Low pH-induced Conformational Changes in Vesicular Stomatitis
Virus Glycoprotein Involve Dramatic Structure Reorganization*
Fabiana A.
Carneiro,
Artur S.
Ferradosa, and
Andrea T.
Da
Poian
From the Departamento de Bioquímica Médica, Instituto
de Ciências Biomédicas, Universidade Federal do Rio de
Janeiro, 21941-590 Rio de Janeiro, Brazil
Received for publication, September 25, 2000, and in revised form, October 5, 2000
 |
ABSTRACT |
Membrane fusion is the key step in the entry of
enveloped animal viruses into their host cells. Fusion of vesicular
stomatitis virus with membranes occurs at acidic pH and is mediated by
its envelope glycoprotein, the G protein. To study the structural transitions induced by acidic pH on G protein, we have extracted the
protein from purified virus by incubation with nonionic detergent. At
pH 6.0, purified G protein was able to mediate fusion of either phospholipid vesicles or Vero cells in culture. Intrinsic fluorescence studies revealed that changes in the environment of Trp residues occurred as pH decreases. In the absence of lipidic membranes, acidification led to G protein aggregation, whereas protein-protein interactions were substituted by protein-lipid interactions in the
presence of liposomes. 1,1'-Bis(4-aniline-5-naphthalene sulfonate) (bis-ANS) binding was utilized to probe the degree of exposure of
hydrophobic regions of G protein during acidification. Bis-ANS binding
was maximal at pH 6.2, suggesting that a hydrophobic segment is exposed
to the medium at this pH. At pH 6.0, a dramatic decrease in bis-ANS
binding was observed, probably due to loss of tridimensional structure
during the conformational rearrangement. This hypothesis was confirmed
by circular dichroism analysis at different pH values, which showed a
great decrease in
-helix content at pH values close to 6.0, suggesting that a reorganization of G protein secondary structure
occurs during the fusion reaction. Our results indicate that G protein
undergoes dramatic structural changes at acidic pH and acquires a
conformational state able to interact with the target membrane.
 |
INTRODUCTION |
Viral infection depends on the transfer of viral genetic material
into the host cell. After binding to its cellular receptor, the virus
must cross the plasma membrane and release its genome into the cellular
milieu for subsequent replication. Enveloped viruses always gain entry
to the cytoplasm by membrane fusion (reviewed in the Refs. 1-3),
whereas nonenveloped viruses must use alternative strategies to cross
the membrane. The membrane of some enveloped viruses, such as
paramyxoviruses, retroviruses, or herpesviruses, fuses directly with
the host cell plasma membrane after virus binding to their cell
receptor. Other enveloped viruses, such as influenza, alphaviruses, or
rhabdoviruses, enter the cells by the endocytic pathway, and fusion
depends on the acidification of the endosomal compartment. In both
cases, membrane fusion is mediated by viral envelope glycoproteins,
which have already been identified for most enveloped animal viruses
(reviewed in Refs. 3 and 4).
The best studied low pH-activated viral fusion protein is the influenza
hemagglutinin (HA).1 This is
the only fusion protein for which atomic resolution structure has been
obtained in neutral (pre-fusogenic) and in low pH (5). The
conformational changes observed suggest that the apolar fusion peptide
moves to the tip of the molecule and is delivered toward the target
membrane. The low pH-induced conformational changes that enable
membrane fusion in Semliki Forest virus (SFV) are also dramatic.
Time-resolved cryoelectron microscope studies showed that low pH
treatment resulted in movement of E1 subunits to
the center of the spike, which initiates the formation of
E1 trimer. Subsequently, the fusion sequence is
extended toward the target membrane (6).
Vesicular stomatitis virus (VSV) is a member of the Rhabdoviridae
family, a group of enveloped negative single strand RNA virus. Both VSV
binding to the cell surface and fusion between viral envelope and
endosomal membrane are mediated by its trimeric surface type I
glycoprotein, the G protein. This protein of 67 kDa is anchored to
viral and cellular membranes via a single transmembrane anchor sequence
close to the C-terminus (7). Unlike other viral fusion proteins, VSV G
protein does not contain an obvious hydrophobic peptide sequence that
can serve as the fusion peptide. Mutation analysis revealed that an
ectodomain segment localized between the amino acids 117 and 136 is
essential for fusion (8, 9). This segment is highly conserved among the
vesiculoviruses and is believed to contain VSV G protein internal
fusion peptide. Another region of G protein, encompassing residues
395-418, has been identified as a segment that affects fusogenic
activity of the protein by influencing the low pH-induced
conformational changes (10). In addition, it was recently shown that
not only the ectodomain segment but also the membrane anchoring domain
is required for VSV fusion activity (11, 12).
