixing of large unilamellar
vesicles
The ability of the peptides to induce lipid mixing of
DOPtdCho/DOPtdEth/Cho (1 : 1 : 1) large unilamellar
vesicles (LUV) was determined by the probe-dilution assay
[27]. As depicted in Fig. 2, WT-palm-19–33 induces lipid
mixing of LUV in a dose-dependent manner, although it is
not as potent as the longer WT 1–33. On the contrary, SC-
palm-19–33 is poorly active and palmitic acid alone did not
induce any significant lipid mixing (not shown), reflecting
that the WT-palm-19–33¢s potency is not solely a conse-
quence of palmitoylation. It has been previously reported
that the mutations G7A and G12A increase the cell–cell
fusion activity of the full-length F protein [30]. Accordingly,

the G12A mutation enhanced the lipid mixing ability of a
peptide corresponding to the N-terminal segment of Sendai
F
1
protein toward negatively charged LUV composed of PS
[31]. As we wanted to determine the role of these mutations
on the mechanism of membrane fusion exerted by Sendai F
1
protein, we also tested here the ability to induce lipid mixing
of zwitterionic DOPtdCho/DOPtdEth/Cho (1 : 1 : 1) LUV
of the mutant G12A 1–33, the double mutant G7A G12A
1–33, and a peptide with the same amino-acid composition
of WT 1–33, but a scrambled sequence (SC 1–33). We found
that successive replacement of Gly 7 and Gly 12 by Ala
results in higher fusogenic activity (Fig. 2), whereas scram-
bling of the wild-type sequence renders the SC 1–33 peptide
inactive, indicating that the fusogenicity of the peptides
Table 1. Sequences of the peptides and lipopeptides.
WT 1–33 FFGAVIGTIALGVATSAQITAGIALAEAR
EAKR
G12A 1–33 FFGAVIGTIALAVATSAQITAGIALAEAR
EAKR
G7A–G12A 1–33 FFGAVIATIALAVATSAQITAGIALAEAR
EAKR
SC 1–33 VILEQRAFAVGGAILTSKFAIGGRTAAIA
TAEA
WT-palm-19–33 palmitoyl – ITAGIALAEAREAKR
SC-palm-19–33 palmitoyl -AERATAELGIKAIAR
Fig. 2. Peptide-promoted membrane fusion of DOPtdCho/DOPtdEth/
Cho (1 : 1 : 1) LUV as determined by lipid mixing. Panel A, dose

dependence of lipid mixing. Peptide aliquots were added to mixtures of
LUV (22 l
M
), containing 0.6% NBD-PtdEth and Rho-PtdEth, and
unlabeled LUV (88 l
M
)inNaCl/KCl/P
i
. The increase in the fluores-
cence was measured 15 min after the addition of the peptide. The
fluorescence intensity upon the addition of reduced Triton-X-100
(0.25% v/v) was referred to as 100%. Symbols: WT 1–33, empty cir-
cles; G12A 1–33, empty triangles; G7A–G12A 1–33, filled squares; SC
1–33, empty squares; WT-palm-19–33, filled circles; SC-palm-19–33,
filled triangles. Panel B, kinetics of lipid mixing for a peptide to lipid
ratio of 0.06.
Ó FEBS 2002 Sendai virus-mediated membrane fusion (Eur. J. Biochem. 269) 4345
depends on their specific sequences. Note that when the
experiments were repeated using different liposome prepa-
rations, differences of 15–20% were observed. However,
with any given single liposome preparation the relative
activities of the different peptides remained unchanged and
the error was never higher than 5–10%.
Lipid mixing is a result of membrane fusion
In order to confirm that the observed intervesicular lipid
mixing was the result of membrane fusion, suspensions of
LUV were directly visualized under an electron microscope,
before and after the treatment with the peptides. Briefly,
DOPtdCho/DOPtdEth/Cho (1 : 1 : 1) LUV of 100 nm-
diameter (200 l

