From the Cell Biology Programme Structural Biology
Programme, European Molecular Biology Laboratory, Postfach 10 2209, D-69012 Heidelberg, Germany and Max-Planck-Institute for Molecular Cell
Biology and Genetics, Dresden 01307, Germany
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ABSTRACT |
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During the budding of enveloped viruses from the
plasma membrane, the lipids are not randomly incorporated into the
envelope, but virions seem to have a lipid composition different from
the host membrane. Here, we have analyzed lipid assemblies in three different viruses: fowl plague virus (FPV) from the influenza virus
family, vesicular stomatitis virus (VSV), and Semliki Forest virus
(SFV). Analysis of detergent extractability of proteins, cholesterol,
phosphoglycerolipids, and sphingomyelin in virions showed that FPV
contains high amounts of detergent-insoluble complexes, whereas such
complexes are largely absent from VSV or SFV. Cholesterol depletion
from the viral envelope by methyl- The hypothesis that lipids from the plasma membrane are not
randomly included into the viral envelope, but that budding could occur
from specialized domains of the membrane, was put forward several years
ago (1, 2). Comparing the lipid composition of various viruses with the
composition of the host cell membrane, clear cut differences have been
reported, although caution has to be exercised because viruses can be
isolated practically pure, whereas plasma membrane fractions cannot be
(2-4). Thus, the role of lipid domains has remained controversial and
has not so far been given functional significance.
New insights into the mechanisms of domain formation in biological
membranes have come from the analysis of detergent-resistant membrane
fractions (5). Recent evidence suggests that laterally associating
sphingolipids and cholesterol form small ordered domains, called rafts,
which are resistant to extraction with Triton X-100 at 4 °C. These
domains can incorporate specific proteins and function as platforms for
intracellular sorting and signal transduction events (6, 7). Recently,
the concentration of
GPI1-anchored proteins in
microdomains has been confirmed in living cells by biophysical and
biochemical methods without the use of detergents (8, 9). However, when
GPI-anchored proteins on the apical surface of epithelial cells were
analyzed, no such concentration could be observed (10). The reason for
this discrepancy is not known, yet one explanation could be that this
membrane represents a continuous raft domain in contrast to the plasma membrane of nonpolarized cells, where raft domains would be
noncontinuous (6, 10, 11). Recent studies have shown that cholesterol promotes the detergent insolubility of GPI-anchored proteins or transmembrane proteins both in cellular membranes and in artificial lipid vesicles (12-15). Lipids recovered in detergent-insoluble glycolipid-enriched complexes (DIGs) were shown to have higher melting
temperatures than the average lipids from the plasma membrane, suggesting that raft-domains might be formed from lipids with preferentially saturated acyl chains (16). Interestingly, for artificial lipid vesicles detergent insolubility could be correlated with the formation of a liquid-ordered phase in the membrane (15, 17),
suggesting that phase separation might be the basis for the formation
of insoluble lipid rafts in biological membranes (7, 18).
Also, specific viral glycoproteins have been described to be associated
with raft domains during transport to the cell surface. Both the
influenza virus neuraminidase and hemagglutinin are recovered in DIGs
after entering the Golgi complex (19, 20). Efficient surface transport
of influenza virus HA requires cholesterol, and the protein stays
raft-associated at the plasma membrane (14, 21).
Here we used membranes of baby hamster kidney (BHK) cells and the viral
envelopes of influenza fowl plague virus (FPV), vesicular stomatitis
virus (VSV), and Semliki Forest virus (SFV) as model systems to analyze
the formation of detergent-resistant lipid domains in a biological
membrane. Our results demonstrate that the viruses contain lipid raft
assemblies to a very different extent. Furthermore, we show that in all
of the membranes analyzed cholesterol is required for the formation of
detergent-insoluble lipid complexes and that such complexes are highly
ordered. Our results therefore support a model in which cholesterol
promotes the formation of lipid domains that become selectively
incorporated into the influenza envelope.
