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,§1
* Department of Paediatrics, Imperial College School of Medicine, Norfolk Place, London W2 1PG, U.K.;
MRC Clinical Sciences Centre, Imperial College School of Medicine, London W12 0NN, U.K.;
Department of Microbiology and Parasitology, University of Queensland, Brisbane, Queensland 4072, Australia; and
§ Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital, Brisbane, Queensland 4029, Australia
1Correspondence: SASVRC, Department of Microbiology and Parasitology, University of Queensland, St. Lucia QLD 4072, Australia. E-mail: p.young{at}mailbox.uq.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: flavivirus • dengue • viral nonstructural proteins • GPI • antibody
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Infection with dengue virus can result in a
self-limiting febrile illness (dengue fever) or in severe disease with
abnormalities in hemostasis and vascular permeability (dengue
hemorrhagic fever-dengue shock syndrome or DHF/DSS). Although there is
now considerable evidence that precirculating anti-dengue antibody is
the predominant risk factor predisposing individuals to DHF/DSS, the
molecular mechanisms of pathogenesis remain unknown (2)
.
Shock is a result of endothelial cell dysfunction rather than cell
death (3)
and is believed to be a consequence of induction
of vasoactive mediators, particularly from infected
monocytes/macrophages (4)
, which are the primary sites of
dengue replication.
Flaviviruses are enveloped, positive-strand RNA viruses encoding a
single 10–11 kb polycistronic message. Co- and post-translational
processing gives rise to three structural and seven nonstructural
proteins, in the gene order
5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5–3' (5)
(Fig. 1A
). Cleavage of the polyprotein to generate the individual
proteins is mediated either by host cell enzymes, signal peptidase
(8
9
10)
, and furin (11)
or a virus-encoded
two-component proteinase, NS2B/NS3 (10
, 12
, 13)
. The
exception is cleavage at the NS1-NS2A junction, which is mediated by an
unidentified enzyme located within the endoplasmic reticulum (ER)
(14)
. NS1 (nonstructural protein 1), which has been shown
to be involved in viral RNA replication (15
, 16)
, is
initially translocated into the ER via a hydrophobic signal sequence
that is encoded by the carboxyl terminus of E (17)
. In the
ER, two N-linked high-mannose carbohydrate moieties are
attached, followed by rapid dimerization. Membrane association then
occurs even though hydropathy plots predict that NS1 is essentially
hydrophilic and lacks a membrane-spanning domain (18
, 19)
(Fig. 1B
). Subsequent transit through the Golgi results in
one of the two high mannose carbohydrates on each NS1 molecule being
trimmed and further processed to a complex form.
|
The sequence immediately downstream of NS1, in the
NH2 terminus of NS2A, comprises a hydrophobic
domain (Fig. 1B
) that is similar to carboxyl-terminal
sequences present in nascent eukaryotic proteins prior to processing to
a glycosyl-phosphatidylinositol (GPI) -anchored form (20)
.
A diverse range of eukaryotic cell-surface proteins are known to be
anchored in the plasma membrane by attachment to GPI (20
, 21)
, a complex glycolipid structure that is highly conserved
among all eukaryotic cells (22)
. GPI anchor addition
involves cleavage of a hydrophobic carboxyl-terminal signal sequence in
the ER, followed by covalent attachment of a preformed GPI precursor
(23)
. GPI-linked proteins are then targeted to the plasma
membrane. The precise role of GPI anchors remains uncertain, but they
are implicated in signal transduction (24
25
26)
and confer
on proteins the ability to transfer between plasma membranes of
different cells (27)
. GPI-anchored proteins that are
transferred remain functional (28
, 29)
and retain the
capacity for signal transduction (30)
.
The present study examined whether the dengue virus NS1 glycoprotein can be processed to a GPI-linked form. Analysis of stable HeLa cell transfectants constitutively expressing NS1 showed that the hydrophobic domain at the NH2 terminus of NS2A could function as a GPI anchor addition sequence for NS1. The expression of a GPI-anchored form of NS1 in dengue virus-infected cells was then confirmed. Last, based on observations made with other GPI-anchored proteins, binding of anti-NS1 antibody to cell surface-expressed NS1 was shown to induce signal transduction, as assessed by tyrosine phosphorylation of cellular proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Anti-NS1 monoclonal antibodies (mAbs) 1H7.4,
5H5.4, 1A12.3, and 1E2.3 were obtained and characterized as described
(32)
. For cell signaling experiments, antibodies were
first purified from ascites preparations by affinity chromatography
using protein A Sepharose (Pharmacia, Piscataway, N.J.). Anti-CD55 mAb
1H4 was a gift from D. Shafren (University of Newcastle, Australia).