M
) were incubated for 15 min alone, or with
each of the peptides (peptide lipid
)1
molar ratio of 0.05) in
NaCl/KCl/P
i
, before examination by electron microscopy.
Figure 3 shows representative micrographs of the LUV
without any peptide (Panel A), with WT 1–33 (Panel B),
with G12A 1–33 (Panel C), with G7A G12A 1–33 (Panel
D), with WT-palm-19–33 (Panel E), and with SC 1–33
(Panel F). The activity of SC-palm-19–33 was similar to that
of SC 1–33 and therefore it is not shown. It is evident from
the micrographs that the lipid mixing observed with G7A
G12A 1–33, G12A 1–33, WT 1–33, and WT-palm-19–33
appear concurrently with an increase in the size of the
vesicles, confirming that the ability of the peptides to induce
lipid mixing is the result of membrane fusion. In order to
shed light into their mechanism of action, we analyzed the
ability of the peptides to lower the T
H
of DiPoPtdEth, to
give rise to isotropic
31
P NMR signals, and determined the
secondary structure of the membrane-bound full-length
peptides by ATR-FTIR spectroscopy.
Peptide effects on DiPoPtdEth transition temperature
T

H
is a measure of the relative stability of the L
a
and H
II
lipid phases. A reduction in T
H
with the addition of a
peptide can be interpreted as a tendency of the peptide to
promote negative curvature of the membrane. As indicated
by shifts in T
H
, at high peptide concentrations, WT 1–33 is
the most potent in lowering the transition temperature,
followed by G12A 1–33, SC 1–33, and by G7A–G12A 1–33.
On the other hand, WT-palm-19–33 slightly increases T
H
(Fig. 4).
31
P NMR spectroscopy
The shape of the
31
P NMR powder pattern of lipid mixtures
serves as a good criterion for their morphology. The mixture
of DOPtdCho:DOPtdEth:cholesterol (1 : 1 : 1) exhibits a
spectrum typical of a membrane bilayer (see Fig. 5). Upon
addition of only 0.05 mol% peptide, WT 1–33 and to a
lower extent G12A 1–33 cause the formation of a structure
that gives rise to an isotropic component at the chemical
shift of phosphoric acid (Fig. 5). This is typical for highly

curved membrane structures and the appearance of such
peaks has been associated with higher rates of membrane
fusion [32,33]. Structures such as hemifusion intermediates
and fusion pores have highly curved surfaces that would
allow for the motional averaging of the chemical shift
anisotropy of the phospholipid. Interestingly, G7A G12A
1–33 and Palm-WT 19–33, although active in lipid mixing,
did not give rise to significant isotropic components. A
similar lack of isotropic component was observed for the
scrambled peptide, SC 1–33, as well as the short and lipid-
mixing inactive peptides.
Fig. 3. Electron micrographs of negatively
stained vesicles. Panel A, DOPtdCho/DOPtd-
Eth/Cho(1:1:1)LUValone;PanelB,WT
1–33; Panel C, G12A 1–33; Panel D, G7A–
G12A 1–33; Panel E, WT-palm-19–33; Panel
F, SC 1–33. The vesicles were incubated with
the peptides ([peptide]/[lipid] 0.05) for 15 min
prior to visualization. The bar represents
200 nm. The effect of SC-palm-19–33 was
similar to that of SC 1–33, therefore it is not
shown.
4346 S. G. Peisajovich et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Replacement of Gly7 and Gly12 by Ala results in higher
a-helical content in the membrane
As structure has been shown to be important for the activity
of fusion peptides [31,34,35] and in order to investigate
whether the different behavior of the peptides in inducing
lipid mixing of LUV, lowering the T
H

of DiPoPtdEth, and
promoting isotropic
31
P NMR signals could be related to
differences in their structures, we determined the secondary
structure of the membrane-bound peptides by ATR-FTIR
spectroscopy. The spectra of the different peptides and the
respective second derivatives calculated to identify the
positions of the components bands are shown in Fig. 6.
The percentages corresponding to the different structures
are listed in Table 2. As expected, the combined G7A and
G12A mutations significantly increase the a-helical content
of the peptides, likely lowering the conformational flexibility
associated with the higher amount of random structures
observed in WT 1–33. On the other hand, scrambling of the
wild-type sequence resulted in a very different spectrum with
a predominance of aggregated b strands.
DISCUSSION
The region consecutive to the N-terminal fusion peptide
participates in the fusion process
In this study we report that the N-terminal domain of
Sendai F
1
protein can be considered as two consecutive
repeats (Fig. 1). Remarkably, the mutations G7A and
G12A, known to enhance F
1
-induced cell-cell fusion [30],
augment also the identity between the two segments.
Interestingly, we found that, when partitioned into the

