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Journal of Virology, July 1999, p. 6104-6110, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Dengue Virus Type 1 Nonstructural Glycoprotein NS1 Is Secreted
from Mammalian Cells as a Soluble Hexamer in a
Glycosylation-Dependent Fashion
Marie
Flamand,1,*
Françoise
Megret,1
Magali
Mathieu,2,
Jean
Lepault,3
Félix A.
Rey,2, and
Vincent
Deubel1
Unité des Arbovirus et Virus des
Fièvres Hémorragiques, Institut Pasteur, 75724 Paris Cedex
15,1 and Laboratoire d'Enzymologie et
de Biochimie Structurales, CNRS UPR 9063,2 and
Centre de Génétique Moléculaire, CNRS UPR
9061,3 91198 Gif-sur-Yvette Cedex, France
Received 21 December 1998/Accepted 1 April 1999
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ABSTRACT |
Nonstructural glycoprotein NS1, specified by dengue virus type 1 (Den-1), is secreted from infected green monkey kidney (Vero) cells in
a major soluble form characterized by biochemical and biophysical means
as a unique hexameric species. This noncovalently bound oligomer is
formed by three dimeric subunits and has a molecular mass of 310 kDa
and a Stokes radius of 64.4 Å. During protein export, one of the two
oligosaccharides of NS1 is processed into an
endo-
-N-acetylglucosaminidase F-resistant complex-type
sugar while the other remains of the polymannose type, protected in the
dimeric subunit from the action of maturation enzymes. Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin. These results, together with observations related to the absence of secretion
of NS1 from Den-infected insect cells, suggest that maturation and
secretion of hexameric NS1 depend on the glycosylation status of the
host cell.
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TEXT |
Dengue (Den) is one of the most
threatening mosquito-borne viral diseases of humans. It is widely
spread in the tropical and subtropical areas of the world, and its
incidence, in terms of morbidity and mortality, has increased
dramatically over the past 20 years. The etiological agent belongs to
the flavivirus genus of the family Flaviviridae, which
comprises other major human pathogens such as Japanese encephalitis,
yellow fever, and tick-borne encephalitis viruses (3, 35).
Flaviviruses are enveloped, single-stranded, positive-sense RNA viruses
formed by three structural proteins. Protein C (capsid), enclosing the
genome, forms a nucleocapsid that is surrounded by a lipid bilayer in
which are anchored proteins M (membrane) and E (envelope). The genome
is approximately 11 kb long and contains a single open reading frame
encoding a polyprotein precursor of about 3,400 amino acid residues.
Individual viral proteins are generated from this precursor by the
action of cellular and viral proteases (44). The three
structural proteins derive from the N-terminal part of the polyprotein
and are followed by seven nonstructural proteins: NS1, NS2A/2B, NS3,
NS4A/4B, and NS5. The two cytosolic proteins, NS3 and NS5, have been
identified as the viral protease/helicase and polymerase, respectively
(44); the roles of the other nonstructural proteins remain
to be determined.
Glycoprotein NS1, present in all flaviviruses, appears to be essential
for virus viability. This protein contains two conserved N-glycosylation sites and 12 invariant cysteine residues (3, 35). NS1 is inserted into the lumen of the endoplasmic reticulum (ER) via a signal peptide that is cleaved cotranslationnally by the
action of a cellular signalase to generate the N terminus of the
protein (3). NS2A is released from the C-terminal end of NS1
by an ER resident host protease (15). Although NS1 does not
contain an identifiable membrane-anchoring domain, NS1 becomes associated with membranous components upon dimerization (52, 53). Recently, intracellular NS1 was shown to be involved in the
early steps of viral replication (31, 32, 36, 37, 51), in
agreement with its retention in intracellular organelles of the
infected cell (33, 40) and its ability to interact with
membranes (8, 16, 18, 52). However, intracellular NS1 faces
the lumen of the ER and may play a role only indirectly in the
replication process. Dimerization is also a prerequisite for NS1
protein export along the secretory pathway to the plasma membrane
(41), where it remains as the unique viral resident protein
of the infected cell surface (19, 45, 50). In mammalian cells, but not in insect cell lines derived from Aedes
albopictus which support flavivirus infection (33, 52),
part of transported NS1 is released into the extracellular milieu.
