Charged residues are involved in membrane fusion mediated by ahydrophilic peptide located in vesicular stomatitis virus G protein

FABIANA A. CARNEIRO1, GUY VANDENBUSSCHE2, MARIA A. JULIANO3,

LUIZ JULIANO3, JEAN-MARIE RUYSSCHAERT2, & ANDREA T. DA POIAN1

1Instituto de Bioquımica Medica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro,

Rio de Janeiro, Brazil, 2Structure et Fonction des Membranes Biologiques, Centre de Biologie Structurale et de

Bioinformatique, Universite Libre de Bruxelles, Brussels, Belgium, and 3Departamento de Biofısica, Escola Paulista de

Medicina, UNIFESP, Sao Paulo, Brazil

(Received 11 October 2005; and in revised form 27 April 2006)

AbstractMembrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus(VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of theendosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequenceis very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, wedemonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formationof a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of thehydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linearfashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion,suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptidescreates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.

Keywords: Membrane fusion, fusion peptide, vesicular stomatitis virus, dicyclohexylcarbodiimide, hydrophobicity

Introduction

Virus replication depends on the transfer of viral

genome and accessory proteins to the cytosol or to

the nucleus of a host cell. In the case of enveloped

virus, this entry process involves the fusion of the

virus envelope with the plasma or the endosomal

membranes of the host cell [1]. The membrane

fusion reaction is catalyzed by viral surface glyco-

proteins, which undergo conformational changes

triggered by either their interaction with a cellular

receptor or by the acidification of the endosomal pH.

Viral fusion glycoproteins contain a short sequence

directly involved in the interaction with the target

membrane during the fusion reaction, known as the

fusion peptide. Fusion peptides from several viruses

have been identified by mutagenesis experiments, in

which a single amino acid change abolished the

fusion activity of the glycoprotein. The sequence of

the fusion peptides is generally conserved within the

viral family, but not among different families.

Based on structural differences, the viral fusion

proteins were classified into two groups. Class I

fusion proteins form trimeric spikes predominantly

folded as a-helices with a hydrophobic fusion pep-

tide located at the N-terminal end of the protein [2].

After binding to a cellular receptor or on exposure to

low pH, the protein forms an extended conforma-

tion and the fusion peptide inserts into the target

membrane. The post-fusion conformation is a hair-

pin-like structure in which the fusion peptide and

the membrane anchor are at the same end [2]. In

class II fusion proteins three domains folded largely

on b-sheets are arranged in a continuous protein

lattice formed by dimers [3]. The fusion peptide is

an internal loop between two b-strands, buried in

the dimer interface. The determination of the post-

Correspondence: Andrea T. Da Poian, Instituto de Bioquımica Medica, Universidade Federal do Rio de Janeiro, Av. Bauhinia, 400,

Bl. H, s 22, Rio de Janeiro, RJ 21941-590, Brazil. Tel: 55 21 22706264. Fax: 55 21 22708647. E-mail: dapoian@bioqmed.ufrj.br

Molecular Membrane Biology, September�October 2006; 23(5): 396�406

ISSN 0968-7688 print/ISSN 1464-5203 online # 2006 Informa UK Ltd

DOI: 10.1080/09687860600780892

fusion structure of class II fusion proteins revealed a

surprising convergence of the class I and class II

fusion mechanisms [4]. The acidic pH of the

endosome induces a disassembly of envelope pro-

teins dimers, which rearrange in trimers with the

fusion peptide loops clustered at one end of an

elongated molecule.

The viruses that belong to the Rhabdoviridae

family are widely distributed in nature and their

hosts range from vertebrates and invertebrates

animals to many species of plants. All the rhabdo-

viruses present a bullet-shaped structure that is

formed by two major components: the nucleocapsid

and the envelope. The envelope is a lipid bilayer

derived from the host cell containing trimeric

transmembrane spikes composed by the viral surface

glycoprotein G. Vesicular stomatitis virus (VSV) is

the prototype of the Rhabdoviridae family. VSV G

protein is involved both in the cell recognition and in

the membrane fusion reaction, which occurs in the

acidic environment of the endosome after virus

internalization. Mutagenesis experiments have

shown that substitution of conserved Gly, Pro, or

Asp located in the region between amino acids 117

and 137 either abolished the fusion ability of G

protein or shifted the optimum pH of fusion [5�7].

This led the authors to propose that this segment

would be the VSV G protein putative fusion peptide,

although direct evidence that this particular region

interacts with the target membrane is still lacking.

Studying the requirement for PS in the target

membrane [8] and the crucial role of G protein

His residues for VSV fusion [9], we found another

candidate to be the VSV fusion peptide, the PS

binding site of the rhabdoviruses G protein [10].

This segment was firstly characterized for viral

hemorrhagic septicemia virus (VHSV), a rhabdo-

virus of salmonids [11,12], and then was found

among all rhabdoviruses [10]. For VSV, it corre-

sponds to the sequence between amino acid 145 and

164 (sequence VTPHHVLVDEYTGEWVDSQF).

We have demonstrated that a synthetic peptide

corresponding to this sequence was as efficient as

the whole virus in catalyzing fusion, whereas the

putative fusion peptide failed to induce fusion [9].

Moreover, as found for VSV-induced membrane

fusion, the fusion induced by the peptide was

dependent on pH and on the presence of PS in the

target membrane.