membrane by palmitoylation of its N-terminus, the most
C-terminal repeat induces fusion of large unilamellar
liposomes (Figs 2 and 3). It should be noted that
membrane anchoring via a C-terminal cysteine coupled
to a modified phospholipid has been shown before to lead
to fusogenic properties in a short synthetic model peptide
[36]. However, the fusogenic activity of WT-palm-19–33 is
not solely a consequence of its palmitoylation, as a
palmitoylated peptide with the same amino-acid compo-
sition of WT-palm-19–33, but a scrambled sequence, as
well as palmitic acid alone, are not active. These results
suggest that the Sendai virus F
1
N-terminal domain is
composed of two repeats, both of which participate in the
actual merging of the viral and cellular membranes. We
do not believe that palmitoylation fulfills all of the
functions of the initial 18 amino acids, as addition of
these residues results in a longer peptide with a substan-
tially increased fusogenic activity. The finding that
homologous regions exist in other paramyxoviruses
(Fig. 1, panels B and C) suggests that the structural
Fig. 5.
31
P NMR spectra of an equimolar
mixture of DOPtdCho:DOPtdEth:cholesterol
with and without the addition of 0.05 mol per-
centage peptide at 25 °C. The samples were
hydrated with 20 m
M

Pipes, 1 m
M
EDTA,
150 m
M
NaCl with 0.002% NaN
3
, pH 7.40.
Fig. 4. Shift of the bilayer to the hexagonal phase transition temperature
of DiPoPtdEth as a function of the mole fraction of peptide. Symbols:
WT 1–33, filled circles; G12A 1–33, filled squares; SC 1–33, filled
diamonds; G7A–G12A 1–33, empty triangles; WT-palm-19–33, empty
circles.
Ó FEBS 2002 Sendai virus-mediated membrane fusion (Eur. J. Biochem. 269) 4347
and functional organization reported here for Sendai
N-terminal fusion peptide, may be common to other
members of the Paramyxoviridae family. Furthermore,
recently it was shown that the polar region consecutive to
the HIV-1 gp41 N-terminal fusion peptide also enhances
its fusogenic activity, presumably by promoting self-
association of the fusion peptide [17]. The similarity
between what was found in HIV-1, a retrovirus, and what
we report here in Sendai, a paramyxovirus, suggests that
the N-terminal fusogenic domains from these distantly
related viruses share a common mechanism.
Secondary structure modulates the fusogenic activity
of the peptides
Changes in the secondary structure of fusogenic peptides
have been shown to alter their activity [34]. Interestingly,
the mutations Gly7 to Ala and Gly12 to Ala were shown to

increase F
1
-induced cell-cell fusion [30]. Here, we analyzed
how these mutations, which increase the identity between
the two repeats, affect the structure and activity of the
N-terminal region of Sendai F
1
protein. We found that
replacement of Gly7 and Gly12 by Ala, which increased the
a helical content of the peptide when bound to DOPtdEth/
DOPtdCho/Cho (1 : 1 : 1) membranes, enhanced the fuso-
genicity of the peptide. On the contrary, scrambling of its
amino-acid sequence resulted in an inactive peptide with a
significantly reduced amount of a helix. The presence of Gly
at position 7 and 12 in the wild-type Sendai F
1
protein
imparts a greater flexibility to this region. Replacement of
the two Gly by Ala, a residue with a higher helical
propensity, may result in a longer or more stable helix. The
G7A and G12A mutations are associated in vivo with a
severe cytopathic effect, thus glycine may have been selected
to balance high fusion activity with successful viral repli-
cation [30]. It should be mentioned that Rapaport and Shai
[31] did not observed a significant difference in the a helical
content of WT 1–33 and its G12A mutant, as determined
by circular dichroism in 70% TFE and methanol. However,
these are only Ômembrane mimeticÕ environments, whereas
here we measured the peptides’ secondary structure in the
presence of phospholipid membranes. Unlike organic