Although much progress has been made on the identification and
characterization of VSV G protein segments involved in membrane fusion
activity using site-directed mutagenesis, information on the structural
changes that occur during acidification are still lacking. In this work
we investigated the conformational changes in G protein during
acidification. Intrinsic fluorescence analysis showed that, when the pH
was decreased, the environment of the G protein Trp residues changed,
allowing interaction of these residues with the target membranes.
Binding of a fluorescent probe revealed that the exposure of
hydrophobic domains was maximal at pH 6.2. Between pH 6.0 and 5.6 a dramatic conformational change occurred, which includes loss of
secondary and tertiary structures.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Virus Propagation--
VSV Indiana was
propagated in monolayer cultures of baby hamster kidney-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 (Cultilab, Campinas, SP, Brazil), 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 5 plaque-forming units/ml. The cultures were kept at 37 °C for
16-20 h, and the viruses were harvested and purified by differential
centrifugation followed by equilibrium sedimentation in a sucrose
gradient as described elsewhere (13). Purified virions were stored at
70 °C.
G Protein Purification--
G protein extraction from purified
native VSV was adapted from Newcomb and Brown (14). Purified
suspensions of VSV in 10 mM Tris-HCl (pH 7.4) at a final
protein concentration of 1 mg/ml were brought to a concentration of 30 mM CHAPS (Sigma) by the addition of an equal volume of 60 mM CHAPS in Tris-HCl buffer. The suspension was allowed to
stand at room temperature for 1 h. The insoluble structures called
"skeletons" (nucleocapsids associated with M protein) were pelleted
by ultracentrifugation through a 1-ml glycerol cushion at 43,000 rpm
for 90 min at 4 °C in a Beckman 50 Ti rotor. The supernatant was
collected and dialyzed against Tris-HCl buffer. For circular dichroism
analysis, G protein was extracted with 30 mM
-D-octyl glucoside.
Preparation of Liposomes--
Equimolar amounts of PC and PS
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) at a final concentration of 1 mM. The
suspension was vortexed vigorously for 5 min. Small unilamellar
vesicles were formed 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-resting intervals until a transparent solution was obtained (~10 cycles).
Liposome Fusion Assay--
A suspension containing PCPS
liposomes in a final concentration of 0.1 mM phospholipids
was incubated at room temperature at pH 7.5 or 6.0. The reaction was
initiated by addition of purified VSV or G protein, and fusion was
monitored by the increase in light scattering.
Cell Fusion Assay--
Cell-to-cell fusion was assayed as
described in White et al. (15), using 5 µl of purified VSV
(protein concentration of 100 µg/ml) or 10 µl of G protein (protein
concentration of 300 µg/ml).
Fluorescence and Light Scattering Measurement--
Intrinsic
fluorescence, binding of 1,1'-bis(4-aniline-5-naphthalene sulfonate)
(bis-ANS) (Molecular Probes, Eugene, OR), and light scattering
measurements were recorded using a Hitachi F-4500 Fluorescence
Spectrophotometer. Intrinsic fluorescence was measured exciting samples
at 280 nm and collecting emission between 300 and 420 nm. Bis-ANS
binding was determined by the increase in fluorescence emission between
400 and 600 nm upon excitation at 360 nm. Light scattering was measured
at 90o in the spectrofluorometer by selecting the same
wavelength for both excitation and emission (280 nm).
Circular Dichroism--
Circular dichroism studies were
performed with an Jasco model J-715-1505 protect class I
spectropolarimeter. Samples were prepared in phosphate buffer 20 mM, pH 7.4, at a concentration of 10 µM.
Spectra were recorded using a 0.2 nm bandwidth, a 0.2 nm step in quartz
cells of 0.1 cm, and a time constant of 8 s. A total of 10 scans
was averaged.
 |
RESULTS |
G Protein Purification
G protein was extracted from purified VSV by treatment with the
nonionic detergent CHAPS. This detergent does not interfere in
fluorescence measurements at the UV range, allowing G protein structural studies using Trp fluorescence. After VSV incubation with 30 mM CHAPS for 1 h, the protein could be completely
removed from the virus, and a purified sample was obtained after
ultracentrifugation, as shown by SDS-polyacrylamide gel electrophoresis
(Fig. 1).