Cell Culture, Virus Stocks--
BHK cells (strain CCL10,
American Culture Collection) were maintained in G-MEM (5% FCS, 10%
tryptosephosphate 10 mM Hepes, 2 mM glutamine,
100 units/ml penicillin, 100 µg/ml streptomycin) in 5%
CO2 at 37 °C in a humidified incubator. Virus stocks of influenza virus (strain A/FPV), VSV, SFV, and the recombinant VSV-HA
(22) were produced as described before (23, 24).
Preparation of Membrane Fractions--
From BHK cells, a light
membrane fraction was prepared essentially as described (25). Briefly,
cells were homogenized with a syringe under a nitrogen atmosphere. A
postnuclear supernatant was adjusted to 1.5 M sucrose and
overlayered with 1.2 and 0.8 M sucrose. After
centrifugation for 4 h (45,000 rpm, 4 °C in a SW40 rotor,
Beckman), membranes were recovered from the 1.2/0.8 M
interface. For radioactive labeling, cells on one 10-cm culture dish
were incubated for 4 h with 25 µCi of
[1 Purification of Viruses--
For the analysis of proteins,
lipids, and DPH polarization, the viruses were grown in BHK cells. For
the production of nonlabeled viruses, cells from three overconfluent
80-cm2 flasks were trypsinized and seeded in 10 275-cm2 flasks. After 36 h, viruses were adsorbed to
the 90% confluent cells (2 × 108 plaque-forming
units/flask) in infection medium (MEM; 0.2% BSA, 10 mM
Hepes, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin) for 1 h. The inoculum was removed and replaced by infection medium. After 24 h, the medium was collected and
centrifuged two times to remove cell debris (1500 rpm, 4 °C, Heraeus
centrifuge). The supernatant was then layered on top of a step gradient
in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl (TN)
consisting of 3 ml of 55% (w/v) sucrose and 10 ml of 10% (w/v)
sucrose in a SW28 centrifuge tube. After a 2-h centrifugation at 25,000 rpm and 4 °C, the 55/10% interface was collected, diluted 3-fold in
TN, and then applied on an 8-ml 50% (w/v) glycerol cushion in TN.
After a 2-h spin (25,000 rpm, 4 °C) in a SW28 rotor, the supernatant
was discarded, and the virus pellet was resuspended in TN buffer
overnight at 4 °C. A preparation from 10 275-cm2 flasks
routinely yielded 1-2 mg of viral protein for FPV and VSV and about
0.2 mg for SFV.
Radiolabeled viruses were produced following essentially the same
protocol in smaller scale using two 10-cm tissue culture dishes. Two
hours postinfection, the cells were labeled with 0.5 mCi of
[35S]methionine in medium containing 5 µM
unlabeled methionine. For labeling with
[1
For labeling with 32P, cells on two 10-cm tissue culture
dishes were incubated with 750 µCi of
[32P]orthophosphate/dish for 30 h in G-MEM without
tryptose phosphate. Then cells were washed, and viruses were adsorbed
and collected.
Detergent and Cyclodextrin Extractions--
Aliquots of virus or
membrane preparations with a total lipid content smaller than 1 µg
were extracted with 50 µl of 1% (w/v) Triton X-100, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA (TXNE) on ice for 20 min. In pelleting experiments,
the extract was spun in a TLA-100 centrifuge (Beckman) in a TLA100
rotor for 20 min at 80,000 rpm and 4 °C. Proteins in supernatant and
pellet fractions were analyzed by SDS-PAGE. Alternatively, lipids from
pellet and supernatant fractions were extracted according to Bligh and
Dyer (26) and subsequently analyzed by thin layer chromatography on
silica plates (Kieselgel 60; Merck) with chloroform/methanol/water (60:35:8, v/v/v) as a solvent. Radiolabeled 32P-containing
lipids were visualized by autoradiography and quantified by
PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). Relative
amounts of [3H]cholesterol were quantified by
scintillation counting.
