From the Departamento de Bioquímica
Médica, Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-590 and
¶ Departamento de Biofísica, Escola Paulista de Medicina,
Universidad Federal de São Paulo, Rua Três de Maio,
100, São Paulo 04044-020, Brazil
Received for publication, October 17, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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Entry of enveloped animal viruses into
their host cells always depends on a step of membrane fusion triggered
by conformational changes in viral envelope glycoproteins. Vesicular
stomatitis virus (VSV) infection is mediated by virus spike
glycoprotein G, which induces membrane fusion at the acidic environment
of the endosomal compartment. VSV-induced membrane fusion occurs at a
very narrow pH range, between 6.2 and 5.8, suggesting that His
protonation is required for this process. To investigate the role of
His in VSV fusion, we chemically modified these residues using
diethylpyrocarbonate (DEPC). We found that DEPC treatment inhibited
membrane fusion mediated by VSV in a
concentration-dependent manner and that the complete
inhibition of fusion was fully reversed by incubation of modified virus
with hydroxylamine. Fluorescence measurements showed that VSV
modification with DEPC abolished pH-induced conformational changes in G
protein, suggesting that His protonation drives G protein interaction
with the target membrane at acidic pH. Mass spectrometry analysis of
tryptic fragments of modified G protein allowed the identification of
the putative active His residues. Using synthetic peptides, we showed
that the modification of His-148 and His-149 by DEPC, as well as
the substitution of these residues by Ala, completely inhibited
peptide-induced fusion, suggesting the direct participation of these
His in VSV fusion.
Membrane fusion is an essential step in the entry of
enveloped viruses into their host cells (1-3). Virus-induced fusion is
always mediated by viral surface glycoprotein and may occur through two
different general mechanisms: (i) surface fusion between viral envelope
and host cell plasma membrane after virus interaction with its cellular
receptor, and (ii) fusion of endosomal membrane with viral envelope
after virus particle internalization by receptor-mediated endocytosis.
In the latter case, fusion is triggered by conformational changes in
viral glycoproteins induced by the decrease in the pH of the endosomal medium.
Vesicular stomatitis virus
(VSV)1 is a member of
Rhabdoviridae family, genus Vesiculovirus.
Rhabdoviruses contain helically wound ribonucleocapisid surrounded by a
lipid bilayer through which spikes project. These spikes are formed by
trimers of a single type of glycoprotein, named G protein. VSV enters
into the cell by endocytosis followed by low pH-induced membrane fusion in the endosome (4, 5), which is catalyzed by VSV G protein (6). A
common feature of viral fusion proteins is that they bear a highly
conserved hydrophobic fusion domain, which is most often located at the
N terminus of the polypeptide chain (7). However, VSV G protein does
not contain an apolar amino acid sequence similar to the fusion
peptides found in other viruses, suggesting alternative mechanisms
involved in VSV-induced membrane fusion.
We have shown recently (8) that VSV-induced fusion depends on a
dramatic structure reorganization of G protein, which occurs within a
very narrow pH range, close to 6.0. In addition, we have found that VSV
binding to membranes, as well as the fusion reaction, were highly
dependent on electrostatic interactions between negative charges on
membrane surface and positively charged amino acids in G protein at the
fusion pH (9). These results suggest the involvement of histidyl
residue(s) in G protein conformational changes required for fusion,
because the protonation of imidazole ring occurs at the fusion pH range
(pK = 6.0).
Hydrophobic photolabeling experiments allowed the identification of a G
protein segment comprising amino acids 59 to 221, which interacts with
membranes at low pH (10). Furthermore, studies using site-directed
mutagenesis in the region spanning amino acids 117 to 137 have shown a
reduction of G protein-induced fusion efficiency (11-13). However,
there is no conclusive evidence that this sequence participates
directly in the fusion reaction. Another region of rhabdovirus G
protein has been implicated in its interaction with anionic
phospholipids. This segment was better characterized for viral
hemorrhagic septicemia virus, a rhabdovirus of salmonids, and it
was named p2 peptide (14, 15). Viral hemorrhagic septicemia virus p2
peptide mediates phospholipid vesicle fusion, lipid mixing, and leakage
of liposome contents and inserts itself into liposome membranes by
adopting a To evaluate the role of G protein His residues in VSV-induced membrane
fusion we modified these residues using diethylpyrocarbonate (DEPC). We
showed that His protonation was essential both for low pH-induced
conformational changes of VSV G protein and for the fusion reaction
itself. Mass spectrometry analysis of G protein fragments obtained by
limited proteolysis allowed the identification of the putative active
His residues. Using synthetic peptides, we found that VSV p2-like
peptide (sequence between amino acids 145 and 168) was as efficient as
the virus in catalyzing membrane fusion at pH 6.0 and that the
modification of His-148 and His-149 by DEPC completely abolished fusion
activity. Substitution of the His by Ala residues inhibits
peptide-mediated fusion, confirming the requirement of His protonation
in VSV-induced membrane fusion.