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Fig. 1.
SDS-polyacrylamide gel electrophoresis
analysis of G protein. Purified VSV (lane 1) was
incubated with CHAPS, and G protein was separated from other viral
components by ultracentrifugation. The supernatant containing G protein
was collected (lane 2). Molecular mass markers
(bovine serum albumin, 66 kDa; chicken egg albumin, 45 kDa; and
-lactoglobulin, 18.4 kDa) are shown to the right. The
positions of VSV G, N, and M proteins
are indicated to the left.
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Purified G Protein Maintains Its Biological Activity
Two different methods were utilized to test the ability of
purified G protein to mediate membrane fusion. First, we developed a
spectroscopic method to follow the kinetics of liposome fusion induced
by the protein. Fusion of vesicles containing equimolar amounts of PC
and PS (PCPS liposomes) was quantified by the increase of the light
scattering of the suspension after protein addition (Fig.
2A). Light scattering directly
correlates with the size of particles in solution (16) and has already
been used to quantify protein aggregation (17, 18). At pH 7.5, no
increase in light scattering was observed either in the absence or in
the presence of G protein. Although a small increase in light
scattering occurred when PCPS liposomes were incubated at pH 6.0 without the protein, a much greater increase in vesicle size was
observed in the presence of purified G protein. After ~30 min of
incubation, fusion was completed. This result suggests that, at low pH,
isolated G protein promotes vesicle fusion. An experiment performed
with the whole virus led to similar results, except that fusion
occurred more rapidly (Fig. 2B). Because cell-to-cell fusion
has been extensively used for the demonstration of fusion activity in
several virus families (15, 19), we used cell fusion assay to confirm
the ability of purified G protein to mediate membrane fusion (Fig. 3). This assay has already been
used to analyze low pH fusion activity of whole VSV (15) or G protein
expressed on the plasmatic membrane of transfected cells (8). Formation
of large polykarions was observed when either purified G protein or
whole virus was incubated at pH 5.8 with monolayers of Vero cell
cultures (Fig. 3, B and C).

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Fig. 2.
G protein-mediated liposome fusion.
A, fusion of liposomes induced by purified G protein or
B, whole VSV. Small unilamellar vesicles composed by
equimolar amounts of phosphatidylcholine and phosphatidylserine in a
final concentration of 0.1 mM were incubated in the
presence ( and ) or in the absence ( and ) of purified G
protein or whole VSV at pH 6.0 ( and ) or 7.5 ( and ).
Fusion was determined by measuring the increase in light scattering in
the spectrofluorometer. Final protein concentration was 30 µg/ml for
G protein and 100 µg/ml for VSV.
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Fig. 3.
pH dependence of G protein-induced cell
fusion. Vero cells were washed twice with the binding medium (pH
6.8) and then incubated with 10 µl of medium (A), G
protein (0.3 mg/ml) (B), or purified VSV (0.1 mg/ml)
(C) for 1 h in ice-cold medium. Prewarmed medium
(pH 5.8) was added for 60 s, and the cells were postincubated with
medium (pH 7.2) for 1 h at 37 °C. After this time, the cells
were observed under the light microscope and photographed. Observation
of polykarions (arrows) when Vero cells was exposed to G
protein or whole VSV at low pH indicates that the isolated purified G
protein maintained its ability to promote membrane fusion.
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pH Induced Conformational Changes
Changes in Trp Environment--
Intrinsic fluorescence spectrum of
purified G protein showed a maximal emission in 334 nm. Fig.
4A shows that G protein
intrinsic fluorescence emission decreased as pH was diminished,
indicating that changes in Trp residues environment occurred during
acidification. This fluorescence quenching was not a direct effect of
pH decrease on Trp residues, since no significant changes in
N-acetyl-L-tryptophanamide (NATA) fluorescence
was observed at this pH range (Fig. 4A). G protein
fluorescence spectrum is more blue-shifted than NATA spectrum, suggesting that Trp residues are not completely exposed to the aqueous
medium (Fig. 4B). However, no red shift in the intrinsic fluorescence spectrum was observed when the pH was lowered, indicating that Trp exposure to the polar solvent did not occur.