For flotation gradients, virus aliquots containing 10 µg of protein
were extracted with 250 µl of TXNE on ice for 20 min. Extracts were
then brought to 40% Optiprep by the addition of 500 µl of Optiprep
stock solution (Nycomed Pharma, Oslo, Norway), overlayered with 1.2 ml
of 30% Optiprep in TXNE and 250 µl of TXNE. Samples were spun for
2 h in a TLS 55 rotor (50,000 rpm, 4 °C), and six fractions
were collected from the top of the gradient. Proteins were precipitated
from the fractions by the addition of one volume of 20%
trichloroacetic acid and analyzed by SDS-PAGE and Western blotting.
Extractions with methyl- Electron Microscopy--
Virions were sedimented by
centrifugation in a TLA45 rotor (20 min, 30,000 rpm, 4 °C), and
negative staining was performed using glow-discharged carbon-coated
Formvar grids and a 1% (w/v) solution of uranyl acetate (Sigma).
DPH Polarization Measurements--
Polarization measurements
were performed essentially as described (16). Virus preparations or
lipid vesicles were resuspended in 100 µl of TN buffer, and 1 µl of
DPH (Sigma) dissolved in tetrahydrofuran was added. Samples were
incubated for 1 h in the dark, and DPH fluorescence intensity was
measured at 25 °C in an AB2 luminescence spectrometer (SLM Aminco)
equipped with Glan-Thompson polarizers (excitation wavelength, 359 nm;
emission at 427 nm; band pass 4 nm). Lipid vesicles were prepared by
sonication of 2 ml of 0.5 mg/ml PC from egg yolk (Sigma) or 0.5 mg/ml
DPPC (Sigma) in TN buffer. Similar results were obtained using viruses
or lipid vesicles at concentrations of 0.05-0.5 µg/ml and DPH at
final concentrations of 50 nM to 1 µM.
Fluorescence detected without DPH or without the addition of viruses
was negligible.
Glycoproteins in the Influenza Virus Envelope, but Not in VSV or
SFV, Are Recovered in DIGs--
To study raft domains in different
envelopes, the viruses were produced in BHK fibroblasts and isolated by
gradient centrifugation. The obtained preparations were essentially
pure as judged by SDS-PAGE and silver staining (Fig.
1A). The detergent solubility
of the viral glycoproteins was analyzed by extraction with Triton X-100 on ice and centrifugation in Triton-containing density gradients. Influenza virus HA was floating to low density as observed before (14),
whereas the VSV G or the SFV glycoproteins stayed in the bottom of the
gradient, indicating that they were solubilized by the detergent (Fig.
1B). Therefore, association of a glycoprotein with rafts in
the viral envelope is specific for influenza viruses and not a general
phenomenon.
Extractability of Cholesterol in Viral Envelopes--
Cholesterol
is strongly enriched in detergent-insoluble fractions from cellular
membranes (5). We therefore compared the Triton solubility of
cholesterol in the different viral envelopes and the host cell
membrane. BHK cells were labeled with [3H]cholesterol,
and viruses were produced and purified. The radiolabeled membranes were
extracted with Triton X-100 on ice and centrifuged, and radioactivity
in supernatant and pellet fractions was quantified by scintillation
counting (material pelleted by this centrifugation at 178.000 × g will be considered "insoluble"). For comparison, a
[3H]cholesterol-labeled light membrane fraction from BHK
cells was prepared and detergent-extracted. Strikingly, the cholesterol solubility showed large differences between the viruses analyzed (Fig.
2A). In the influenza viruses
(FPVs) 41 ± 8% of the cholesterol was pelleted, in VSV 13 ± 6%, and in SFV only 5 ± 5%. Compared with the BHK light
membrane fraction (enriched for the plasma membrane, endosomes, and
Golgi membranes (27)), cholesterol solubility in the influenza virus
envelope is decreased and increased in VSV and SFV. It is important to
note that the cholesterol solubility in membranes from virally infected
BHK cells was not significantly changed in comparison with uninfected
cells (data not shown). We next used CD to further analyze the
extractability of cholesterol in the different viral envelopes. In
contrast to detergents, incubation with low concentrations of CD leaves
membranes intact and mediates selective efflux of sterols from the
bilayer (28, 29). When [3H]cholesterol-labeled influenza
viruses and VSV were treated with increasing concentrations of CD,
cholesterol was more easily removed from the VSV envelope (Fig.