Extracellular NS1 is secreted either as a soluble protein, which may be
present in a higher oligomeric form than a dimer (4, 5), or
in association with microparticles but not with virions (14, 16,
29, 30, 33). In addition, NS1 has been found circulating in sera
from Den virus-infected patients (13), suggesting that
secretion of NS1 may be an important event in flavivirus infection in
the human host.
In this study, we were interested in the biochemical characterization
of the major extracellular form of NS1 released from Den virus type 1 (Den-1)-infected Vero cells. Vero cells were infected at a multiplicity
of infection (m.o.i) of 1 focus forming unit of Den-1, strain FGA/89
(7), per cell. The supernatant was harvested at 5 days
postinfection, clarified by centrifugation to eliminate cell debris,
and treated with 7.5% polyethylene glycol (PEG) 6000 to precipitate
particles, such as virions and any remaining membraneous components.
The PEG-precipitable fraction contained less than 5% of the total
amount of NS1 released in the extracellular fluid. NS1 found in this
fraction could represent a microparticulate form of NS1, as has been
noted for Japanese encephalitis virus infection of Vero cells
(33). Soluble NS1, accumulating at concentrations of 5 to 10 µg/ml (as determined by enzyme-linked immunosorbent assay [data not
shown]), was purified from the PEG-clarified supernatant by
immunoaffinity chromatography with a column prepared with the anti-NS1
Den virus-specific monoclonal antibody (MAb) 3D1.4, kindly provided by
A. Falconar. For this purpose, antibodies contained in a MAb-enriched
ascitic fluid were recovered by ammonium sulfate precipitation and
ion-exchange chromatography (DEAE-Trisacryl M matrix; Sepracor) and
further coupled to CNBr-activated Sepharose 4B (Pharmacia), according
to the manufacturer's instructions. The PEG-treated supernatant was
passed over the immunoadsorbant at a low flow rate, and bound protein
was subsequently eluted at a basic pH of 11.2, conditions that were
previously shown to preserve the dimeric state of the protein
(14). The eluted protein was concentrated by
ultrafiltration, the buffer was exchanged for phosphate-buffered saline
(PBS), and the sample was subjected to size exclusion chromatography
(SEC) to determine both the apparent molecular mass and the degree of
homogeneity of the purified product (Fig.
1). One major peak that solely contains
the NS1 protein is apparent on the chromatogram, as detected by
Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (Fig. 1a). NS1 exhibits a Stokes radius
(RS) of 64.4 Å (Fig. 1b), corresponding to an
apparent molecular mass of about 300 kDa. In SDS-PAGE, however, only
the SDS-resistant dimer of NS1, migrating with an apparent molecular
mass of 80 kDa, can be visualized (Fig. 1a), and it is converted into
the monomer (50 kDa) by heat denaturation. No species with an apparent
molecular mass of 300 kDa can be detected on the gel (Fig. 1a),
suggesting that if a higher oligomeric species is initially present, it
dissociates into its dimeric subunits in SDS-PAGE. We used analytical
ultracentrifugation to determine unequivocally the molecular mass of
purified soluble NS1 (data not shown), since a high
RS value could be due to an elongated form of
the molecule with a smaller mass than expected. The sedimentation
coefficient obtained by this method for NS1 recovered from the SEC
column is 11.2S (data not shown). This, together with an
RS value of 64.4 Å, yields a molecular mass for NS1 of 310 kDa, consistent with the presence of a hexamer. If a
hexamer, the molecular mass of NS1, estimated from its amino acid
sequence, would be 240.9 kDa. Since each monomer is glycosylated at two
positions, we can roughly add 8 to 10 kDa per monomer (see Fig. 4a),
reaching a value of about 300 kDa as an upper limit. Given that
hydration effects usually tend to increase the molecular mass
determined experimentally, the results obtained are concordant with the
existence of a hexamer of NS1, as was suggested previously for NS1 from
tick-borne encephalitis virus (4, 5).
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FIG. 1.