An interesting feature of VSV peptide145�164 is

that it contains four acidic amino acid residues, two

aspartic and two glutamic acids, which would be

negatively charged at the fusion pH. Using the

carboxyl-modifying agent dicyclohexylcarbodiimide

(DCCD) and several mutant peptides, we showed in

this work that the acidic residues, especially Asp153

and Glu158, participate in the formation of a hydro-

phobic domain at pH 6.0, which is necessary to the

peptide-induced membrane fusion. In addition, the

formation of this hydrophobic region as well as the

membrane fusion itself, are highly dependent on

peptide concentration, suggesting the involvement of

peptide oligomerization.

Materials and methods

Chemicals

Phosphatidylserine (PS) and phosphotidylcholine

(PC) from bovine brain, and dicyclohexylcarbodii-

mide (DCCD) were purchased from Sigma Chemi-

cal Co., St Louis, MO, USA. N-(lissamine

Rhodamine B sulfonyl) phosphatidylethanolamine

(Rh-PE), N-(7- nitro-2,1,3-benzoxadiazol-4-yl)

phosphatidylethanolamine (NBD-PE) and 8-anili-

nonaphthalene-1-sulfonate (ANS) were purchased

from Molecular Probes Inc., Eugene, OR, USA. All

other reagents were of analytical grade.

Peptides synthesis. VSV peptide145�164 (sequence

VTPHHVLVDEYTGEWVDSQF) and histidine

and acid residues mutants were synthesized by solid

phase using the Fmoc methodology and all protected

amino acids were purchased from Calbiochem-

Novabiochem (San Diego, CA) or from Neosystem

(Strasbourg, France). The syntheses were done in an

automated bench-top simultaneous multiple solid-

phase peptide synthesizer (PSSM 8 system from

Shimadzu, Tokyo, Japan). The final deprotected

peptides were purified by semipreparative HPLC

using an Econosil C-18 column (10 mm, 22.5�/250

mm) and a two-solvent system: (A) trifluoroacetic

acid/H2O (1:1000, v/v) and (B) trifluoroacetic acid/

acetonitrile/H2O (1:900:100, v/v/v). The column

was eluted at a flow rate of 5 ml.min�1 with a 10

or 30 to 50 or 60% gradient of solvent B over 30 or

45 min. Analytical HPLC was performed using a

binary HPLC system from Shimadzu with a SPD-

10AV Shimadzu UV/vis detector, coupled to an

Ultrasphere C-18 column (5 mm, 4.6�/150 mm),

which was eluted with solvent systems A1 (H3PO4/

H2O, 1:1000, v/v) and B1 (acetonitrile/H2O/H3PO4,

900:100:1, v/v/v) at a flow rate of 1.7 ml.min�1 and

a 10�80% gradient of B1 over 15 min. The

HPLC column eluted materials were monitored

by their absorbance at 220 nm. The molecular

mass and purity of synthesized peptides were

checked by MALDI-TOF mass spectrometry (Tof-

Spec-E, Micromass) and/or peptide sequencing

using a protein sequencer PPSQ-23 (Shimadzu

Tokyo, Japan).

G protein charged residues in VSV-induced membrane fusion 397

Peptide modification with DCCD. A solution of

dicyclohexylcarbodiimide (DCCD) was freshly pre-

pared by dilution of the reagent in ethanol.

Peptide145�164 was diluted in 20 mM MES, 30

mM Tris buffer, pH 6.0 and incubated for 1 h at

room temperature with DCCD, in a molar ratio of

DCCD/peptide of 40.

Preparation of liposomes. PC and PS at a molar ratio

of 1:3 were dissolved in chloroform and evaporated

under nitrogen. The lipid film formed was resus-

pended in 20 mM MES, 30 mM Tris buffer (pH

indicated in the Figure legends) at a final concentra-

tion of 1 mM. The suspension was vortexed

vigorously for 5 min. Small unilamellar vesicles

(SUV) were obtained by sonicating the turbid

suspension using a Branson Sonifier (Sonic Power

Company, Danbury, CT) equipped with a titanium

microtip probe. Sonication was performed in an ice

bath, alternating cycles of 30 sec at 20% full power,

with 60-sec resting intervals until a transparent

solution was obtained (approx. 10 cycles). For

fusion assays, 1 mol% of each Rh-PE and NBD-

PE was incorporated in the lipid films.

Liposome fusion assay. Liposomes composed of PC-

PS (1:3) containing equal amounts of unlabeled

vesicles and vesicles labeled with Rh-PE and NBD-

PE were prepared in 20 mM MES, 30 mM Tris

buffer (pH indicated in the Figure legends), at a final

phospholipid concentration of 0.1 mM. The fusion

reaction was initiated by addition of the peptide.

Fusion was followed by the resonance energy

transfer assay as described in Struck et al. (1981)

[13]. The samples were excited at 470 nm and the

fluorescence intensity was collected at 530 nm, using

a Hitachi F-4500 Fluorescence Spectrophotometer.

Mass spectrometry. A stock solution of VSV

peptide145�164 in ethanol was diluted at different

concentrations (as indicated in the Figure legends)

in 20 mM MES, 30 mM Tris buffer pH 6.0, and

incubated with DCCD for 1 h at room temperature.

Before analysis by mass spectrometry, the buffer was

removed by loading the peptide on a ZipTip C18

(Millipore, Billerica, USA). The peptide was washed

with TFA 0.1% (v/v) and eluted in 5 ml acetonitrile

50%/formic acid 1% (v/v). The samples were loaded

into a nanoflow capillary (Proxeon, Odense, Den-

mark). ESI mass spectra were acquired on a quad-

rupole time-of-flight instrument (Q-Tof Ultima �Micromass/Waters, Manchester, UK) operating in

the positive ion mode, equipped with a Z-spray

nanoelectrospray source. Capillary voltages of

1.1�1.5 kV and cone voltage of 50 V typically were

used. The source temperature was held at 808C. The

spectra represent the average of 1 sec scans. Data

acquisition was performed with a MassLynx 4.0

system. The exact mass of the peptide was deter-

mined after processing of the spectra by the software

Transform (Micromass/Waters, Manchester, UK).