Chemicals--
DEPC, phosphatidylserine (PS) and
phosphotidylcholine (PC) from bovine brain, trypsin from bovine
pancreas, and phenylmethylsulfonyl fluoride were purchased from
Sigma.
1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (10-PyPC) was purchased from Molecular Probes Inc., Eugene, OR. All
other reagents were of analytical grade.
Virus Propagation and Purification--
VSV Indiana was
propagated in monolayer cultures of BHK-21 cells. The cells were grown
at 37 °C in roller bottles containing 150 ml of Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, 100 µg/ml
ampicillin, 5 µg/ml gentamicin. When the cells reached confluence,
the medium was removed, and the cell monolayer was infected with VSV at
a multiplicity of 0.1 plaque-forming unit/ml. The cultures were kept at
37 °C for 16-20 h, and the virus were harvested and purified by
differential centrifugation followed by equilibrium sedimentation in a
sucrose gradient as described elsewhere (18). Purified virions were stored at Preparation of Liposomes--
Phospholipids were dissolved in
chloroform and evaporated under nitrogen. The lipid film formed was
resuspended in 20 mM MES, 30 mM Tris buffer, pH
7.5 or 6.0, at a final concentration of 1 mM. The
suspension was vortexed vigorously for 5 min. Small unilamellar
vesicles 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 s at 20% full power with 60-s resting
intervals until a transparent solution was obtained (~10 cycles). The
vesicles used in this study were composed of PC and PS at a 1:3 ratio.
For fusion assays, 1% 10-PyPC was incorporated in PC:PS vesicles by
vortexing for 10 min.
Sample Modification with DEPC--
DEPC solutions were freshly
prepared by dilution of the reagent in cold ethanol. The concentration
of stock DEPC solution was determined by reaction with 10 mM imidazole (19). For modification with DEPC, VSV was
diluted in 20 mM MES, 30 mM Tris buffer, pH 7.5. At fixed time intervals, aliquots of DEPC were added to the mixture, and the reaction was monitored by the increase of absorbance at 240 nm because of the formation of N-carbethoxyhistidine
using a Hitachi U-2001 spectrophotometer. The final concentration of DEPC ranged from 0.005 to 0.05 mM.
To study the kinetics of modification, VSV was diluted in 20 mM MES, 30 mM Tris buffer, pH 7.5, and the
reaction was initiated by the addition of 0.02 mM DEPC at
25 °C. The time course of the reaction was monitored by an increase
of absorbance at 240 nm.
Reversal of DEPC Inactivation--
VSV was reacted with 0.02 mM DEPC at 25 °C. After 3 min, the mixture was incubated
with 400 mM hydroxylamine (from a 3 M stock solution of hydroxylamine in 20 mM MES, 30 mM Tris buffer, adjusted to pH 7.5) for 15 min at 25 °C.
For demodification of peptides, the experiment was carried out at the
same conditions except that the concentrations of DEPC and
hydroxylamine used were 0.2 and 500 mM, respectively. For
the control, a solution that contained the same concentration of
hydroxylamine without DEPC was used.
Liposome Fusion Assay--
A suspension of liposomes of
different phospholipid composition containing equal amounts of
unlabeled vesicles and vesicles labeled with 10-PyPC were prepared in
20 mM MES, 30 mM Tris buffer, pH 6.0 or 7.5, with a final phospholipid concentration of 0.1 mM. The
emission spectrum of pyrene-labeled vesicles exhibited a broad excimer
fluorescence peak with maximal intensity at 480 nm and two sharp peaks
at 376 and 385 nm because of monomer fluorescence emission (not shown).