Analysis of immunoaffinity-purified soluble
extracellular NS1 by SEC. (a) Immunoaffinity-purified NS1 was submitted
to SEC on an S300 gel filtration column, and the single peak was
concentrated by ultrafiltration and analyzed on a 4 to 20% gradient
SDS-polyacrylamide gel stained by Coomassie blue. The protein was
either unheated or heated to 95°C for 3 min prior to electrophoresis.
(b) By comparison with protein standards used to calibrate the SEC
column, the NS1 protein exhibited a Stokes radius
(RS) of 64.4 Å, corresponding to an apparent
molecular mass of 300 kDa.
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Purified NS1 is a unique hexameric species.
To further
characterize the hexameric form of NS1, we carried out chemical
cross-linking on purified NS1 (Fig. 2a)
with the bifunctional cross-linker dimethylsuberimidate (DMS) (Pierce). DMS, solubilized in 200 mM triethanolamine, was added to a cold solution of NS1 in 50 mM triethanolamine (pH 8.5)-100 mM NaCl, and the
reaction was carried out for 1 h at room temperature. The
resulting products were subjected to SDS-PAGE (4 to 20% gradient gel)
and stained with Coomassie blue (Fig. 2a). At 0.5 mM DMS, we observed
the formation of tetrameric intermediates and cross-linked hexamers,
the latter species accumulating with increasing concentrations of DMS,
as would be expected for a hexameric species formed by dimeric building
blocks. Boiling the sample treated with the highest concentration of
DMS prior to electrophoresis generates species ranging from monomers to
hexamers. Hexamers, as opposed to dimeric subunits, have been shown to
be sensitive to very low concentrations of SDS (5). We
analyzed the nature of the interfaces between dimers by subjecting
hexameric NS1 to the action of the nonionic detergent
n-octylglucoside (nOG) (Fig. 2b). Purified NS1 was incubated overnight at 37°C in the absence or presence of nOG (0.5 or 1%) and
subsequently treated with 25 mM DMS. The resulting products were
separated on a 4 to 20% acrylamide gradient gel, transferred to a
nitrocellulose membrane, and revealed with MAb 3D1.4. In the samples
that did not contain nOG, bands corresponding to dimers, tetramers, and
hexamers were produced after DMS treatment, although it can be noted
that the DMS-treated dimer was poorly recognized by MAb 3D1.4 compared
to the cross-linked hexamer or to the noncovalently bound dimer. The
hexamer was found to be partially dissociated by 0.5% nOG, as shown by
the decrease in hexameric species and the concomitant increase in the
amount of tetrameric intermediates. Levels of tetramers and hexamers
were undetectable after treatment of the purified product with 1% nOG
(Fig. 2b), suggesting that the protein is converted into dimers under
these conditions. This suggests that the interfaces stabilizing the
dimeric subunits are essentially weak hydrophobic interactions.
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FIG. 2.
Purified extracellular NS1 is a hexamer that can be
converted to its dimeric subunit in the presence of the nonionic
detergent nOG, as demonstrated by chemical cross-linking. (a)
Subsequent to SEC, the protein was concentrated to 0.5 mg/ml by
ultrafiltration and treated with final concentrations of 0, 0.5, 5, and
50 mM DMS. The resulting products were placed in nonreducing Laemmli
sample buffer, separated on a 4 to 20% gradient acrylamide gel, and
stained with Coomassie blue. One sample, treated with 50 mM DMS, was
treated for 3 min at 95°C prior to electrophoresis to dissociate
noncovalently linked oligomers. (b) Purified NS1 was treated overnight
at 37°C with 0, 0.5, or 1% nOG and submitted or not submitted to 25 mM DMS for 1 h. Proteins were separated without heat denaturation
on a 4 to 20% gradient acrylamide gel and detected by immunoblotting
with MAb 3D1.4.
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We examined the homogeneity of our purified NS1 preparation by electron
microscopy, both by negative staining (Fig.
3a) and
by rotary shadowing (data not
shown). In both cases, we observed
a homogeneous distribution of
isometric particles of a diameter
of 11 ± 2 nm. Most of the
particles did not display any identifiable
symmetry. A few of them,
however, seemed to show a twofold rotational
symmetry (large circle in
Fig.