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Fig. 4.
Effects of pH in G protein intrinsic
fluorescence. A, G protein was diluted in 30 mM MES, 10 mM Tris-HCl (pH 7.5), to a final
concentration of 30 µg/ml. Tryptophan fluorescence emission at 334 nm
( ) was recorded, whereas pH was gradually acidified by addition of
HCl. As a control, fluorescence spectra of NATA ( ) were also
recorded upon acidification. NATA fluorescence emission was collected
at 350 nm. The excitation wavelength was 280 nm in both cases.
B, fluorescence spectra of G protein ( and ) and NATA
( and ) at pH 7.5 ( and ) and 5.4 ( and ). The
excitation wavelength was 280 nm. C, interaction of G
protein with liposomes. G protein was diluted to a final concentration
of 30 µg/ml in 30 mM MES, 10 mM Tris-HCl (pH
7.5), in the presence of 0.1 mM PCPS vesicles. Tryptophan
fluorescence emission at 334 nm was recorded, and pH was gradually
acidified by addition of HCl. The excitation wavelength was 280 nm.
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Conversely, G protein intrinsic fluorescence greatly increases upon
acidification in the presence of PCPS vesicles. Trp fluorescence quantum yield reaches a maximal value at pH 6.2, suggesting that the
interaction with the quencher was substituted by interaction with
phospholipid molecules in the membranes (Fig. 4C).
Aggregation--
Acidification promotes G protein aggregation, as
observed by light scattering measurements (Fig.
5). Aggregation and the conformational changes probed by Trp fluorescence were not simultaneous processes. The
greater changes in intrinsic fluorescence occurred between pH 7.5 and
6.4, whereas significant increase in light scattering initiates at pH
6.5, where quenching phenomenon is almost completed. G protein
aggregation probably occurred due to exposure of hydrophobic regions,
which results in intermolecule interactions.

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Fig. 5.
Effects of pH on G protein aggregation.
Aggregation was measured by the increase in light scattering, and pH
was gradually acidified by addition of HCl. G protein was diluted in 30 mM MES, 10 mM Tris-HCl (pH 7.5), to a final
concentration of 30 µg/ml. The dotted line corresponds to
the intrinsic fluorescence quenching at the same pH range.
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Exposure of Hydrophobic Domains--
Exposure of hydrophobic
segments of G protein at acidic pH was probed by measuring binding of
bis-ANS. This fluorescent compound binds noncovalently to nonpolar
segments in proteins, especially in proximity to positive charges (20).
Bis-ANS binding is accompanied by a large increase in its fluorescence
quantum yield, and it has been used to probe protein structural changes
(21, 22). Purified G protein was incubated with bis-ANS at pH 7.5, and
spectra were obtained as the solution was acidified (Fig.
6). Bis-ANS binding increased when the pH
was lowered from 7.5 to 6.2, suggesting the exposure of hydrophobic
segments. No change in bis-ANS fluorescence maximum was observed as pH
decreased. Intriguingly, a great decrease in bis-ANS binding occurred
at pH 6.0. Bis-ANS binding requires not only the presence of nonpolar
amino acids but a hydrophobic environment. Denatured proteins or very
nonorganized structures do not bind this probe (21, 23). The pH
threshold for membrane fusion mediated by VSV is close to 6.0 (15), and
the fusogenic activity of expressed wild-type G protein initiates at pH
6.3, reaching the maximum activity between pH 6.0 and 5.6 (8, 9). Conceivably, the major conformational changes in G protein occur at
this pH range. Titration of bis-ANS binding to purified G protein was
performed at different pH values (Fig.
7A). Fluorescence emission of
bis-ANS increased gradually as the probe was added to the protein sample (final protein concentration of 0.1 µM). Binding
was saturated at 0.8 µM bis-ANS, suggesting the presence
of ~8 binding sites. When the pH decreased from 7.5 to 6.2, less
bis-ANS was necessary for maximal emission increase, confirming that at
mildly acidic pH values hydrophobic regions became more exposed to the
solvent (Fig. 7B). Our results suggest that G protein
undergoes a dramatic conformational change, which involves loss of the
hydrophobic domain structure before its reorganization in the
fusion-inducing conformation. To confirm that the conformational
changes observed for purified G protein also occur when it is
integrated in virus envelope, we performed titration of bis-ANS binding
with purified VSV (Fig. 7, C and D). Accordingly,
the results were similar to those obtained for purified protein, except
by the absence of a defined plateau at higher concentrations of the
probe, probably due to nonspecific binding of bis-ANS to viral
membrane.