solvents, aqueous dispersions of phospholipids allow seg-
ments of peptides to partition simultaneously into both
aqueous and nonpolar solvent environments. As observed
with HIV-1 gp41 fusion peptide, we cannot rule out that
other secondary structures play also some role during the
fusion process [17,35,37,38].
The mechanism of membrane fusion
According to the stalk model [36] in its modified form
[39,40], both a membrane fusion pore and the inverted
Fig. 6. FTIR spectra deconvolution of the fully deuterated amide I band
(1600–1700 cm
1
) and their respective second derivatives. PanelA,WT
1–33; panel B, G12A 1–33; panel C, G7A–G12A 1–33; panel D, SC
1–33. The component peaks are the result of a curve fitting using a
Gauss line shape. The amide I frequencies characteristic of the various
secondary-structure elements were taken from [41]. The sums of the
fitted components superimpose on the experimental amide I region
spectra. The solid lines represent the experimental FTIR spectra after
Savitzky-Golay smoothing; the broken lines represent the fitted com-
ponents of the spectra. A 100 : 1 lipid:peptide molar ratio was used.
Table 2. Secondary structure of the membrane-bound peptides according to FTIR spectroscopy. A 100 : 1 lipid:peptide molar ratio was used. The
amide I frequencies characteristic of the various secondary-structure elements were taken from Jackson and Mantsch [41], mean values ± standard
deviation are given.
Sample
Secondary Structure (%)
a helix Random coil Aggregated strands Other structures
WT 1–33 50 ± 8 32 ± 4 17 ± 4 1 ± 1
G12A 1–33 65 ± 1 13 ± 2 18 ± 1 4 ± 2
G7A–G12A 1–33 72 ± 1 13 ± 1 10 ± 1 5 ± 1

SC 1–33 31 ± 3 16 ± 2 43 ± 4 10 ± 4
4348 S. G. Peisajovich et al. (Eur. J. Biochem. 269) Ó FEBS 2002
hexagonal phase arise through a common intermediate. The
first step is hemifusion between the outer leaflets of two
opposing membranes that results in a stalk with high
negative curvature. Subsequently, joining of the opposing
monolayers leads to formation of the more stable trans
monolayer contact (TMC) intermediate. The pathway after
formation of TMCs diverges, leading either to membrane
fusion or to the formation of an inverted hexagonal phase.
Rupture of a single TMC produces a fusion pore, whereas
transition to inverted hexagonal phase requires aggregation
of numerous TMCs [40]. Several fusion peptides have been
shown to lower the transition temperature from lamellar to
inverted hexagonal phases (reviewed in [16]), indicating that
they promote negative curvature in the membrane, thus
favoring formation of the highly curved stalk intermediate.
This property correlated well with the infectivity of influenza
virus containing single amino-acid mutations in the fusion
peptide segment of hemagglutinin [16]. Here we observed an
inverse correlation between the lipid mixing ability of WT
1–33, G12A 1–33, and G7A G12A 1–33 and their ability to
lower T
H
or to give rise to an isotropic peak in the
31
PNMR
spectra. It should be noted that in the NMR and DSC
experiments the peptide was added to both sides of the
bilayer starting from a solution in organic solvent. This is

different from the procedure for the lipid mixing assay in
which the peptide is added to one side of the bilayer in
buffer. This difference in the methodology that had to be
used could contribute to a different behaviour of the
peptides in the different system. An additional factor,
however, that could contribute to the higher fusogenic
activity of the single mutant G12A and the double mutant
G7A G12A, despite their smaller effect on T
H
and on the
31
P NMR isotropic peak, as compared to the wild-type
peptide, may be related to their more shallow penetration
into the membrane, as shown for different constructs of the
HIV-1 fusion peptide [17]. This possibility is supported by
the effect of WT-palm-19–33, which due to the polar nature
of its amino-acid composition, is likely to be located on the
surface and, indeed, causes a slight increase in T
H
.As
noticed before for other viral fusion peptides [1], when the
amino-acid sequence of Sendai F
1
N-terminal region is
represented as an a helix, it forms a sided helix, with most of
the Gly and Ala residues lying on the same face. We can
speculate that the presence of Ala at position 7 and/or 12
reduces the flexibility of G12A 1–33 and G7A G12A 1–33
by extending an a helix that runs closer to the membrane
surface, thus diminishing the insertion into the membrane.