3a) or a threefold rotational symmetry
(small circle). The fact
that the electron micrographs show twofold
and threefold rotational
symmetries for some of the particles
suggests that the NS1 hexamer has
32-point symmetry. Since the
particles are oriented randomly on the
electron microscope grid,
it is expected that symmetry will be apparent
only for those particles
that happen to lie with one symmetry axis
normal to the plane
of the grid. There is evidence of a space between
subunits in
the hexamer, in particular in particles seen along the
twofold
rotational axis.
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FIG. 3.
Hexameric NS1 appears as a unique species by electron
microscopy and SAXS analysis. (a) Negatively stained sample of purified
NS1 at 0.2 mg/ml observed by electron microscopy at 80 kV. The image
shows a field containing NS1 particles in random orientations.
Dimensions are quite homogeneous, with a diameter of roughly 11 nm.
Occasionally, particles lying with a twofold (large circle) or a
threefold (small circle) axis of symmetry perpendicular to the plane of
the figure are seen. Bar, 0.1 µm. (b) Guinier plot of purified NS1 at
a protein concentration of 6.2 mg/ml. The radius of gyration (see text)
calculated from the slope, Rg, is 5 ± 0.1 nm. Note that the curve is linear from very small angles (lower
s values), showing that there is a single form of NS1
protein in solution. The molecular mass estimated from extrapolation to
s = 0 is 300 ± 50 kDa.
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To confirm the monodispersity of the protein in solution, we tested our
purified NS1 preparation by small-angle X-ray scattering
(SAXS) on a
synchrotron source (Fig.
3b). The detection instrument
(
6),
the data acquisition system (
2) and the thermostated
cell
under vacuum (
10) have been described previously. This
method provides a very sensitive test to check the homogeneity
of a
given macromolecule in solution. In addition, it provides
the radius of
gyration,
Rg, of the molecule (which is the
second
moment of the electron density distribution of the particle),
as
well as the molecular weight. At small angles, the scattering
pattern
of a monodisperse solution can be approximated by a Gaussian
curve the
width of which yields the radius of gyration of the
macromolecule
(
21). A plot of intensity as a function of the
square of the
scattering vector
s,
s = (2sin
)/
(where 2
is
the scattering angle and
is the wavelength of the
incident beam),
called "Guinier plot," should thus give a straight
line if the
solution is monodisperse. The slope provides the
Rg, and extrapolation
of this straight line to
s = 0 provides the total intensity,
I(0),
scattered in the direction of the incident beam. This value is
proportional to the molecular mass of the protein and to the
concentration
of the protein in solution. The data were collected at
different
protein concentrations by using synchrotron radiation at a
wavelength
of 1.488 Å and a sample-to-detector distance of 2,580 mm.
Figure
3b shows the Guinier plot of NS1 at a concentration of 6.2 mg/ml
in solution. The scattering curve is indeed Gaussian, even at
very
small angles. This plot proves that purified NS1 is a unique
species
and does not form any oligomeric structures larger than
a hexamer. The
value we obtained for
Rg, 5 ± 0.1 nm, is
in agreement
with the diameter found by electron microscopy and with
the Stokes
radius,
RS, obtained by SEC. The
molecular mass of NS1 was estimated
by using a reference solution of
rotavirus protein VP6 (which
forms a trimer of 120 kDa) at defined
concentrations and yielded
a value of roughly 300 kDa, in agreement
with the data obtained
from analytical centrifugation and SEC. The
fairly large radius
of gyration of hexameric NS1
(
Rg of 5 nm) indicates that the electron
distribution is away from the center of the particle. A compact
spherical molecule with a uniform electron density distribution
and an
identical
Rg would have a molecular mass of
10
6 Da, more than three times the actual mass of NS1. All
of this
is in agreement with electron micrographs that show a space
between
subunits down the twofold symmetry axis of the particle, as
explained
above. This case is reminiscent of the hexameric enzyme
aspartyl
transcarbamylase, which has a molecular mass of 300 kDa and
32-point
symmetry. The relaxed state of this allosteric enzyme has an
Rg of 5 nm (
22) and contains a big
cavity in the center, as observed
by X-ray crystallography
(
23). The presence of such cavities
in the structure of
hexameric NS1 would thus explain the discrepancy
we find between the
second moment of electron density distribution
and the molecular weight
of the
hexamer.