2B). For example, 1 mM CD extracted 70 ± 3% of cholesterol from VSV but only 43 ± 2% from the influenza virus particles. It should be noted that practically no further cholesterol efflux was observed when the extraction times were extended
beyond 15 min (data not shown). During the extraction, the viral
particles remained intact and did not show any dramatic structural
changes as judged by negative staining and electron microscopy (Fig.
3). The extractability of cholesterol by
CD from the SFV envelope was found to be intermediate compared with the VSV and FPV envelope; however, the results were somewhat variable (data
not shown). One possible explanation came from the morphological analysis of the extracted particles. In contrast to FPV and VSV, the
structure of the SFV particles was affected upon cholesterol removal,
and the particles were penetrated by the stain (Fig. 3F).
This might indicate a perturbation of the membrane and result in
fragmentation of the particles, especially at higher CD concentrations. Instability of SFV particles has been observed before, when virions were produced in cholesterol-free insect cells (30). However, clearly
cholesterol in influenza viruses is in a detergent-resistant state,
whereas this is not the case for VSV and SFV.
The Influenza Envelope Is Enriched for
Cholesterol-dependent Detergent-insoluble Lipid
Assemblies--
The observed differences in cholesterol extractability
could result from the inclusion of raft domains into the viral
envelopes to differing extents. To test this hypothesis, viruses were
produced in 32P-labeled BHK fibroblasts and
detergent-extracted at 4 °C. Lipids from the soluble and insoluble
fractions were separated by thin layer chromatography and quantified by
PhosphorImager analysis. The major 32P-labeled lipids
detected in the BHK light membrane fraction were phosphatidylethanolamine (PE), phosphatidylcholine (PC), and
sphingomyelin (SM) (Fig. 4A).
While PE and PC were largely solublized by Triton X-100, a fraction of
the SM (41 ± 7%) was insoluble (as for the cholesterol
extractability, no significant changes in the Triton X-100 solubility
of 32P-labeled lipids were observed 6 h after viral
infection; data not shown). However, the SM solubility in the viral
envelopes showed dramatic differences. While in influenza viruses
70 ± 5% of the SM could be sedimented after detergent
extraction, only 27 ± 7 or 15 ± 8% could be pelleted from
VSV or SFV, respectively (Fig. 4A). To analyze the
cholesterol dependence of the lipid solubility, membranes or virus
particles were preextracted with 5 mM CD. We chose this
concentration because it allows the removal of 70-90% of the
cholesterol but leaves the particles intact and results only in a
minimal loss of 32P-labeled lipids (<5%) from the samples
(data not shown). Insolubility of SM was
cholesterol-dependent, since CD preextraction of the membranes resulted in increased solubilization, both in the FPV and VSV
envelopes and in the BHK membrane fraction (Fig. 4B, Table I). Also, the solubility of PC in the FPV
envelope was strongly increased upon cholesterol removal (Table I).
Therefore, cholesterol is not only required for the association of
proteins with DIGs, but generally for the formation of
detergent-insoluble lipid assemblies in a biological membrane. This
also confirms that DIGs are not complexes coalescing from intrinsically
insoluble components but that an interplay between different lipid
species is required to form a detergent-resistant membrane.