For MS/MS studies, the quadrupole was used to

select the charged parent ion, which was subse-

quently fragmented in a hexapole collision cell using

argon as collision gas and an appropriate collision

energy. MS/MS data were processed by a maximum

entropy data enhancement program, MaxEnt 3

(Micromass/Waters, Manchester, UK). Amino acid

sequence was semi-automatically deduced with the

peptide sequencing program, PepSeq.

Infrared spectroscopy. ATR-FTIR (Attenuated Total

Reflection Fourier Transform infrared spectroscopy)

spectra were recorded on a Bruker IFS-55 FTIR

spectrophotometer (Bruker, Karlsruhe, Germany)

equipped with a liquid nitrogen-cooled mercury-

cadmium-telluride detector [14�16]. The spectro-

photometer was continuously purged with dried air.

The internal reflection element (ATR) was a germa-

nium plate (50�/20�/2 mm) with an aperture angle

of 458, yielding 25 internal reflections. Samples were

deposited on the germanium element. Films were

formed by slowly evaporating the sample on one side

of the ATR plate under a stream of nitrogen.

Samples were rehydrated by flushing D2O-saturated

N2 for 30 min at room temperature.

Results

Effects of Glu and/or Asp modification on peptide-

induced membrane fusion

We have previously shown that fusion induced by

peptide145�164 depends on the protonation of its

His148 and His149 residues, which confer positive

charges to the peptide at pH below 6.0 [12].

However, the peptide also contains four negatively

charged amino acid residues (Asp153, Glu154, Glu158

and Asp161) and no other positively charged residue.

To investigate the role of these negative charges in

peptide-induced fusion, we used DCCD, a com-

pound that labels Asp and Glu residues located in

hydrophobic environments [17]. Peptide-induced

membrane fusion was quantified by NBD-PE/Rh-

PE energy transfer assay (Figure 1a). DCCD label-

ing completely abolished the ability of the peptide to

mediate membrane fusion. To confirm the impor-

tance of the negative charges of these residues,

fusion was assayed at pH 4.0, a pH close to the

pKa of the carboxyl groups of lateral chain of the

acidic residues (Figure 1b). Fusion did not occur at

pH 4.0, even when the peptide was not incubated

398 F. A. Carneiro et al.

with DCCD, confirming the importance of the

negative charges. No lipid mixing was observed at

pH 7.5, confirming the role of the positively charged

histidines.

Mass spectrometry analysis revealed the presence

of different populations containing up to three

modified residues (Figure 2). The computed mono-

isotopic mass of each population agrees with the

addition of one, two or three DCCD (monoisotopic

mass�/206.17 Da) molecules: 2356.13 (�/0),

2562.26 (�/1), 2768.44 (�/2) and 2974.62 (�/3),

respectively.

Acidic residues are involved in the formation of a

hydrophobic region necessary for peptide-induced fusion

It is well established that negatively charged phos-

pholipids, especially PS, are required for VSV

binding to membranes and fusion [8,18]. This

suggests that the acidic amino acids of the

peptide145�164 might not be involved directly in the

interaction with the membrane. Since the carboxyl

modification by DCCD is highly favored in hydro-

phobic environment [17], one possibility is that the

negative charges could participate in the formation

of a hydrophobic fusion-active structure maintained

either by intra-molecule or by inter-molecule inter-

actions. To investigate the formation of hydrophobic

regions, we used the fluorescent probe ANS, whose

binding to non-polar segments in proteins is accom-

panied by a large increase in its fluorescence

quantum yield. Figure 3a shows that the peptide

binds ANS in a pH-dependent manner. ANS bind-

ing was maximal at pH 6.0, showing that the

formation of the hydrophobic domain was maximal

at the fusogenic pH. This result is quite similar to

those previously obtained by us for the whole virus

and purified G protein [19]. The hydrophobic

domain is lost at lower pHs, suggesting that the

protonation of negatively charged residues impairs

the formation of this domain. Indeed, peptide

modification with DCCD led to a great decrease in

ANS binding (Figure 3b), confirming that the acidic

amino acids are important for the formation of the

hydrophobic domain.

To investigate which were the residues directly

involved in peptide hydrophobicity and fusogenic

activity, we synthesized a number of mutants

(Figure 4a). Substitution of His149, Glu154 and

Asp161 did not affect the exposure of the hydro-

phobic domain, while His148, Asp153 and Glu158

were essential for the hydrophobicity (Figure 4b).

Peptide hydrophobicity greatly correlates to peptide

fusogenic activity, except for the His149 mutant,

which binds ANS but is not fusogenic (Figure 4c).

At pH 6.0, in which the ANS binding is maximal,

the His might be protonated, and positively-charged,

whereas the Glu and Asp are probably non-proto-

nated, and negatively-charged. One possibility is that

electrostatic interactions between one of the acid

residues from one peptide and one of the His

residues of other peptide drive the formation of an

oligomeric hydrophobic structure at pH 6.0.

time (min)0 5 10 15 20

NB

D-P

E fl

uore

scen

ce in

tens

ity (

a.u.