Maturation of NS1 carbohydrates is site dependent and may be
required in the secretion process.
The glycosylation status of
extracellular NS1 was determined by using the endoglycosidases
endo--N-acetylglucosaminidase H (endo H),
endo--N-acetylglucosaminidase F (endo F), and
peptide-N-(acetyl--glucosaminyl)asparagine amidase (NPGase F) (Fig.
4a). Purified NS1 was incubated for 3 min
at 95°C in the presence of 1% SDS prior to digestion to convert dimeric subunits into monomers. It was then necessary to add 1% Nonidet P-40 to the sample before adding endoglycosidase in order to
avoid inactivation of the enzyme by SDS. At the end of the 1-h
incubation period at 37°C, samples were subjected to SDS-PAGE in
nonreducing Laemmli sample buffer and detected by immunoblotting with
anti-NS1 MAb 3D1.4 (Fig. 4a). Although NPGase F is able to cleave both
sugars, yielding the unglycosylated form of NS1 [NS1(p)0] (Fig. 4a), endo H and endo F cleave only one of the two glycans, leaving the complex-type sugar (NS1c) attached to the polypeptide chain
(Fig. 4a). The experiment with endo F was also carried out at an acidic
pH of 5.5, which was reported to be more favorable to the action of the
enzyme, but no further digestion was observed (data not shown). These
digestion profiles show that the observed microheterogeneity of
extracellular NS1 on SDS-PAGE is due exclusively to the complex-type
carbohydrate moiety. More importantly, they suggest that only one sugar
is modified into a multibranched
at least triantennarycomplex chain
(47), the second glycan being maintained as a high-mannose
type. This indicates that the latter is protected from the action of
enzymes of N-glycan biosynthesis during transport of the protein to the
cell surface. This particular feature has been described for the NS1
glycoprotein of several flaviviruses (8, 33, 42, 53).
Examples of site-specific glycosylation, in which maturation of the
glycan depends on the particular environment in the three-dimensional
structure of the protein or in the quaternary structure of a protein
complex, have been reported (25, 26, 34, 38).
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FIG. 4.
Analysis of the nature and accessibility of the two
oligosaccharides of extracellular NS1. (a) Extracellular NS1 was mock
treated (mt) or treated for 1 h at 37°C with endo H (H), endo F
(F), or NPGase F (N) in its monomeric form. Samples were placed in
nonreducing Laemmli sample buffer and separated by SDS-12% PAGE, and
the resulting products were detected by immunoblotting after transfer
to nitrocellulose. "p" indicates the presence of a polymannose-type
oligosaccharide; "c" indicates a complex-type oligosaccharide on
the protein. NS1(p)0 is the deglycosylated form of the
protein. (b) Purified NS1 was either mock treated or treated with
NPGase F for 1 h at 37°C in 10 mM sodium phosphate buffer, pH
7.5. One sample was further treated by endo H after the pH was
readjusted to 5.5 with 50 mM citrate buffer. All samples were subjected
to SDS-12% PAGE with or without preliminary heat denaturation in
nonreducing Laemmli sample buffer.
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Accessibility of the carbohydrate side chains on the oligomeric
structures of NS1 was assessed in vitro by NPGase F, the reactivity
of
which appears to be highly dependent on the free exposure of
its
substrate (
47,
48). Purified NS1 was treated with NPGase
F
in its native state or in the presence of 1% nOG, known from
the
experiment reported in Fig.
2b to disrupt interactions between
the
dimeric subunits. At the end of the 1-h incubation period,
samples were
placed in nonreducing Laemmli buffer and either boiled
or not boiled
prior to analysis by Western blotting (Fig.
4b).
The polymannose-type
sugar (designated p) is not accessible to
the enzymatic cleavage of
NPGase F when the protein is present
in a dimeric form (NS1p) (Fig.
4b)
and is hydrolyzed only when
the protein is subsequently subjected to
endo H, which still recognizes
its polymannose-rich substrate under
these conditions [NS1(p)
0]
(Fig.