DPH Fluorescence Polarization Reveals Ordered Domains in the FPV
Envelope--
Studies with artificial lipid vesicles demonstrated that
membranes in the liquid-ordered phase and GPI-anchored proteins
incorporated into these membranes are insoluble in Triton X-100 at
4 °C (16, 17). It was therefore proposed that DIGs isolated from
biological membranes might also be derived from domains in the
liquid-ordered phase (7). Fluorescence polarization of the dye DPH
incorporated into membranes has been widely used as a measure of acyl
chain order. Lipid vesicles containing detergent-insoluble lipids
showed significantly higher fluorescence polarization than lipid
vesicles from detergent-soluble lipid mixtures, supporting the idea of ordered domains as a basis for insolubility (16). While cellular membranes are heterogenous, viral envelopes provide a unique
possibility to analyze DPH fluorescence polarization in a biological
lipid bilayer. When DPH was incorporated into the envelopes of FPV, VSV, and SFV, the influenza virus showed the highest fluorescence polarization, whereas the lowest values were obtained for VSV (Table
II). For comparison, DPPC and egg yolk PC
vesicles prepared by sonication were analyzed. Clearly, all viral
membranes show a higher fluorescence polarization than the egg yolk PC
lipid vesicles, which are in the liquid crystalline phase. At 25 °C, the DPPC vesicles are in the gel phase (Tm = 41 °C) and accordingly show a very high fluorescence polarization (0.382). The
values obtained for the FPV envelope are only slightly lower, suggesting a high degree of order in this envelope and strongly supporting the hypothesis that DIGs are derived from ordered domains in
biological membranes. Rather surprisingly, the SFV envelope showed
intermediate values between the VSV and FPV indicating a limited
"fluidity," although according to the detergent solubility of the
lipids highly ordered lipid complexes were not detected (Table I). Most
likely, this can be explained by the fact that SFV is an alphavirus,
which is structurally different from the two negative strand RNA
viruses FPV and VSV. Alphaviruses have a very high protein content, and
the icosahedral lattices of the spike and capsid proteins are connected
by strong protein-protein interactions (31, 32). These tightly packed
protein assemblies might cause the ordered structure of the membrane,
sensed by the lipid probe.
HA Incorporated into the VSV Envelope Is
Detergent-soluble--
In vitro detergent-resistant
membranes can be formed solely by lipids (16). However, it is not
understood how proteins influence these lipid assemblies. Proteins
might be required to induce or stabilize such domains in biological
membranes. We used a recombinant vesicular stomatitis virus, which
incorporates influenza virus HA into the envelope (VSV-HA (22)) to
analyze the detergent solubility of HA in the lipid environment of VSV.
BHK cells were infected with VSV-HA, and
[35S]methionine-labeled viruses were collected. The ratio
of HA to VSV G in the envelope was approximately 1:5, as previously
reported (Ref. 22, Fig. 5A).
Virions were extracted with Triton X-100 on ice and centrifuged.
Proteins in the supernatant and pellet were analyzed by autoradiography
or Western blotting with HA or VSV G antibodies (Fig. 5A).
Strikingly, both glycoproteins were largely solubilized by the
detergent. Consistently, no significant amounts of HA were floating in
density gradients after Triton X-100 extraction (Fig. 5B,
compare with Fig. 1B). Furthermore, cholesterol in the
VSV-HA envelope was efficiently solubilized in Triton X-100 (8 ± 6% insoluble), indicating that the presence of HA in a viral envelope
is not sufficient to create insoluble domains and that the lipid
environments incorporated into VSV and FPV during budding are
different.
Viruses as Model System to Study Membrane Domains--
We have
analyzed detergent-resistant lipid complexes in cellular membranes and
different viral envelopes. The use of the viral envelope as a model
membrane has several advantages over artificial lipid vesicles. Since
the envelope is derived from the cellular plasma membrane, it has a
natural lipid composition. Furthermore, bilayer asymmetry is preserved.
Finally, the membrane contains proteins that will have a profound
influence on the membrane structure. Our results demonstrate that the
three viral envelopes contain lipid raft domains to a very different
extent as judged by the detergent solubility of their constituent
proteins and lipids. For artificial lipid vesicles, it was demonstrated
that such detergent-insoluble complexes are derived from lipids in the
liquid-ordered or gel phase (15, 17). Several of our observations
suggest that influenza viruses contain lipids in a state similar to the
liquid-ordered phase. First, cholesterol, sphingomyelin, and
phosphatidylcholine in the influenza envelope are detergent-insoluble
to a large extent. Second, cholesterol is required for the insolubility
of the lipids. We have previously demonstrated that cholesterol is
required for detergent insolubility of HA in cells as well in the
isolated virions (14). The cholesterol dependence of SM and PC
insolubility shows that cholesterol is generally required for the
formation of insoluble complexes in biological membranes rather than
only for a specific interaction between HA and the sterol. This is consistent with the requirement for cholesterol for the formation of
ordered domains in the membrane (33). Finally, the DPH fluorescence polarization measurements demonstrate that acyl chains in the influenza
virus envelope are highly ordered, whereas acyl chains in the VSV
envelope are more mobile. In lipid vesicles, detergent-resistant membranes in the gel phase can be formed independently of cholesterol, simply by the addition of high amounts of sphingolipid (15). Instead,
the lipid composition of the membranes in mammalian cells appears to be
such that cholesterol is essential to promote the formation of the
liquid-ordered phase, whereas a gel phase does not exist in cell
membranes (34). However, it has been observed that mammalian cells can
remodel their lipid acyl chains when grown under sterol-limiting
conditions. Insect cells, on the other hand, remain viable when
cellular cholesterol levels are reduced to 1-3% (35). Therefore,
cells appear capable of changing their lipid composition to allow the
formation of laterally organized domains even in the absence of
cholesterol as has been observed for glycosphingolipids. However, the
properties of such cholesterol-poor domains have not been characterized
(36).