The fusion reaction was initiated by addition of purified VSV
preincubated with different concentrations of DEPC for 3 min at
25 °C, ranging from 0.005 to 0.02 mM. Fusion was
followed by the decrease in the 10-PyPC excimer/monomer fluorescence intensity ratio, which was measured by exciting the sample at 340 nm
and collecting the fluorescence intensities of excimer and monomer at
480 and 376 nm, respectively. A control experiment using equivalent
volumes of ethanol (without DEPC) was performed under comparable
conditions. For peptide-induced fusion, the concentration of DEPC used
was 0.02 and 0.2 mM.
Intrinsic Fluorescence Measurements--
G protein
conformational changes during VSV interaction with membranes of
different phospholipid composition were monitored by the changes in
virus intrinsic fluorescence. VSV (final protein concentration of 15 µg/ml) was incubated with a liposome suspension containing 1 mM phospholipid in 20 mM MES, 30 mM
Tris buffer, pH 6.0. Intrinsic fluorescence data were recorded using a
Hitachi F-4500 fluorescence spectrometer, exciting the samples at 280 nm, and collecting emission between 300 and 420 nm.
MALDI-TOF Mass Spectrometry of Modified VSV G Protein--
VSV
(0.3 mg/ml) was reacted with 0.02 mM DEPC for 15 min at
25 °C. After modification, G protein was denatured by virus
incubation with 8 M urea for 1 h. Then, the sample was
diluted 4-fold in 10 mM Tris buffer, pH 7.4, and incubated
with trypsin (final concentration of 11 mg/ml) for 4 h at
37 °C. The reaction was stopped by addition of 0.1 mM
phenylmethylsulfonyl fluoride. The tryptic peptides were separated from
the remaining virus by filtration. For mass spectrometry analysis,
aliquots of 1 µl of the digested sample mixed with 1 µl of the
matrix solution (a saturated solution of Peptides Synthesis--
All peptides were synthesized by solid
phase using the Fmoc (N-(9-fluorenyl)methoxycarbonyl)
methodology, and all protected amino acids were purchased from
Calbiochem-Novabiochem 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). The
final deprotected peptides were purified by semipreparative HPLC using
an Econosil C-18 column (10 µm, 22.5 × 250 mm) and a
two-solvent system, Solvent A (trifluoroacetic acid/H2O)
(1:1000) (v/v) and Solvent B (trifluoroacetic acid/acetonitrile/H2O) (1:900:100) (v/v/v). The column was
eluted at a flow rate of 5 ml·min Role of G Protein His Residues in VSV-induced Membrane
Fusion--
VSV was incubated with increasing concentrations of DEPC,
which reacts with His-forming N-carbethoxyhistidyl
derivatives (19), and the virus-mediated membrane fusion was quantified
by measuring the decrease in pyrene phospholipid excimer/monomer
fluorescence ratio (9, 20) (Fig. 1).
Incubation of 0.02 mM DEPC with VSV (15 µg/ml) completely
abolished virus ability to mediate membrane fusion, whereas lower
concentrations of DEPC partially inhibits it. The formation of
N-carbethoxyhistidine was followed spectrophotometrically by
the absorbance increase in 240 nm (19). The major changes observed in
absorbance occurred when the virus was incubated with DEPC in final
concentrations up to 0.03 mM (Fig.
2A). Kinetics of VSV
modification with 0.02 mM DEPC revealed that the reaction was completed after 3 min (Fig. 2B). To further test whether
modification of His residues was responsible for inhibition of virus
fusion activity, hydroxylamine, which removes the carbethoxy group from imidazole group (19), was added 3 min after VSV incubation with 0.02 mM DEPC. Virus incubation with hydroxylamine after
modification with 0.02 mM DEPC completely restored its
ability to catalyze membrane fusion (Fig.
3). This set of results indicates that
His protonation is required for membrane fusion catalyzed by VSV, suggesting a central role of His in pH-induced conformational changes
in VSV G protein.