4b). We observed a
similar profile for the intracellular
form of the protein (data not
shown), suggesting that the dimeric
association may by itself protect
one of the N-glycans against
further maturation in the Golgi. It
corroborates previous studies
showing that a mutation at the second
glycosylation site on the
polypeptide chain, accordingly lacking the
polymannose-type sugar,
significantly reduced dimer stability and
secretion of the protein
(
42). Interestingly, NPGase F
appeared to be unable to reach
either of its two substrates on native
hexameric NS1 (NS1pc) (Fig.
4b). The fact that the complex-type
carbohydrate moiety has acquired
endo F resistance during transport of
NS1 along the secretory
pathway favors the view of prolonged
accessibility of this particular
oligosaccharide to the specific
enzymes of glycan biosynthesis,
as opposed to the polymannose-type
glycan. When processing of
the complex glycan is completed, it could
become somewhat cryptic
after a change in protein conformation. This
would suggest that
the complex-type sugar may be involved in triggering
or stabilizing
a mature form of the hexamer, a role similar to that of
the polymannose-type
sugar in the dimeric association (
42).
In favor of this hypothesis
are the results obtained with the
N-glycosylation inhibitors swainsonine
and deoxymannojyrimicin
(DMJ).
To evaluate the importance of maturation of the complex sugar chain on
the processing and secretion of NS1, infected cells
were treated with
glycosylation inhibitors that specifically block
steps in the mannose
trimming of N-glycans in the ER or Golgi
(
11,
12,
24) but
that do not interfere with the preceding
glucose trimming required for
chaperone-mediated protein folding
(
49). Swainsonine blocks
Golgi mannosidase II and leads to the
generation of hybrid glycans
instead of complex-type sugars; DMJ
inhibits mannosidase I and gives
rise to polymannose-rich carbohydrates.
After treatment with the
glycosylation inhibitors, the presence
of radiolabeled extracellular
NS1 was analyzed by immunoprecipitation
of proteins from total culture
fluids or from supernatants recovered
after 7.5% PEG precipitation and
containing only the soluble form
of NS1 (Fig.
5a). Experiments were carried out with
polyclonal
ascitic fluid (PAb) directed against Den virus antigens, MAb
13A1
(not shown), or MAb 3D1.4 (not shown), and immunoprecipitated
products were boiled prior to electrophoresis. When the PAb was
used on
total supernatants, two proteins other than NS1 were precipitated
(Fig.
5a). These proteins migrate at molecular masses of about
20 and 60 kDa
and are neither present in the MAb immunoprecipitates
nor in the PEG
supernatants precipitated with the PAb. The natures
of the two proteins
are unknown, but they could be structural
proteins C (or prM) and E. The molecular weight of NS1 released
from swainsonine-treated cells is
slightly lower than that of
NS1 in mock-treated supernatants, according
to a hybrid-type structure
replacing the complex-type sugar. In
DMJ-treated supernatants,
NS1 migrates with a significantly lower
molecular weight and is
a homogeneous species in SDS-PAGE, in agreement
with the presence
of polymannose-rich glycans only.
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FIG. 5.
Altered secretion of NS1 from infected cells treated
with N-glycan processing inhibitors. (a) Cells were infected with Den
virus at an m.o.i. of 3. At 7 h postinfection, swainsonine (Sw)
and DMJ were added for 20 h at concentrations of 5 µM and 1 mM,
respectively. Proteins were then radiolabeled for 3.5 h;
immunoprecipitated with Den virus-specific PAb from cell lysates, total
supernatants (spnt), or PEG-treated supernatants; separated by
SDS-PAGE; and analyzed by autoradiography. (b) NS1-related radioactive
signals from PEG-treated supernatants immunoprecipitated with the PAb
were quantified directly from the gels with a PhophorImager. Average
values of at least three independent experiments and corresponding
standard deviations are reported on the graph. mt, mock treatment.
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Both glycosylation inhibitors appear to have a down-regulating effect
on secretion of NS1 but not of the two others proteins
recognized by
the PAb. This effect was quantified by measuring
radioactive signals
with a PhosphorImager. The average value of
at least three independent
experiments, in which the amount of
soluble NS1 released from
mock-treated Den virus-infected cells
was defined as 100%, is reported
in Fig.