)

10

20

30

40

50

DCCD

no modification

time (min)0 5 10 15 20

10

20

30

40

50

pH 6.0

pH 7.5

pH 4.0

A B

Figure 1. Membrane fusion induced by peptide145 �164 depends on the negative charges of its acidic residues. (A) Effect of the modification

of the acidic residues with DCCD. Equal amounts of unlabeled vesicles and vesicles labeled with Rh-PE and NBD-PE were incubated with

the peptide145 �164 or the peptide pre-incubated with DCCD. The vesicles were composed of PC:PS (1:3) and were prepared in 20 mM

MES, 30 mM Tris buffer, pH 6.0, in a final phospholipid concentration of 0.1 mM. Peptide-induced membrane fusion activity was

measured by the increase in the NBD-PE fluorescence. NBD-PE was excited at 470 nm, and the intensity was collected at 530 nm, during

10 min. The final peptide concentration was 20 mg/ml. (B) Effect of pH. Fusion reaction was measured as described above except that the

pH was adjusted to 7.5, 6.0 or 4.0, as indicated in the Figure.

G protein charged residues in VSV-induced membrane fusion 399

Peptide oligomerization

To evaluate the requirement of peptide oligomeriza-

tion for hydrophobicity and fusion, we checked the

dependence on peptide concentration (Figure 5).

The extent of lipid mixing varied with the peptide

concentration in a higher than linear fashion (Figure

5a and 5b), supporting the hypothesis that peptide

promotes membrane fusion as oligomers. A drastic

increase in ANS binding occurred at the peptide

concentration range required for fusion (Figure 5c),

suggesting that peptide hydrophobicity is a conse-

quence of its oligomerization. The threshold of

aggregation was neither altered for the fusogenic

mutants E154Q and D161N nor for the non-

fusogenic H149Q mutant (Figure 5c).

Analyses by mass spectrometry indicated that

DCCD labels the peptide only at concentrations

that favor oligomerization (Figure 6). Since DCCD

labeling occurs in hydrophobic environments, this is

another evidence that oligomerization contributes to

the formation of a hydrophobic region that is

probably necessary for the interaction with the

membrane and consequently, for fusion.

We analysed peptide secondary structure at pH

7.5 and 6.0 by infrared spectroscopy (Figure 7). The

1600�1700 cm�1 region (amide I) corresponding to

the C�/O stretching vibration is the most sensitive to

the secondary structure of the proteins and each

secondary structure absorbs at different wave-

lengths. The peak at 1650 cm�1 is characteristic of

a peptide folded in an a-structure [14]. More

importantly, the fold is not affected by pH (Figure

7a), suggesting that the hydrophobicity is a conse-

quence of reorganization of the oligomeric state of

the peptides rather than due to changes in their

secondary structure. IR spectra of the non-fusogenic

mutants did not reveal any structural changes as

compared with wild type spectrum (Figure 7b),

excluding the possibility that the mutations disrupt

the structure and not only the generation of the

hydrophobicity.

pH2345678

AN

S fl

uore

scen

ce in

tens

ity (

a.u.

)

200

250

300

350

400

450B

wavelength (nm)440 480 520 560 600

0

100

200

300

400

500A

DCCD

no modification

Figure 3. Acidic residues are involved in the formation of a hydrophobic region. (A) ANS binding as a function of pH. Peptide145 �164 was

diluted to a final concentration of 20 mg/ml in 20 mM MES, 30 mM Tris buffer, pH as indicated in the Figure, and incubated with 1 mM

ANS. ANS was excited at 360 nm and the emission was collected at 492 nm. (B) Peptide145 �164 without modification (*) or modified with

DCCD (. . .) were incubated with 1 mM ANS in 20 mM MES, 30 mM Tris buffer, pH 6.0, and the ANS fluorescence spectra were collected

after excitation at 360 nm.

Figure 2. Labeling of the peptide145 �164 with DCCD. ESI-MS

spectra were recorded to monitor the addition of DCCD groups

to the Asp and Glu residues of the peptide145 �164. The peptide

(20 mg/ml) was incubated for 1 h with DCCD in 20 mM MES, 30

mM Tris buffer, pH 6.0. Before analysis, the buffer was removed

on ZipTip C18 and the peptide was solubilized in 50% acetoni-

trile/ 1% formic acid (v/v). The different populations observed

(arrows) correspond to the addition of 1, 2 and 3 DCCD

molecules, respectively. The accuracy of mass measurement was

in the order of 8 ppm.

400 F. A. Carneiro et al.

Discussion

All enveloped viruses have transmembrane glyco-

proteins that mediate fusion between the virus

membrane and host cell membrane, initiating the

viral replication cycle. Viral fusion proteins vary in

their mode of activation and their structural features,

and based on these differences, they were grouped in

two classes. It has been proposed that both class I

and class II fusion proteins are synthesized in a

metastable state and the native state is prevented

from achieving the lower-energy fusogenic confor-

mation by a kinetic barrier imposed during the

folding and/or maturation [20]. In the case of

Influenza virus, for example, the hemagglutinin

(HA) folds within the cell as the fusion-incompetent

precursor that undergoes proteolytic cleavage to

generate the mature, two-chain native state [2].

This metastability allows the coupling of an energe-

tically expensive membrane-fusion reaction to an

energetically favorable conformational change, what

could drive the reaction toward complete membrane

fusion [20]. However, several results suggest that the

glycoproteins of the rhabdoviruses catalyze fusion

through a different mechanism.

A striking difference between VSV fusion and

other viruses-induced membrane fusion is the rever-

sibility of G protein conformational changes induced

by low pH, even after the virus interaction with the

membrane [21�23]. This suggests that the metast-

ability is not absolutely required for viral membrane

fusion. Another finding revealing a different me-

chanism of membrane recognition by VSV was the

demonstration of an essential role of electrostatic

interactions in VSV binding to membranes [8]. The

electrostatic nature of VSV interaction with mem-

branes is an interesting observation considering that

most of the viral fusion proteins studied so far bind

to membrane through hydrophobic interactions

[24�26]. Indeed, it is believed that, in a certain

stage of fusion process, the fusion peptide is exposed

wt 145 V T P H H V L V D E Y T G E W V D S Q F 164

H148Q . . . Q . . . . . . . . . . . . . . . .