His Protonation Is Involved in pH-induced Conformational Changes on
G Protein--
We have shown recently (8) that G protein interaction
with liposomes at pH 6.0 resulted in dramatic protein conformational changes, which can be followed by intrinsic fluorescence. In the presence of vesicles composed of PC and PS, a great increase in tryptophan fluorescence of G protein occurred upon acidification of the
medium, whereas pH decrease led to intrinsic fluorescence quenching in
the absence of liposomes (8). VSV incubation with DEPC inhibited
intrinsic fluorescence quenching during acidification, suggesting the
involvement of His protonation in G protein conformational changes
(Fig. 4A). Time course of
fluorescence increase after VSV incubation with liposomes, at pH 6.0, is shown in Fig. 4B. The increase in fluorescence was
completely inhibited when the virus was incubated with 0.02 mM DEPC. These results indicate that the G protein
conformational changes that take place during protein-lipid interaction
are mediated by His protonation at pH 6.0.
Mass Spectrometry Analysis of Modified G Protein--
VSV G
protein contains a total of 16 His residues. Previous investigations
have revealed that a specific domain spanning residues 59 to 221, which
contains 6 His residues, interacted with the target membrane at
low pH (10). To determine whether DEPC treatment modified the His
residues within this sequence, the peptides obtained after
limited proteolysis of modified G protein were analyzed by MALDI-TOF
mass spectrometry. Seven fragments could be identified as VSV G protein
peptides (Table I). These peptides cover
64% of G protein (329/511 amino acids). We also analyzed the data considering the increase in mass because of DEPC modification, and four
modified peptides could be identified (Table
II). Two of these peptides are included
in the membrane-interacting domain (32-87 and 110-168), suggesting
that the active His are located within this segment.
Role of p2-like Peptide in VSV-induced Membrane Fusion--
The
putative fusion peptide (region 117-137) and the p2-like peptide
(region 145-168) are located within one of the modified segments of G
protein identified by mass spectrometry. To evaluate the ability of
both the p2-like peptide and the putative fusion peptide in catalyzing
fusion in vitro, we synthesized a number of peptides (Fig.
5). Besides the putative fusion peptide
and the p2-like peptide, we synthesized three other His-containing sequences to be used as controls. The peptides corresponding to the
sequences between amino acids 65-85 and 170-190 contain two His
residues and are located within the sequence that was identified as the
membrane-interacting segment by photolabeling experiments (10). The
peptide between amino acids 395-418 was also chosen, because it was
found to be modified by DEPC treatment by mass spectrometry analysis
(Table II).
Fig. 6 shows that the p2-like peptide was
as efficient as the whole virus to catalyze fusion of PC:PS vesicles.
Using p2-like peptide in a 50-fold lower concentration, which gives a
peptide molar concentration similar to G protein concentration used in virus-induced fusion, we obtained a very similar profile (Fig. 6B). In addition, peptide-induced fusion presented the same
requirements of VSV-mediated fusion. It occurs at pH 6.0 but not at pH
7.5 and depends on the presence of PS on the target membrane (Fig. 6B). These data suggest a direct participation of p2-like
peptide in VSV-induced membrane fusion. On the other hand, when the
synthetic peptide corresponding to the VSV putative fusion peptide was
assayed for liposome fusion, it failed to induce a decrease in pyrene excimer/monomer fluorescence ratio (Fig. 6C). This result
shows that this sequence alone is not able to catalyze fusion reaction and reinforces the involvement of p2-like peptide in VSV fusion.
The pH dependence of membrane fusion mediated by p2-like peptide
suggests the participation of His in the process. To evaluate whether
His protonation was also necessary for peptide-induced fusion, as
observed for the virus, the effect of peptide incubation with DEPC on
the membrane fusion was analyzed. As shown on the Fig.
7A, His modification by DEPC
abolished peptide activity, suggesting that His residues are crucial
for membrane recognition and fusion. Hydroxylamine treatment reversed
fusion inhibition by DEPC modification (Fig. 7A). In
addition, substitution of both His-148 and His-149 for Ala residues on
the peptide sequence completely abolished fusion, whereas removing one
of the His residues led to a less efficient fusion (Fig.
7B). All other G protein amino acid sequences containing two
His residues used as controls did not present fusion activity (Fig.
7C). These results together suggest that VSV p2-like peptide
directly participates in membrane fusion mediated by G protein and that
protonation of His is necessary for peptide fusion activity.