Then, G12A 1–33 and more markedly G7A G12A 1–33
protrude from the membrane more than WT 1–33; thus, at
high mole fraction, G12A 1–33 and even more G7A G12A
1–33 may sterically prevent aggregation of TMCs and the
concomitant transition to the inverted hexagonal phase.
Fusion between two opposing membranes requires the
formation of only one fusion pore and therefore it is not
affected by a protruding peptide. Alternatively, this intrigu-
ing observation might be related to their different potency in
facilitating the rupture of the dimple in the center of the
TMC. The peptide that better promotes the rupture of the
TMC will favor formation of fusion pore-like structures
more easily, therefore lowering the chances of TMC
aggregation and subsequent hexagonal phase formation.
The current study generalizes the finding that, consecutive
to their N-terminal fusion peptides, the envelope glycopro-
teins from Paramyxo- and Lentiviruses have a relatively
polar helical segment that facilitates membrane fusion but
do not insert deeply into the membrane, suggesting that
unrelated viral families share common mechanisms of cell
entry. How such surface seeking helices promote fusion
remains to be determined but could include lowering the
degree of hydration of the membrane surface.
ACKNOWLEDGEMENTS
Sergio G. Peisajovich is supported by fellowships from The Mifal
Ha’paiys Foundation of Israel and the Feinberg Graduate School of
the Weizmann Institute of Science. This work was supported in part by
the Canadian Institutes of Health Research (grant MT-7654).
REFERENCES
1. White, J., Kielian, M. & Helenius,A. (1983) Membrane fusion pro-

teins of enveloped animal viruses. Q. Rev. Biophys. 16, 151–195.
2. Bullough, P.A., Hughson, F.M., Skehel, J.J. & Wiley, D.C. (1994)
Structure of influenza haemagglutinin at the pH of membrane
fusion. Nature 371, 37–43.
3. Chan, D.C., Fass, D., Berger, J.M. & Kim, P.S. (1997) Core
structure of gp41 from the HIV envelope glycoprotein. Cell 89,
263–273.
4. Weissenhorn, W., Dessen, A., Harrison, S.C., Skehel, J.J. & Wiley,
D.C. (1997) Atomic structure of the ectodomain from HIV-1
gp41. Nature 387, 426–430.
5. Malashkevich, V.N., Chan, D.C., Chutkowski, C.T. & Kim, P.S.
(1998) Crystal structure of the simian immunodeficiency virus
(SIV) gp41 core: conserved helical interactions underlie the broad
inhibitory activity of gp41 peptides. Proc. Natl Acad. Sci. USA 95,
9134–9139.
6. Yang, Z.N., Mueser, T.C., Kaufman, J., Stahl, S.J., Wingfield,
P.T. & Hyde, C.C. (1999) The crystal structure of the SIV gp41
ectodomain at 1.47 A resolution. J. Struct. Biol. 126, 131–144.
7. Kobe, B., Center, R.J., Kemp, B.E. & Poumbourios, P. (1999)
Crystal structure of human T cell leukemia virus type 1 gp21
ectodomain crystallized as a maltose-binding protein chimera
reveals structural evolution of retroviral transmembrane proteins.
Proc. Natl Acad. Sci. USA 96, 4319–4324.
8. Baker, K.A., Dutch, R.E., Lamb, R.A. & Jardetzky, T.S. (1999)
Structural basis for paramyxovirus-mediated membrane fusion.
Mol. Cell. 3, 309–319.
9. Harter, C., James, P., Bachi, T., Semenza, G. & Brunner, J. (1989)
Hydrophobic binding of the ectodomain of influenza hemagglu-
tinin to membranes occurs through the Ôfusion peptideÕ. J. Biol.
Chem. 264, 6459–6464.