H149Q . . . . Q . . . . . . . . . . . . . . .

D153N . . . . . . . . N . . . . . . . . . . .

E154Q . . . . . . . . . Q . . . . . . . . . .

E158Q . . . . . . . . . . . . . Q . . . . . .

D161N . . . . . . . . . . . . . . . . N . . .

A

wavelength (nm)

440 480 520 560 600

AN

S fl

uore

scen

ce in

tens

ity (

a.u.

)

0

100

200

300

400

500

wt

H149QE154QD161N

D153NE158Q

H148Q

B C

time (min)

0 2 4 6 8 10

NB

D-P

E fl

uore

scen

ce in

tens

ity (

a.u.

)

10

20

30

40

50

60

70 wtE154QD161N

H148QH149QD153NE158Q

Figure 4. His148, Asp153 and Glu158 are important for peptide hydrophobicity and fusion. (A) Amino acid sequences of the peptides used in

this study. Peptide wt used in this study corresponds to VSV G protein residues between 145 and 164. Six different mutants were also

synthesized: His148, His149, Glu154 or Glu158 were substituted by Gln residues, and Asp153 or Asp161 were substituted by Asn residues. The

dots represent wt residues. (B) Peptide145 �164 or the mutant peptides were incubated with 1 mM ANS in 20 mM MES, 30 mM Tris buffer,

pH 6.0, and the ANS fluorescence spectra were collected after excitation at 360 nm. (C) Kinetics of membrane fusion by peptide145 �164 or

mutant peptides. Equal amounts of unlabeled vesicles and vesicles labeled with Rh-PE and NBD-PE were incubated with each of the

peptides and the membrane fusion was measured monitoring the increase in NBD-PE fluorescence. The vesicles were composed of PC:PS

(1:3) and were prepared in 20 mM MES, 30 mM Tris buffer, pH 6.0, in a final phospholipid concentration of 0.1 mM. NBD-PE was

excited at 470 nm, and fluorescence intensity was collected at 530 nm during 10 min.

G protein charged residues in VSV-induced membrane fusion 401

and inserted into the membrane of the target cell

[27] and several studies using isolated fusion pep-

tides have evaluated peptide insertion and orienta-

tion into the lipid bilayer [28�32]. However, in the

case of the peptide145�164, a linear peptide used in

this study, the mode of interaction with the lipid

bilayer might probably be different since this peptide

is very hydrophilic. This is clearly shown in Figure 8,

which compares the plots of average hydropathies of

the amino acid residues between VSV peptide and

HIV-1 fusion peptide using the hydropathy scale of

Kyte and Doolittle [33]. While HIV-1 fusion peptide

is very hydrophobic, presenting most of the hydro-

pathies above zero, VSV peptide hydropathies are

time (min)0 2 4 6 8 10

NB

D-P

E fl

uore

scen

ce in

tens

ity (

a.u.

)

10

20

30

40

50

60

70

12 µ g

6 µ g

3 µ g

100 µ g

30 µ g

18 µ g

A C

[peptide] (µg/ml)0 20 40 60 80 100

% fu

sion

0

20

40

60

80 B

[peptide] (µg/ml)0 20 40 60 80 100

AN

S fl

uore

scen

ce in

tesi

ty (

a.u.

)

100

200

300

400

500

H148Q

D153N

E158Q

wt

H149Q

E154Q

D161N

Figure 5. Peptide oligomerization is required for hydrophobicity and fusion. (A) Kinetics of membrane fusion at different peptide

concentration. Equal amounts of unlabeled vesicles and vesicles labeled with Rh-PE and NBD-PE were incubated with the peptide145 �164

in a final concentration of 3, 6, 12, 18, 30 and 100 mg/ml, and the membrane fusion was measured monitoring the increase in NBD-PE

fluorescence. The vesicles were composed of PC:PS (1:3) and were prepared in 20 mM MES, 30 mM Tris buffer, pH 6.0, in a final

phospholipid concentration of 0.1 mM. NBD-PE was excited at 470 nm, and the fluorescence intensity was collected at 530 nm during 10

min. (B) Percentage of fusion after 10 min as a function of peptide concentration. (C) ANS binding as a function of peptide concentration.

Peptide145 �164 (m) and the mutants H148Q (k), H149Q (j), D153N (%), E154Q (^), E158Q (%), and D161N (\) were diluted in 20

mM MES, 30 mM Tris buffer, pH 6.0, to a final concentration as indicated in the Figure, and incubated with 1 mM ANS. ANS was excited

at 360 nm and the emission was collected at 492 nm.

Figure 6. DCCD labeling is dependent on peptide concentration. Peptide145 �164 diluted to a final concentration of 3 mg/ml (A) or

100 mg/ml (B) was modified for 1 h with DCCD at pH 6.0 and ESI-MS spectra were recorded. No labeling was observed with 3 mg/ml and a

labeling comparable to the one described in Figure 3 was observed for 100 mg/ml.

402 F. A. Carneiro et al.

mostly below zero, what characterizes a hydrophilic

peptide.

An important question raised from these data is:

how could this very hydrophilic peptide mediate

membrane fusion? We believe that the answer to this

question was found when we showed that the

oligomerization of the peptide confers hydrophobi-

city to the oligomer. The increase in hydrophobicity

correlates to the peptide fusogenic activity. Both are

maximal at pH 6.0 and decrease as pH is increased

to 7.5 or decreased to 4.0. Thus, we propose that the

self-association of the peptides at pH 6.0 promotes

the formation of a hydrophobic region important for

the interaction with the target membrane.