In this work, we describe two main findings concerning VSV-induced
membrane fusion. First, we showed that fusion is driven by His
protonation at the pH range of endosomal medium. Although several
residues have already been implicated in G protein fusion ability, to
our knowledge His has never been considered. Second, we found that VSV
p2-like peptide was as efficient as the whole virus in catalyzing
fusion, whereas the putative fusion peptide failed to induce fusion.
VSV p2-like peptide contains two His residues, whose protonation are
required for its fusion activity.
The identification of the amino acid residues essential for membrane
fusion mediated by viral glycoproteins might contribute to the
elucidation of the molecular mechanisms underlying the fusion event. In
the case of VSV, mutational analysis have shown that substitution of
conserved Gly, Pro, or Asp present in the region between amino acids
117 and 137 either abolished fusion ability of G protein or shifted the
optimum pH of fusion (11-13). Based on these results, the authors
proposed that this segment was the putative fusion domain of VSV G
protein. However, direct evidence that this particular region interacts
with the target membrane is still lacking. VSV-induced membrane fusion
occurs in a very narrow pH range, between 5.8 and 6.2 (4, 8). This indicates that the protonation of a small number of ionizable groups is
required for G protein structural changes. His is the only amino acid
whose ionization pKa is in the range of VSV
fusion, suggesting that fusion is driven by His protonation. Using
DEPC, we showed that His modification abolished pH-induced
conformational changes on G protein and the fusion reaction catalyzed
by the virus. VSV putative fusion peptide contains no His, and thus it cannot be modified by DEPC. In addition, we found that a synthetic peptide corresponding to the VSV putative fusion sequence failed to
induced phospholipid vesicle fusion, although several studies have
reported that synthetic fusion peptides of different viruses promote
fusion independent of the remainder protein (21-25). Further investigation will be necessary to answer whether the segment between
amino acids 117 and 137 of G protein directly participates in VSV
fusion or whether the substitution of its conserved amino acids affects
the conformation or the exposure of other membrane-interacting sequences in G protein.
Another question to be answered is how general is the requirement of
His protonation for pH-dependent viral membrane fusion. In
the case of influenza virus, for example, the participation of
hemagglutinin N-terminal peptide in fusion is very well established, although this peptide does not contain His residues. In this case, however, the fusion occurs at pH 5.0, in which protonation of acidic
amino acids could take place. Another possibility that could not be
discarded so far is that the protonation of His residues in other
regions of the fusion protein could affect the overall protein
structure leading to the exposure of the fusion peptide.
We have shown recently (9) that G protein-membrane interaction is
highly dependent on the presence of PS, a negatively charged
phospholipid, in the target membrane. In addition, we have found that G
protein conformational changes, as well as VSV-mediated fusion, are
driven by electrostatic interactions. Based on the results showed here,
we believe that the protonation of His residues could generate positive
charges on G protein, which might contribute to the electrostatic
interactions required for protein insertion in membrane during fusion.
Heptad repeats play an important role in many viral membrane fusion
processes. Three-dimensional structures of fragments from several viral
fusion proteins, including influenza hemagglutinin, Moloney leukemia
virus transmembrane (TM) subunit, HIV-1 glycoprotein 41, Ebola
virus GP2, and simian immunodeficiency virus glycoprotein 41, have been
determined (26-30). The results obtained revealed that these proteins
adopt a post-fusion hairpin structure formed by the interaction of
N-terminal and C-terminal heptad-repeat segments, which generate a
trimeric coiled-coil (31). For Sendai virus, heptad repeats were shown
to bind phospholipid membranes with high affinity, probably assisting
in bringing viral and cellular membranes closer (32, 33). Indeed,
studies using synthetic peptides supported a direct role of the
N-terminal heptad repeat in Sendai virus fusion event (34). The G
protein from all rhabdoviruses also presents heptad repeats (14), which
were mapped as the PS binding domain of this protein (17). We showed
here that a synthetic peptide corresponding to VSV G protein heptad
repeat, the p2-like peptide, was very efficient in mediating
pH-dependent fusion of PS-containing vesicles, which, as
found for the whole virus, was inhibited by treatment of the peptide
with DEPC. p2-like peptide from viral hemorrhagic septicemia virus,
another rhabdovirus, was also able to induce membrane fusion (16).