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Fig. 6.
Binding of bis-ANS to G protein. A G
protein was diluted in 30 mM MES, 10 mM
Tris-HCl (pH 7.5), to a final concentration of 6 µg/ml, and incubated
with bis-ANS (final concentration of 1.0 µM).
Fluorescence emission at 485 nm was recorded when pH was gradually
acidified by addition of HCl. The excitation wavelength was 360 nm.
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Fig. 7.
Titration of bis-ANS binding. Binding of
bis-ANS to G protein (A and B) or whole VSV
(C and D) was measured. A and
C, fluorescence intensity at 485 nm was collected upon
addition of increasing concentrations of the probe at pH 7.5 ( ), 6.2 ( ), 6.0 ( ), and 5.6 ( ). B and D, binding
of bis-ANS (in a final concentration of 0.8 µM) to G
protein or whole VSV at different pH values. The protein final
concentration was 6 µg/ml for G protein and 18 µg/ml for whole VSV.
The excitation wavelength was 360 nm.
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Changes in the Secondary Structure--
To examine the alterations
in G protein secondary structure during acidification, we analyzed
circular dichroism spectra of the protein at different pH values (Fig.
8). In these experiments, the protein was
extracted using
-D-octyl glucoside, to avoid CHAPS
interference in the circular dichroism spectra. The secondary structure
content decreased as pH was lowered from 7.5 to 6.4 (Fig.
8B). Further acidification led to an increase in the
secondary structure content, but protein probably acquires a different
conformation, as seen by the different spectra at pH 7.5 and 5.6. These
results confirm that a G protein undergoes structure reorganization
during the fusion reaction.

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Fig. 8.
Effect of pH on the secondary structure of G
protein. Circular dichroism of G protein. G protein was diluted in
20 mM phosphate buffer (pH 7.5). pH was gradually acidified
by addition of HCl. A, CD spectra at pH values of 7.5 (---),
6.2 (-·-), and 5.6 (···). B, effect of pH in the
-helix content, expressed by the inverse of differential absorption
at 222 nm. Final protein concentration was 300 µ g/ml.
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 |
DISCUSSION |
G protein mediates membrane fusion at the acidic environment of
the endosomal compartment after virus entry into the cell by
endocytosis. In this work we described conformational changes of
purified VSV G protein during acidification, using light scattering and
intrinsic fluorescence measurements, binding of the fluorescent probe
bis-ANS, and circular dichroism.
To measure the kinetics of G protein-induced fusion, we developed an
assay based on the increase in the light scattering of a suspension
containing small unilamellar vesicles in the presence of the protein.
The kinetics of whole VSV-induced fusion measured by the light
scattering assay presented a half-time of 2.5 min, similar to the
2.0-min half-time measured in vitro by fluorescence energy
transfer, using either G protein reconstituted into phospholipid vesicles (24) or whole VSV (25). The kinetics of in situ
fusion of VSV and the endosomal membranes during infection has also
been determined, showing a half-time of ~25 min (26). In this case, the longer half-time of fusion might be due to the gradual
acidification of the endosomal compartment. The light scattering assay
revealed that the kinetics of fusion mediated by purified G protein was slower than VSV fusion (Fig. 2). Fusion between the PCPS liposomes mediated by G protein at pH 6.0 presented a half-time of 10 min and was
virtually completed within 25 min. This result may be explained by the
fact that G protein in the intact virus is clustered, therefore
increasing the cooperativity of protein membrane-interaction, since
ectodomain insertion into the target membrane has been shown to be
reversible (27).
In the absence of membranes, intrinsic fluorescence was quenched as the
pH decreased, although no red shift in the fluorescence spectrum was
observed (Fig. 4, A and B). This result suggests that the pH-induced conformational change probably involves
approximation between the Trp and a His or an Arg residue, rather than
exposure of the Trp to the aqueous medium. Alternatively, the absence
of red shift might be a consequence of simultaneous increase and decrease of different Trp residues exposure to the solvent, since G
protein presents 13 Trp residues (7).