It has already been shown that other fusion

peptides associate at the membrane surface. For

example, comparing the membrane interaction

properties of a synthetic coiled-coil Influenza he-

magglutinin fusion peptide with the monomeric

peptide, Lau et al. [34] showed that the trimeriza-

tion of the peptide increased lipid mixing, liposome

leakage and membrane destabilization, suggesting an

important role for the oligomerization of fusogenic

peptides in the fusion mechanism. Studies using

different peptide constructs suggested that the

oligomers are formed as a result of membrane

association and that small oligomers may be more

fusogenic than monomers or large aggregates [35].

For HIV-1 fusion peptide, the ability of the peptide

to form aggregates was also correlated to its ability to

induce membrane fusion [28].

In most cases, the oligomerization of the fusion

peptides is driven by changes in their secondary

structure. Influenza hemagglutinin fusion peptide

exists in at least two interconvertible forms at

membrane surface: monomeric a-helical peptides

that insert into the bilayer, and self-associated

peptides adopting a b-sheet structure [36]. The

equilibrium between these two forms is dependent

on the pH and on the ionic strength. HIV-1 fusion

peptides form oligomeric b-strand structures when

associated to membranes [37]. GALA, a synthetic

fusogenic peptide, undergoes a conformational

change to an amphipathic helix when the pH is

reduced [38]. IR spectroscopic data demonstrated

that the peptide inserted deeply into the lipid bilayer,

oriented parallel with respect to the lipid acyl chains

[39]. In our case, however, we demonstrated by

infra-red spectroscopy that there is no change in

peptide secondary structure induced by lowering the

Figure 7. Secondary structure measurements. (A) ATR-FTIR

spectra were recorded from thin films obtained by slowly

evaporating a sample containing 10 mg of peptide145 �164 (at pH

7.5 or 6.0, as indicated in the Figure) on an attenuated total

reflection element. The samples were rehydrated by flushing D2O-

saturated N2 for 30 min at room temperature. (B) The wild-type

spectrum was compared with the spectra obtained, at pH 6.0, for

the mutants H148Q, H149Q, D153N and E158Q, as indicated in

the Figure.

0 2 4 6 8 10 12 14 16 18 20-3

-2

-1

0

1

2

3VSV peptide

amino acid sequence0 2 4 6 8 10 12 14 16 18 20

-3

-2

-1

0

1

2

3HIV peptide

hydr

opho

bici

ty

Figure 8. Kyte-Doolittle plots of fusion peptide hydropathy.

Hydrophobic profile of VSV peptide[145�164] and HIV-1 fusion

peptide (sequence VGIGALFLGFLGAAGSTHGA). The hydro-

pathy of these peptides was plotted from the amino terminus to

the carboxy terminus by averaging hydropathy values over a

window of 5 residues. More positive values are assigned to more

hydrophobic residues.

G protein charged residues in VSV-induced membrane fusion 403

pH from 7.5�6.0. Thus, rather than favored by

conformational changes, the oligomerization of

peptide145�164 seems to be triggered by the proto-

nation of His residues at the pH range of fusion,

which creates positive charges in the peptides

probably involved in electrostatic interactions with

the negatively-charged residues. This explanation is

in agreement with our previous results showing the

importance of the electrostatic interactions for VSV

fusion [8], and adds to our previous findings the idea

that these electrostatic interactions are responsible

for the formation of a hydrophobic region directed

involved in VSV interaction with the membrane

during fusion. This mechanism is different from that

observed for WAE, an amphipathic negatively

charged peptide that induces fusion of liposomal

phosphatidylcholine membranes. Pecheur et al.

(1999) provided evidence that it is the peptide

penetration rather than peptide oligomerization

that modulates peptide-induced fusion [40].

Using peptide mutants we demonstrated that

His148, Asp153 and Glu158 are essential for the

hydrophobicity and fusion. Asp153 is conserved

among at least 13 different animal rhabdoviruses

representing four recognized genera [41]. His148 is

also conserved except that it is substituted in the

Novirhabdovirus genus for a lysine, which should

also be protonated at the fusion pH (pK 10.53), or

for a serine in the Lyssavirus genus. Glu158 probably

has a specific role in VSV fusion since it is not

conserved among the rhabdovirus G proteins. We

found that His149 is important for fusion but not for

hydrophobicity, suggesting that this residue is prob-

ably involved in the direct binding to PS in the target

membrane.

Taken together, our data suggest that the negative

charges of peptide acidic residues participate in

intermolecular electrostatic interactions with posi-

tively-charged His residues, leading to the formation

of a hydrophobic domain at pH 6.0, which is

necessary to the peptide-induced membrane fusion.

This suggests a mechanism of membrane interaction

and destabilization resembling that one promoted by

the antimicrobial peptides acting through a carpet

model [42]. This model predicts an initial interac-

tion between the hydrophilic portion of the peptide

and negatively-charged membranes through electro-

static interactions. Then a reorientation of the

hydrophobic residues of the peptide toward the

hydrophobic core of the membrane occurs, causing

membrane disintegration by disrupting the bilayer

curvature.