These results together suggest that p2-like peptides play an active
role in the rhabdoviral fusogenic process. Whether they can be
considered the actual rhabdovirus fusion peptides depends on further investigation.
A common feature of several viral fusion glycoproteins is that they are
synthesized as a fusion-incompetent precursor that is cleaved to
generate the fusogenic protein. The fusion machinery from rhabdovirus
is completely different. The fusion occurs through reversible
conformational changes that do not require activation by proteolytic
cleavage (35, 36). Our previous results showed that VSV G protein
underwent a dramatic loss of secondary structure at the fusogenic pH,
which was shown to be necessary for fusion (8). The loss of secondary
structure during fusion seems to be another particular feature of
rhabdovirus fusion, because most of viral fusion peptides adopt an
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet conformation (16). p2-like peptide was found
among all rhabdoviruses and contains two histidyl residues in VSV G
protein (17).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C.
-cyano-4-hydroxycinnamic
acid in 50% acetonitrile, 0.1% trifluoroacetic acid) were applied on
the plate and allowed to dry at room temperature. Mass profiles of
digested G protein were obtained on a Voyager-DE PRO (Applied
Biosystems) equipped with a nitrogen laser (
= 337 nm). Fifty
laser shots were summed per sample spectrum, and an average of five
spectra was used. The masses obtained were searched against a protein
data base containing the sequence of bovine trypsin and VSV G protein
using the ExPASy Molecular Biology Server (www.expasy.org).
Searches were also done with the DEPC modification option turned on.
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-visible detector, coupled to an Ultrasphere C-18 column (5 µm, 4.6 × 150 mm), which was eluted with a two-solvent system,
Solvent A1 (H3PO4/H2O) (1:1000) (v/v) and Solvent 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 (TofSpec-E; Micromass) and/or peptide sequencing using a
protein sequencer PPSQ-23 (Shimadzu, Tokyo, Japan).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
His modification by DEPC impairs VSV-induced
membrane fusion. Equal amounts of unlabeled vesicles and vesicles
labeled with 10-PyPC were incubated with purified VSV ( ) or VSV
pre-incubated with 0.005 (
), 0.01 (
), and 0.02 (
)
mM DEPC. 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.
VSV-induced membrane fusion was measured by the decrease in the 10-PyPC
excimer/monomer fluorescence ratio. 10-PyPC was excited at 340 nm, and
the intensities were collected at 376 and 480 nm for monomer and
excimer, respectively. The final protein concentration was 15 µg/ml.
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Fig. 2.
VSV modification with DEPC.
A, purified VSV was diluted in 20 mM MES, 30 mM Tris buffer, pH 7.5, and incubated with different
concentrations of DEPC. The formation of carbethoxyhistidyl residues
was followed by the increase in the absorbance at 240 nm. The final
protein concentration was 35 µg/ml. B, kinetics of VSV His
modification with 0.02 mM DEPC. The final protein
concentration was 140 µg/ml.
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Fig. 3.
Reversal of DEPC-induced modification in VSV
G protein His residues with hydroxylamine. Equal amounts of
unlabeled vesicles and vesicles labeled with 10-PyPC were incubated
with purified VSV ( ), VSV pre-incubated with DEPC 0.02 mM (
), or VSV pre-incubated with DEPC 0.02 mM for 3 min and then incubated with hydroxylamine 400 mM for 15 min (
). The final protein concentration was 15 µg/ml. Other experimental conditions were as in Fig. 1.
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Fig. 4.
pH-induced conformational changes on VSV G
protein involve His protonation. A, purified VSV ( )
or VSV pre-incubated with 0.002 mM DEPC (
) or 0.02 mM DEPC (
) were diluted in 20 mM MES, 30 mM Tris buffer, pH 7.5, to a final protein concentration of
25 µg/ml. Tryptophan fluorescence emission at 334 nm was recorded
whereas pH was gradually acidified by addition of HCl. The excitation
wavelength was 280 nm. B, kinetics of G protein interaction
with liposomes at low pH was measured by intrinsic fluorescence of
purified VSV (
) or VSV pre-incubated with 0.02 mM DEPC
(
). Vesicles composed of PC:PS (1:3) were prepared in 20 mM MES, 30 mM Tris buffer, pH 6.0, in a final
phospholipid concentration of 0.1 mM. The excitation
wavelength was 280 nm, and the emission was collected at 334 nm. The
final protein concentration was 25 µg/ml.