10. Stegmann, T., Doms, R.W. & Helenius, A. (1989) Protein-medi-
ated membrane fusion. Annu. Rev. Biophys. Biophys. Chem. 18,
187–211.
11. Wharton, S.A., Martin, S.R., Ruigrok, R.W., Skehel, J.J. &
Wiley, D.C. (1988) Membrane fusion by peptide analogues of
influenza virus haemagglutinin. J. Gen. Virol. 69, 1847–1857.
12. Ruigrok, R.W., Aitken, A., Calder, L.J., Martin, S.R., Skehel, J.J.,
Wharton, S.A., Weis, W. & Wiley, D.C. (1988) Studies on the
structure of the influenza virus haemagglutinin at the pH of
membrane fusion. J. Gen. Virol. 69, 2785–2795.
13. Guy, H.R., Durell, S.R., Schoch, C. & Blumenthal, R. (1992)
Analyzing the fusion process of influenza hemagglutinin by
mutagenesis and molecular modeling. Biophys. J. 62, 95–97.
14. Hughson, F.M. (1995) Structural characterization of viral fusion
proteins. Curr. Biol. 5, 265–274.
15. Zimmerberg, J., Curran, M. & Cohen, F.S. (1991) A lipid/protein
complex hypothesis for exocytotic fusion pore formation. Ann.
NY Acad. Sci. 635, 307–317.
Ó FEBS 2002 Sendai virus-mediated membrane fusion (Eur. J. Biochem. 269) 4349
16. Epand, R.M. (1998) Lipid polymorphism and protein–lipid
interactions. Biochim. Biophys. Acta. 1376, 353–368.
17. Peisajovich, S.G., Epand, R.F., Pritsker, M., Shai, Y. & Epand,
R.M. (2000) The polar region consecutive to HIV-1 fusion peptide
participates in membrane fusion. Biochemistry. 39, 1826–1833.
18. Collins, P.L., Chanock, R.M. & McIntosh, K. (1996) Parainflu-
enza Viruses. In Virology (Fields, B.N., Knipe, D.M. & Howley,
P.M., eds), pp. 1205–1241. Lippincott-Raven, Philadelphia, PA,
USA.
19. Lamb, R.A. & Kolakofsky, D. (1996) Paramyxoviridae: The
VirusesandTheirReplication.InVirology (Fields, B.N., Knipe,

D.M. & Howley, P.M., eds), pp. 1177–1204. Lippincott-Raven,
Philadelphia, PA, USA.
20. Homma, M. & Ohuchi, M. (1973) Trypsin action on the growth of
sendai virus in tissue culture cells. J. Virol. 12, 1457–1465.
21. Suarez, T., Gallaher, W.R., Agirre, A., Goni, F.M. & Nieva, J.L.
(2000) Membrane interface-interacting sequences within the
ectodomain of the human immunodeficiency virus type 1 envelope
glycoprotein: putative role during viral fusion. J. Virol. 74, 8038–
8047.
22. Peisajovich, S.G., Samuel, O. & Shai, Y. (2000) The F
1
protein of
paramyxoviruses has two fusion peptides: implications for
the mechanism of membrane fusion. J. Mol. Biol. 296, 1353–1365.
23. Samuel, O. & Shai, Y. (2001) Participation of two fusion peptides
in measles virus-induced membrane fusion: emerging similarity
with other Paramyxoviruses. Biochemistry 40, 1340–1349.
24. Ghosh,J.K.,Peisajovich,S.G.&Shai,Y.(2000)Sendaivirus
internal fusion peptide: structural and functional characterization
and a plausible mode of viral entry inhibition. Biochemistry 39,
11581–11592.
25. Chen, L., Gorman, J.J., McKimm-Breschkin, J., Lawrence, L.J.,
Tulloch, P.A., Smith, B.J., Colman, P.M. & Lawrence, M.C.
(2001) The structure of the fusion glycoprotein of Newcastle dis-
ease virus suggests a novel paradigm for the molecular mechanism
of membrane fusion. Structure 9, 255–266.
26. Merrifield, R.B., Vizioli, L.D. & Boman, H.G. (1982) Synthesis of
the antibacterial peptide cecropin A(1–33). Biochemistry 21, 5020–
5031.
27. Struck, D.K., Hoekstra, D. & Pagano, R.E. (1981) Use of