Secondary structure prediction using the program

PSIPRED Protein Structure Prediction Server

(us.expasy.org) suggests that a loop is formed

between the residues Glu154 and Val160. The forma-

tion of this loop is supported by the presence of

residues Tyr155 and Trp159, which have large side

chains, and by the Gly157 in the middle, allowing the

structure to bend at this point. A similar loop was

found in the fusion peptide of dengue virus E

glycoprotein between residues 99 and 105 [43],

suggesting that some similarity might occur between

VSV and class II fusion peptides. One important

question to be posed is whether the data obtained

using the peptide145�164 could be correlated to the

role of the peptide when taking part of the whole G

glycoprotein. The peptide145�164 is located within

the domain shown to interact with the target

membrane at the fusogenic pH [44]. In addition,

its His residues (His148 and His149) were shown to

be modified after treatment of the whole virus with

DEPC, leading to the inhibition of virus-induced

fusion [9]. These observations support the hypoth-

esis that the peptide145-164 is also active within the

whole protein.

Acknowledgements

We would like to thank Adriana S. de Melo for

technical assistance, Dr Erik Goormaghtigh for

helpful assistance with ATR-FTIR experiments

and Dr Fabio C. Almeida for helpful suggestions.

This work was supported by grants from Conselho

Nacional de Desenvolvimento Cientıfico e Tecnolo-

gico (CNPq), Centro Argentino-Brasileiro de Bio-

tecnologia (CABBIO), Fundacao Carlos Chagas

Filho de Amparo a Pesquisa do Estado do Rio de

Janeiro (FAPERJ) and Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (FAPESP). F. A.

C. was recipient of PDEE fellowship from Coorde-

nacao de Aperfeicoamento de Pessoal de Nıvel

Superior (CAPES). G.V. thanks Action de Re-

cherches Concertees (ARC) for financial support.

References

[1] Hernandez LD, Hoffman LR, Wolfsberg TG, White JM.

Virus-cell and cell-cell fusion. Ann Rev Cell Dev Biol

1996;12:627�661.

[2] Skehel JJ, Wiley DC. Receptor binding and membrane

fusion in virus entry: the influenza hemagglutinin. Ann Rev

Biochem 2000;69:531�569.

[3] Heinz FX, Allison SL. The machinery for flavivirus fusion

with host cell membranes. Curr Opin Microbiol

2001;4:450�455.

[4] Jardetzky TS, Lamb RA. Virology: a class act. Nature

2004;427:307�308.

[5] Li Y, Drone C, Sat E, Ghosh HP. Mutational analysis of the

vesicular stomatitis virus glycoprotein G for membrane

fusion domains. J Virol 1993;67:4070�4077.

[6] Zhang L, Ghosh HP. Characterization of the putative

fusogenic domain in vesicular stomatitis virus glycoprotein

G. J Virol 1994;68:2186�2193.

404 F. A. Carneiro et al.

[7] Fredericksen B, Whitt MA. Vesicular stomatitis virus glyco-

protein mutations that affect membrane fusion activity and

abolish virus infectivity. J Virol 1995;69:1435�1443.

[8] Carneiro FA, Bianconi ML, Weissmuller G, Stauffer F, Da

Poian AT. Membrane recognition by vesicular stomatitis

virus involves enthalpy-driven protein-lipid interactions. J

Virol 2002;76:3756�3764.

[9] Carneiro FA, Stauffer F, Lima CL, Juliano MA, Juliano L,

Da Poian AT. Membrane fusion induced by vesicular

stomatitis virus depends on histidine protonation. J Biol

Chem 2003;278:13789�13794.

[10] Coll JM. Synthetic peptides from the heptad repeats of

the glycoproteins of rabies, vesicular stomatitis and

fish rhabdoviruses bind phosphatidylserine. Arch Virol

1997;142:2089�2097.

[11] Estepa A, Coll JM. Pepscan mapping and fusion-related

properties of the major phosphatidylserine-binding domain

of the glycoprotein of viral hemorrhagic septicemia virus, a

salmonid rhabdovirus. Virology 1996;216:60�70.

[12] Coll JM. Heptad-repeat sequences in the glycoprotein of

rhabdoviruses. Virus Genes 1995;10:107�114.

[13] Struck DK, Hoekstra D, Pagano RE. Use of resonance

energy transfer to monitor membrane fusion. Biochemistry

1981;20:4093�4099.

[14] Goormaghtigh E, Raussens V, Ruysschaert J-M. Attenuated

total reflection infrared spectroscopy of proteins and

lipids in biological membranes. Biochim Biophys Acta

1999;1422:105�185.

[15] Grimard V, Li C, Ramjeesingh M, Bear CE, Goormaghtigh

E, Ruysschaert J-M. Phosphorylation-induced conforma-

tional changes of cystic fibrosis transmembrane conductance

regulator monitored by attenuated total reflection-Fourier

transform IR spectroscopy and fluorescence spectroscopy. J

Biol Chem 2004;279:5528�5536.

[16] Manciu L, Chang X-B, Buyse F, Hou YX, Gustot A,

Riordan JR, Ruysschaert J-M. Intermediate structural states

involved in MRP1-mediated drug transport. Role of glu-

tathione. J Biol Chem 2003;278:3347�3356.

[17] Carraway KL, Koshland DE Jr. Carbodiimide modification

of proteins. Meth Enzymol 1972;25:616�623.

[18] Eidelman O, Schlegel R, Tralka TS, Blumenthal R. pH-

dependent fusion induced by vesicular stomatitis virus

glycoprotein reconstituted into phospholipid vesicles. J Biol

Chem 1984;259:4622�4628.

[19] Carneiro FA, Ferradosa AS, Da Poian AT. Low pH-induced

conformational changes in vesicular stomatitis virus glyco-

protein involve dramatic structure reorganization. J Biol

Chem 2001;276:62�67.

[20] Carr CM, Chaudhry C, Kim PS. Influenza hemagglutinin is

spring-loaded by a metastable native conformation. Proc

Natl Acad Sci USA 1997;94:14306�14313.