MALDI-TOF mass spectrometry analysis of peptides from VSV G protein
MALDI-TOF mass spectrometry analysis of DEPC-modified peptides from VSV
G protein
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Fig. 5.
Amino acid sequences of the peptides used in
this study. The putative VSV fusion peptide corresponds to the G
protein sequence between residues 117 and 137. VSV p2-like peptides
used in this study correspond to VSV G protein residues between 145 and
168. His-148 or His-149 or both were substituted for Ala residues.
Dots represent wild-type residues. Peptides corresponding to
other G protein sequences between residues 65 and 85, 170 and 190, and
395 and 418 were used as control peptides containing two
histidines.
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Fig. 6.
Role of p2-like peptide in VSV-induced
membrane fusion. Equal amounts of unlabeled vesicles and vesicles
labeled with 10-PyPC were incubated with purified VSV (A),
VSV p2-like peptide (B), or VSV peptide 117-137
(C). The vesicles were prepared in 20 mM MES, 30 mM Tris buffer in a final phospholipid concentration of 0.1 mM. Membrane fusion was measured by the decrease in the
10-PyPC excimer/monomer fluorescence ratio. 10-PyPC was excited at 340 nm, and the intensities were collected at 376 and 480 nm for monomer
and excimer, respectively. The vesicles used were composed of PC:PS
(1:3) at pH 6.0 ( ), PC:PS (1:3) at pH 7.5(
), and PC only at pH
6.0 (
). The final viral protein concentration was 15 µg/ml, and
peptide concentration was 10 µg/ml (
,
,
) or 0.2 µg/ml
(
).
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Fig. 7.
His-148 and His-149 are important for
peptide-induced membrane fusion. A, membrane fusion
induced by p2-like peptide ( ), peptide pre-incubated with 0.02 mM DEPC (
) or 0.2 mM DEPC (
), or peptide
pre-incubated with 0.2 mM DEPC for 3 min and then incubated
with 500 mM hydroxylamine for 15 min (
). B,
effect of His substitution on p2-like peptide-induced fusion. Membrane
fusion activity was evaluated for wild-type p2-like peptide (
),
H148A (
), H149A (
), and H148A,H149A double-mutant peptide (
).
C, membrane fusion activity of VSV peptide 65-85 (
),
170-190 (
), and 395-418 (
). The final peptide concentrations
was 10 µg/ml. Other experimental conditions were as in Fig. 1.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical structure when inserted in the lipid bilayer, which is
necessary for their fusogenic activity (21-24, 37). In the case of
HIV-1, however, it is hypothesized that the fusion peptide underwent
conformational transitions from
-helix to
-structures when bound
to the target membrane (38-40), suggesting that fusion may require
conformational flexibility of the fusion peptide itself. The results
described here suggests that, at least in the case of VSV, the
structural transitions that drive fusion reaction depend on His protonation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gonzalo de Prat-Gay for encouragement, Dr. Ronaldo Mohana-Borges for critical reading of the manuscript, Dr. Russolina B. Zingali for helpful suggestions, and Simone C. L. Leão and Denis L. S. Dutra for technical assistance. We also thank Dr. Adalberto R. Vieyra for use of the fluorometer and the "Rede Proteômica do Estado do Rio de Janeiro" for use of the mass spectrometry facility.
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FOOTNOTES |
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* This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Fundação de Amparo Pesquisa do Estado de São Paulo, and Human Frontiers for Science Progress (RG 00043/2000-M).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Contributed equally to this work.
To whom correspondence should be addressed. Tel.:
55-21-22706264; Fax: 55-21-22708647; E-mail:
dapoian@bioqmed.ufrj.br.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M210615200
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ABBREVIATIONS |
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The abbreviations used are: VSV, vesicular stomatitis virus; DEPC, diethylpyrocarbonate; PS, phosphatidylserine; PC, phosphotidylcholine; 10-PyPC, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HIV-1, human immunodeficiency virus, type 1; MES, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography.
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