Influence of Proteins on Membrane Domain Organization--
Viral
envelopes have a very high protein:lipid ratio. It is therefore
conceivable that, in addition to the lipid composition, proteins exert
an influence on the membrane structure. Influenza virus HA contains in
its membrane-spanning domain a determinant that is required for the
association with lipid raft domains (14, 37). Also, the palmitoylation
of HA might contribute to the detergent insolubility, because a mutant
lacking the cytoplasmic tail with the palmitoylation sites (38) is
fully soluble.2 Both
determinants interact with the lipid bilayer and might affect the order
of the lipids. However, the fact that HA incorporated into the VSV
envelope was mostly soluble suggests that this membrane does contain
fewer lipids in an ordered phase and that the inclusion of HA is not
sufficient to organize such a detergent-resistant environment. Most
likely, the VSV and influenza viruses acquire membranes with different
lipid composition from the plasma membrane. The cholesterol to
phospholipid ratios in the envelopes of influenza viruses, VSV, and SFV
appear to be similar (1, 3, 39). Since sphingolipids and saturated
phospholipids are known to promote the formation of raft domains (15,
16), these lipids might be preferentially incorporated into influenza
viruses. In the future, a detailed analysis of the lipid head groups
and acyl chains in the different viral envelopes should clarify this
issue. Such an analysis might also allow the identification of
raft-promoting lipids without the use of detergents.
It has recently been shown that raft domains in the plasma membrane are
small and dispersed but that they can coalesce upon cross-linking of
their lipid or protein constituents (8, 9, 40). Similarly, large
oligomeric proteins like caveolin could stabilize rafts into
macroscopic domains in the liquid-ordered phase. Inversely, the size
and stability of the caveolin complexes are modulated by raft domains,
illustrating an interdependence of raft size and protein
oligomerization (41). Similarly, rafts could be stabilized by proteins
binding to the cytoplasmic leaflet of the domains (42). We speculate
that lipids in cellular membranes are kept in an equilibrium state in
which specific protein interactions can modulate the dynamics of rafts,
leading to coalescence or dispersion of domains in the liquid-ordered
phase, depending on the oligomeric state of the protein. Coalescence of
domains in the liquid-ordered phase could induce bending of the
membrane (43). In agreement with this view, caveolae form invaginations on the cell surface but are flattened out when cholesterol is removed
(44). Similarly, coalescence of a liquid-ordered phase might facilitate
budding of influenza viruses from the plasma membrane. In this latter
case, the membrane is curved outwards, whereas in the former it is
curved inwards. The topology of the invagination might depend on the
properties of the specific proteins involved.