resonance energy transfer to monitor membrane fusion. Bio-
chemistry 20, 4093–4099.
28. Gazit, E., Miller, I.R., Biggin, P.C., Sansom, M.S. & Shai, Y.
(1996) Structure and orientation of the mammalian antibacterial
peptide cecropin P1 within phospholipid membranes. J. Mol. Biol.
258, 860–870.
29. Surewicz, W.K., Mantsch, H.H. & Chapman, D. (1993) Determi-
nation of protein secondary structure by Fourier transform
infrared spectroscopy: a critical assessment. Biochemistry 32,389–
394.
30. Horvath, C.M. & Lamb, R.A. (1992) Studies on the fusion
peptide of a paramyxovirus fusion glycoprotein: roles of conserved
residues in cell fusion. J. Virol. 66, 2443–2455.
31. Rapaport, D. & Shai, Y. (1994) Interaction of fluorescently
labeled analogues of the amino-terminal fusion peptide of Sendai
virus with phospholipid membranes. J. Biol. Chem. 269, 15124–
15131.
32. Siegel, D.P., Banschbach, J., Alford, D., Ellens, H., Lis, L.J.,
Quinn, P.J., Yeagle, P.L. & Bentz, J. (1989) Physiological levels of
diacylglycerols in phospholipid membranes induce membrane
fusion and stabilize inverted phases. Biochemistry 28, 3703–3709.
33. Ellens, H., Siegel, D.P., Alford, D., Yeagle, P.L., Boni, L., Lis,
L.J., Quinn, P.J. & Bentz, J. (1989) Membrane fusion and inverted
phases. Biochemistry 28, 3692–3703.
34. Lee, S., Aoki, R., Oishi, O., Aoyagi, H. & Yamasaki, N. (1992)
Effect of amphipathic peptides with different alpha-helical con-
tents on liposome-fusion. Biochim. Biophys. Acta. 1103, 157–162.
35. Nieva,J.L.,Nir,S.,Muga,A.,Goni,F.M.&Wilschut,J.(1994)
Interaction of the HIV-1 fusion peptide with phospholipid vesicles:
different structural requirements for fusion and leakage. Bio-

chemistry 33, 3201–3209.
36. Markin, V.S., Kozlov, M.M. & Borovjagin, V.L. (1984) On the
theory of membrane fusion. The stalk mechanism. Gen. Physiol.
Biophys. 5, 361–377.
37. Pritsker, M., Jones, P., Blumenthal, R. & Shai, Y. (1998) A syn-
thetic all
D
-amino acid peptide corresponding to the N-terminal
sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in
the membrane and inhibits HIV-1 envelope glycoprotein-mediated
cell fusion. Proc. Natl Acad. Sci. USA 95, 7287–7292.
38. Pritsker, M., Rucker, J., Hoffman, T.L., Doms, R.W. & Shai, Y.
(1999) Effect of nonpolar substitutions of the conserved Phe
11
in
the fusion peptide of HIV-1 gp41 on its function, structure, and
organization in membranes. Biochemistry 38, 11359–11371.
39. Siegel, D.P. & Epand, R.M. (1997) The mechanism of lamellar-to-
inverted hexagonal phase transitions in phosphatidylethanol-
amine: implications for membrane fusion mechanisms. Biophys. J.
73, 3089–3111.
40. Siegel, D.P. (1999) The modified stalk mechanism of lamellar/
inverted phase transitions and its implications for membrane
fusion. Biophys. J. 76, 291–313.
41. Jackson, M. & Mantsch, H.H. (1995) The use and misuse of FTIR
spectroscopy in the determination of protein structure. Crit. Rev.
Biochem. Mol. Biol. 30, 95–120.
4350 S. G. Peisajovich et al. (Eur. J. Biochem. 269) Ó FEBS 2002