Light scattering results showed that G protein aggregated between pH
6.6 and 5.6. Acid-induced aggregation has already been observed for
rabies virus glycoprotein (28). In both cases the aggregation could be
explained by exposure of hydrophobic domains at mildly acidic
conditions. Indeed, we have demonstrated that hydrophobic domains were
gradually exposed as pH decreased from 7.5 to 6.2 (Figs. 6 and 7).
Although an obvious hydrophobic fusion peptide was not identified in
rhabdovirus glycoproteins, the increase in bis-ANS binding is a direct
evidence of the exposure of a hydrophobic domain.
The exposed hydrophobic domain is probably involved in the interaction
of G protein and the target membrane. G protein Trp fluorescence
greatly increased upon acidification when the protein was incubated in
the presence of membranes (Fig. 4C). The increase in
fluorescence quantum yield indicated that Trp residues moved from the
proximity of a quencher to interact with the membrane environment. The
segment between amino acids 117 and 136, which is believed to interact
with the target membrane during acidification, contains one Trp residue
(Trp 119). The increase in intrinsic fluorescence might be explained by
the insertion of this residue into the liposome as pH decreased. NMR
and fluorescence studies with a synthetic peptide corresponding to the
sequence between amino acids 118 and 136 of G protein indicated that
Trp-119 penetrates into PS-containing model membrane (29). These
experiments were done at pH 7.4, showing that the fragment is able to
interact with the membrane even at neutral pH. The pH dependence of
Trp-membrane interaction observed here suggests that the segment is
hidden in the three-dimensional structure of the protein and becomes exposed during acidification. Indeed, Durrer et al. (30)
showed that interaction of a segment in the ectodomain containing the amino acids 59-221 with membranes increased when the pH was lowered from 7.0 to 6.0.
An even more interesting observation was the dramatic conformational
rearrangement at pH 6.0. At this pH, a great decrease in bis-ANS
binding occurred, indicating loss of the hydrophobic domain structure.
In addition, secondary structure was also lost at this pH, as shown by
circular dichroism results. The conformational changes that occur when
influenza virus HA and SFV spike glycoprotein are exposed to low pH are
equally dramatic (5, 6). A comparison of the three-dimensional
structure of the soluble trimers of HA and the trimeric fragment
obtained by acid treatment and proteolysis of the trimers revealed a
great secondary structure rearrangement resulting in the movement of
the fusion peptide toward the tip of the molecule. In the case of SFV,
the spike oligomer was completely reorganized. The stable heterodimers
formed by E1 and
E2 glycoprotein dissociates to form an
E1 homotrimer (31, 32). The pH thresholds of
membrane fusion mediated by several enveloped virus are sharp (15, 28).
VSV fusion has a threshold between pH 6.2 and 5.8 (15), exactly the pH
range where structural reorganization was observed by intrinsic bis-ANS
fluorescence and circular dichroism.
Although at lower pH values, the protein recovered the ability to bind
bis-ANS and acquire secondary structure, it is likely that structural
rearrangements result in a different conformation, as indicated by the
circular dichroism spectra obtained at pH 7.4 and 5.6 (Fig.
8A). Taken together, the results presented herein suggest
that G protein undergoes extensive conformational changes that lead to
the formation of a hydrophobic domain that might play the role of the
fusion peptide.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Adalberto R. Vieyra for use the
fluorometer and Dr. Jerson L. Silva for use of the circular dichroism
facility. We also thank Dr. Mauricio Luz and Dr. Herman Wolosker for
critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from Conselho Nacional de
Desenvolvimento Científico e Tecnológico,
Fundação de Amparo à Pesquisa do Estado do Rio de
Janeiro, and Programa de Apoio ao Desenvolvimento Científico e
Tecnológico.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.
To whom correspondence should be addressed. Tel.: 55-21-2706264;
Fax: 55-21-2708647; E-mail: dapoian@bioqmed.ufrj.br.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008753200
 |
ABBREVIATIONS |
The abbreviations used are:
HA, hemagglutinin;
SFV, Semliki Forest virus;
VSV, vesicular stomatitis virus;
CHAPS, 3[(3-cholamidoprobyl) dimethylammonio]-1-propanesulfonate;
PC, phosphatidylcholine;
PS, phosphatidylserine;
bis-ANS, 1,1'bis(4-aniline-5-naphthalene sulfonate);
NATA, N-acetyl-L-tryptophanamide;
MES, 4-morpholineethanesulfonic
acid.
 |
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