Lipid Domains in Virus Assembly--
We have shown that lipids in
the influenza virus envelope are in an ordered state and less ordered
in the VSV envelope. This suggests that influenza virus is assembled
from raft domains in the plasma membrane. For VSV, fluorescence digital
microscopy has revealed the formation of domains during the budding
step. Both the glycoprotein and the matrix protein were shown to induce lateral organization of lipid within the membrane (39, 45). Consistently, the lipid composition of the VSV envelope was shown to
differ from the host cell membrane, indicating selective inclusion of
specific lipid components (2). For influenza viruses, a detailed
comparison of the lipid head groups and the acyl chains in the envelope
and the host cell membrane has not been performed. The incorporation of
raft domains into the viral envelope may explain some puzzling features
of influenza virus assembly. It has been observed that lateral
cross-linking of HA by antibodies creates large patches with raft
characteristics (40). Concomitantly, cytoplasmic proteins with raft
affinity can be recruited to the raft patches without requiring direct
interactions with the influenza spike protein (40). It has been a
surprising finding that influenza viruses in which the cytoplasmic
tails of both glycoproteins have been deleted still form infectious
progeny (46). This could be explained by connecting the influenza spike
proteins over the rafts with the cytoplasmic matrix protein. Assuming
that the spike proteins interact laterally with each other, this would
lead to exclusion of cellular glycoproteins. A role for raft domains
during virus assembly is also highlighted by the fact that the
membrane-spanning domain of influenza HA is required for both its
incorporation into the envelope and raft association (14, 37, 47).
Therefore, coalescing lipid domains might be employed at sites of
particle assembly to facilitate the exclusion of cellular proteins and to favor specific incorporation of the viral components.
-cyclodextrin results in increased
solubility of sphingomyelin and of the glycoproteins in the FPV
envelope. This biochemical behavior suggests that so-called raft-lipid
domains are selectively incorporated into the influenza virus envelope.
The "fluidity" of the FPV envelope, as measured by the fluorescence
polarization of diphenylhexatriene, was significantly lower than
compared with VSV or SFV. Furthermore, influenza virus hemagglutinin
incorporated into the envelope of recombinant VSV was largely
detergent-soluble, indicating the depletion of raft-lipid assemblies
from this membrane. The results provide a model for lipid selectivity
during virus budding and support the view of lipid rafts as
cholesterol-dependent, ordered domains in biological membranes.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
,2
-3H]cholesterol (48 Ci/mmol) in infection
medium (see below) or for 36 h with 80 µCi of
[32P]orthophosphate in G-MEM without tryptose phosphate.
To analyze membranes after viral infection, the cells were harvested
6 h postinfection and processed as above.
,2
-3H]cholesterol (48 Ci/mmol), cells were
preincubated for 4 h with 50 µCi of
[3H]cholesterol/dish in Dulbecco's modified Eagle's
medium (0.1% ethanol, 10 mM Hepes, 2 mM
glutamine, 100 units/ml, and 100 µg/ml streptomycin). Subsequently,
cells were washed twice with infection medium, and viruses were
adsorbed and collected as described above.
-cyclodextrin (CD; Sigma) were performed for
15 min at 37 °C in TN containing 5 mM EDTA and 10 µg/ml defatted BSA with gentle agitation. Subsequently, samples were cooled on ice and spun for 30 min in a TLA100 rotor (80,000 rpm, 4 °C).
RESULTS
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Fig. 1.
A, purification of viruses from BHK cell
cultures. BHK cells were infected with FPV, VSV, or SFV, and viruses
were collected for 24 h. 3 µg of protein of each preparation
obtained by gradient centrifugation were analyzed by SDS-PAGE and
silver staining. B, glycoproteins in FPV, but not VSV and
SFV, are associated with DIGs. Viral preparations were extracted with
1% Triton X-100 on ice, and extracts were adjusted to 40% Optiprep.
The extract (750 µl) was overlayered with 1.2 ml of 30% Optiprep and
250 µl of buffer, both containing 1% Triton X-100, and centrifuged
for 2 h at 55,000 rpm (TLS 55 rotor, 4 °C). Fractions were
collected from the top of the gradient (lanes
1-6) and analyzed by Western blotting with antibodies
against the viral glycoproteins.
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Fig. 2.