5b. If secretion of soluble
NS1 is quantified from supernatants
recovered after PEG precipitation
and is immunoprecipitated by the PAb
(Fig.
5b), the intensity
of the signals is reduced by 35% in
swainsonine-treated samples
and by 65% in DMJ-treated samples. Similar
results can be obtained
after immunoprecipitations of the same samples
with the two MAbs
(data not shown). Thus, both inhibitors seem to
repress the secretion
of soluble NS1 from Den virus-infected cells
(Fig.
5), the inhibitory
effect being the highest when N-glycans are
maintained in a polymannose-type
structure in the presence of DMJ. The
variation in the amount
of NS1 secreted under the different conditions
of cell treatment
does not seem to be due to a difference in the level
of protein
expression, as radioactive signals that are measured from
cell
lysates immunoprecipitated either with the PAb (Fig.
5a) or with
MAbs (not shown) vary in a range lower than 5%. The smaller amount
of
extracellular soluble NS1 in the supernatants of swainsonine-
or
DMJ-treated cells may be a direct effect on the cell secretory
machinery, but the proteins P20 and P60, recognized by the PAb
(Fig.
5a), appear not to be affected by the inhibitors; examples
where the
secretion of viral or cellular proteins was not impaired
by inhibition
with swainsonine or DMJ have been reported (
1,
20,
43,
46,
54). This down-regulation could be the result
of specific
targeting of intracellular NS1 to the degradation
pathway or reduced
stability of the protein secreted in the presence
of inhibitors.
Alternatively, the oligosaccharides of NS1 could
be important for the
protein to reach a mature quaternary conformation
(
26,
38)
and/or in regulating protein transport along the
secretory pathway
(
9,
17).
Whatever the mechanism involved, proper processing of N-glycans appears
to be essential for the NS1 protein to be efficiently
matured and
released from Den virus-infected cells. Concordant
with these
observations, we (data not shown) and others (
33,
52) have
noted the absence of NS1 secretion in insect cell lines
derived from
A. albopictus. As oligosaccharides synthesized in
insect
cells may accumulate as polymannose-rich structures (
27,
28), similar to those produced in DMJ-treated cells, we propose
that the discrepancy in glycoconjugate biosynthesis between mammalian
and invertebrate cells may be one of the host-specific factors
finely
tuning the maturation, transport, and secretion of NS1.
Interestingly,
previous experiments showed that ablation of the
complex-type
oligosaccharide correlated with a decrease in mouse
neurovirulence
(
36,
39), emphasizing the fact that the complex-type
sugar
may have a specific role to play in the formation of a mature
extracellular form of NS1 which could specifically contribute
to
virulence. Identifying the site of NS1 hexamer formation, the
signals
triggering cell membrane association or release, and at
which steps the
carbohydrate moieties may be involved remains
a prerequisite for the
more general concern of the biological
relevance of the NS1 protein in
flavivirus infection and
pathology.
| |
ACKNOWLEDGMENTS |
We thank Andrew Falconar for the generous gift of 3D1.4 hybridoma
cells, Jean-Claude Mazié for preparing 3D1.4 MAb-containing ascitic fluid, Patrice Vachette for the SAXS experiment (beam line D24;
LURE, Orsay, France), Gérard Batelier for the analytical ultracentrifugation study, Hany Goudran-Botros for early help in
characterizing NS1 by SEC, Robert Putnak for providing MAb 13A1, Franz
Heinz for advice on the DMS cross-linking experiments, Friedrich Piller
for advice on the use of glycosylation inhibitors, and Michele Wien for
helpful comments on the manuscript.
This work was supported in part by grant RG-509 from the Human
Frontiers Science Program (RG-509) to F.A.R. M.M. was the
recipient of an EMBO long-term postdoctoral fellowship during this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité des
Arbovirus et Virus des Fièvres Hémorragiques, Institut
Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: (33) 1 40 61 35 63. Fax: (33) 1 40 61 37 74. E-mail:
mflamand{at}pasteur.fr.
Present address: Laboratoire de Génétique des Virus,
CNRS UPR 9053, 91198 Gif-sur-Yvette Cedex, France.
| |
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