[21] Gaudin Y, Tuffereau C, Segretain D, Knossow M, Flamand

A. Reversible conformational changes and fusion activity of

rabies virus glycoprotein. J Virol 1991;65:4853�4859.

[22] Pak CC, Puri A, Blumenthal R. Conformational changes

and fusion activity of vesicular stomatitis virus glycoprotein:

[125I]iodonaphthyl azide photolabeling studies in biological

membranes. Biochemistry 1997;36:8890�8896.

[23] Gaudin Y. Rabies virus-induced membrane fusion pathway. J

Cell Biol 2000;150:601�611.

[24] Harter C, James P, Bachi T, Semenza G, Brunner J.

Hydrophobic binding of the ectodomain of influenza he-

magglutinin to membranes occurs through the ‘‘fusion

peptide’’. J Biol Chem 1989;264:6459�6464.

[25] Rabenstein M, Shin Y-K. A peptide from the heptad repeat

of human immunodeficiency virus gp41 shows both mem-

brane binding and coiled-coil formation. Biochemistry

1995;34:13390�13397.

[26] Adam B, Lins L, Stroobant V, Thomas A, Brasseur R.

Distribution of hydrophobic residues is crucial for the

fusogenic properties of the Ebola virus GP2 fusion peptide.

J Virol 2004;78:2131�2136.

[27] Nieva JL, Agirre A. Are fusion peptides a good model to

study viral cell fusion? Biochim Biophys Acta

2003;1614:104�115.

[28] Kliger Y, Aharoni A, Rapaport D, Jones P, Blumenthal R,

Shai Y. Fusion peptides derived from the HIV type 1

glycoprotein 41 associate within phospholipid membranes

and inhibit cell-cell Fusion. Structure-function study. J Biol

Chem 1997;272:13496�13505.

[29] Tatulian SA, Jones LR, Reddy LG, Stokes DL, Tamm LK.

Secondary structure and orientation of phospholamban

reconstituted in supported bilayers from polarized attenu-

ated total reflection FTIR spectroscopy. Biochemistry

1995;34:4448�4456.

[30] Martin I, Dubois MC, Defrise-Quertain F, Saermark T,

Burny A, Brasseur R, Ruysschaert JM. Correlation between

fusogenicity of synthetic modified peptides corresponding

to the NH2-terminal extremity of simian immunodeficiency

virus gp32 and their mode of insertion into the lipid

bilayer: an infrared spectroscopy study. J Virol

1994;68:1139�1148.

[31] Martin I, Schaal H, Scheid A, Ruysschaert JM. Lipid

membrane fusion induced by the human immunodeficiency

virus type 1 gp41 N-terminal extremity is determined

by its orientation in the lipid bilayer. J Virol 1996;70:298�304.

[32] Ghosh JK, Shai Y. Direct evidence that the N-terminal

heptad repeat of Sendai virus fusion protein participates in

membrane fusion. J Mol Biol 1999;292:531�546.

[33] Kyte J, Doolittle RF. A simple method for displaying the

hydropathic character of a protein. J Mol Biol

1982;157:105�132.

[34] Lau WL, Ege DS, Lear JD, Hammer DA, DeGrado WF.

Oligomerization of fusogenic peptides promotes membrane

fusion by enhancing membrane destabilization. Biophys J

2004;86:272�284.

[35] Yang J, Prorok M, Castellino FJ, Weliky DP. Oligomeric

beta-structure of the membrane-bound HIV-1 fusion pep-

tide formed from soluble monomers. Biophys J

2004;87:1951�1963.

[36] Han X, Tamm LK. pH-dependent self-association of

influenza hemagglutinin fusion peptides in lipid bilayers. J

Mol Biol 2000;304:953�965.

[37] Yang J, Weliky DP. Solid-state nuclear magnetic resonance

evidence for parallel and antiparallel strand arrangements in

the membrane-associated HIV-1 fusion peptide. Biochem-

istry 2003;42:11879�11890.

[38] Li W, Nicol F, Szoka FC Jr. GALA: a designed synthetic

pH-responsive amphipathic peptide with applications in

drug and gene delivery. Adv Drug Deliv Rev

2004;56:967�985.

[39] Goormaghtigh E, De Meutter J, Szoka F, Cabiaux V,

Parente RA, Ruysschaert J-M. Secondary structure and

orientation of the amphipathic peptide GALA in lipid

structures. An infrared-spectroscopic approach. Eur J Bio-

chem 1991;195:421�429.

[40] Pecheur EI, Sainte-Marie J, Bienvenue A, Hoekstra D. Lipid

headgroup spacing and peptide penetration, but not peptide

oligomerization, modulate peptide-induced fusion. Bio-

chemistry 1999;38:364�373.

G protein charged residues in VSV-induced membrane fusion 405

[41] Walker PJ, Kongsuwan K. Deduced structural model for

animal rhabdovirus glycoproteins. J Gen Virol 1999;80:

1211�1220.

[42] Shai Y. Mechanism of the binding, insertion and destabiliza-

tion of phospholipid bilayer membranes by alpha-helical

antimicrobial and cell non-selective membrane-lytic pep-

tides. Biochim Biophys Acta 1999;1462:55�70.

[43] Modis Y, Ogata S, Clements D, Harrison SC. Structure of

the dengue virus envelope protein after membrane fusion.

Nature 2004;427:313�319.

[44] Durrer P, Gaudin Y, Ruigrok WH, Graf R, Brunner J.

Photolabeling identifies a putative fusion domain in the

envelope glycoprotein of rabies and vesicular stomatitis

viruses. J Biol Chem 1995;270:17575�17581.

This paper was first published online on prEview on 27 June 2006.

406 F. A. Carneiro et al.