Cholesterol extractability in viral and
cellular membranes. A,
[3H]cholesterol-labeled virions or BHK membranes were
extracted with 1% Triton X-100 on ice and centrifuged (30 min, 80,000 rpm, 4 °C, TLA100). Relative amounts of
[3H]cholesterol in supernatant and pellet were quantified
by scintillation counting. Free counts not sedimented without detergent
extraction (3-10% of total) were subtracted from the supernatant
samples. B, [3H]cholesterol-labeled virions
were extracted with increasing amounts of methyl- -cyclodextrin for
15 min at 37 °C. Viruses were sedimented by centrifugation (30 min,
80,000 rpm, 4 °C, TLA100), and relative amounts of
[3H]cholesterol in supernatant and pellet were quantified
by scintillation counting. Free counts not sedimented without
cyclodextrin treatment (3-10% of total) were subtracted from the
supernatant samples.
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Fig. 3.
Morphological analysis of viral particles
after cholesterol depletion. FPV (A and B),
VSV (C and D), and SFV (D and
E) were incubated without (A, C, and
E) or with 5 mM CD (B, D,
and F) for 20 min at 37 °C and subsequently pelleted by
centrifugation for 30 min (80,000 rpm, 4 °C, TLA100). The
preparations were then observed in an electron microscope after
negative staining.
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Fig. 4.
Triton X-100 solubility of lipids in FPV,
VSV, SFV, and BHK membranes. A, BHK cells were labeled
with [32P]orthophosphate, and a light membrane fraction
or radiolabeled viruses were isolated. Samples were treated with 1%
Triton X-100 in TNE buffer on ice and centrifuged (30 min, 80,000 rpm,
4 °C, TLA100). Lipids from the supernatant (S) and the
pellet (P) fractions were extracted and analyzed by thin
layer chromatography. Labeled lipids were detected by autoradiography
and quantified by PhosphorImager analysis. The positions of PE, PC, and
SM are indicated. B, for cholesterol depletion, the
membranes were treated with 5 mM cyclodextrin, pelleted,
subsequently extracted with Triton X-100, and analyzed as in
A.
Triton X-100 solubility of cholesterol (Chol), SM, PC, and PE in BHK
membranes and viral envelopes, without or after (+CD) cholesterol
depletion by 5 mM cyclodextrin
Polarization of DPH fluorescence in viruses or lipid vesicles at
25 °C
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Fig. 5.
Triton X-100 extractability of HA in the
envelope of VSV. A, VSV-HA virions labeled with
[35S]methionine were produced in BHK cells, purified, and
subsequently extracted with Triton X-100 on ice. Proteins of the
supernatant (S) and pellet (P) fractions were
analyzed by autoradiography (35S) or Western blotting with
antibodies against HA ( HA) or VSV G (
VSVG).
B, VSV-HA virions were extracted with 1% Triton X-100 on
ice, and extracts were adjusted to 40% Optiprep. The extract (750 µl) was overlayered with 1.2 ml of 30% Optiprep and 250 µl of
buffer, both containing 1% Triton X-100, and centrifuged for 2 h
at 55,000 rpm (TLS 55 rotor, 4 °C). Fractions were collected from
the top of the gradient (lanes 1-6) and analyzed
by Western blotting with antibodies against HA or VSV G,
respectively.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Kim Ekroos for cell culture, Drs. Marcus Furch and Andreas Herrmann for help and hospitality during the DPH polarization measurements, Dr. J. K. Rose for the recombinant VSV-HA virus, Dr. Derek Toomre for preparation of a VSV-HA stock, and Dr. Jacomine Krijnse-Locker for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 352 and the European Commission TMR program.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: European Molecular Biology Laboratory, Postfach 10 2209, D-69012 Heidelberg, Germany. Tel.: 6221-387-334; Fax: 6221-387-512; E-mail: Simons{at}emblHeidelberg.de.
The abbreviations used are:
GPI, glycosylphosphatidylinositol; DIG, detergent-insoluble
glycolipid-enriched complex; HA, hemagglutinin; FPV, fowl plague virus; VSV, vesicular stomatitis virus; SFV, Semliki Forest virus; BHK, bovine
hamster kidney; SM, sphingomyelin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PAGE, polyacrylamide gel electrophoresis; CD, methyl--cyclodextrin; DPH, diphenylhexatriene; DPPC, dipalmitoylphosphatidylcholine.
2 P. Scheiffele and K. Simons, unpublished data.
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REFERENCES |
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