Anti-E mAb 4G2 was prepared from a hybridoma obtained from the American
Type Culture Collection. Dengue virus serotype 2 stocks were derived by
infecting C6/36 cells (multiplicity of infection 0.001) and collecting
the medium at the onset of visible cytopathic effects (usually day 4
postinfection). The medium was then clarified (4000 g at
4°C for 15 min) and aliquots were stored at -80°C.
cDNA constructs and transfections
cDNA was generated by reverse-transcriptase polymerase chain
reaction amplification using total RNA extracted from dengue 2 virus
(strain PR-159) -infected Vero cells as template. The NS1 sequence of
our isolate of dengue 2 strain PR159 has been reported previously
(33)
. Two constructs were made, each containing the NS1
coding sequence preceded by the coding sequence for the last 28 amino
acids of E (Fig. 1C
), which is sufficient for translocation
of the NS1 protein into the ER (17)
. The two constructs
differed at the carboxyl terminus, encoding either NS1 alone (NS1) or
NS1, followed by the first 26 amino acids of NS2A (NS1H) (Fig. 1C
). Each construct was cloned into the BamHI
site of the bicistronic vector pCIN4 (provided by S. Rees, Glaxo
Wellcome, Amersham, U.K.), in which both the construct and neomycin
phosphotransferase gene were under the transcriptional control of a
single CMV promoter but separated by the encephalomyocarditis virus
internal ribosome entry site. Cloned cDNA nucleotide sequences were
confirmed by automated sequencing (ABI). HeLa cells
(1x106) were transfected with 1 µg of each
construct by liposome-mediated transfection (Lipofectamine, Life
Technologies, Inc., Grand Island, N.Y.), and single G418-tolerant
colonies were picked and expanded to establish stable cell lines.
Infection with dengue virus
Growing cells were washed twice in serum-free medium, then
incubated at 37°C for 30 min with dengue 2 virus (strain New Guinea
C) at a multiplicity of infection of 0.01. The inoculum was then
removed and replaced with growth medium. Metabolic labeling and flow
cytometry (see below) were performed on day 2 and day 3 postinfection,
respectively.
PI-PLC treatment
Cells (1x106) were washed twice in
phosphate-buffered saline (PBS) containing 2 mM EDTA, 100 U/ml
aprotinin, and 1 mM Pefabloc SC (Boehringer Mannheim, Mannheim,
Germany). They were then resuspended in the same buffer (100 µl)
either with or without 1.5 U/ml recombinant PI-PLC and incubated at
37°C for 30 min. At the end of the incubation period, cells were
washed twice in ice-cold PBS and analyzed by fluorescein-activated cell
sorter (FACS) or sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) as required.
Flow cytometric analyses of cell-surface expression of viral
proteins
All steps were performed on ice. Cells were blocked using 20%
pooled, heat-inactivated human serum in PBS containing 0.3% gelatin
and 0.1% sodium azide. Cells were incubated in the same buffer with
either anti-NS1 mAb 1H7.4 or anti-E mAb 4G2, followed by goat
anti-mouse R-phycoerythrin-conjugated F(ab')2
fragments (Jackson Immunochemicals, West Grove, Pa.), and analyzed on a
FACScaliber flow cytometer (Becton Dickinson, Rutherford, N.J.) using
CellQuest software.
Immunoblot analysis of NS1 released from the cell surface
After PI-PLC treatment, cells were pelleted and lysed in SDS
loading buffer (62.5 mM phosphate, pH 7.0, 10% glycerol, 2% SDS,
0.001% bromphenol blue) prewarmed to 60°C; supernatants were
clarified (10,000 g at room temperature for 15 min) and
added to 4x SDS loading buffer. Proteins were separated by 10%
SDS-PAGE and transferred by electroblotting to a polyvinylidene
difluoride membrane (Immobilon-P, Millipore, Bedford, Mass.). NS1 was
detected using anti-NS1 mAb 5H5.4, followed by goat anti-mouse
horseradish peroxidase-conjugated antibody (Jackson Immunochemicals)
and enhanced chemiluminescence reagents (Amersham, Amersham, U.K.).
Metabolic labeling of NS1
Cells were grown to subconfluence in 6-well plates and incubated
with either 50 µCi/ml
[35S]methionine/cysteine, 100 µCi/ml
3H-ethanolamine, or 100 µCi/ml
3H-inositol (Amersham) in Dulbecco’s modified
Eagle medium (DMEM) containing 20% dialyzed fetal calf serum for
18 h. Cells were washed twice in ice-cold PBS and lysed in n-octyl
glucoside buffer [10 mM tris-HCl (pH 7.4), 150 mM NaCl, 60 mM n-octyl
glucoside, 100 U/ml aprotinin, and 1 mM Pefabloc SC]. Cell lysates
were clarified (10,000 g at 4°C for 15 min), precleared
and then immunoprecipitation was performed with anti-NS1 mAb 1H7.4 and
protein A Sepharose (Pharmacia). Immunoprecipitated proteins were
analyzed by 10% SDS-PAGE and fluorography on RX film (Fuji).
Protein tyrosine phosphorylation induced by anti-NS1 antibody
Growth medium was removed from a flask of transfected or
infected HeLa cells, replaced with serum-free DMEM, and incubated at
37°C for 30 min prior to harvest. 4 x 105
cells in 200 µl serum-free DMEM were incubated with 10 µg/ml of an
anti-NS1 mAb mixture (1A12.3, 1E2.3, and 5H5.4), an anti-CD55 mAb, or
an isotype-matched (IgG1) control (Dako, Carpinteria, Calif.) on ice
for 30 min After washing, the cells were resuspended in 200 µl
serum-free DMEM and incubated at 37°C for 10 min (previously
determined to provide an optimal phosphorylation signal; data not
shown). The reactions were terminated by the addition of 1 ml of
ice-cold stop solution (PBS containing 5 mM EDTA, 10 mM NaF, 10 mM
Na4P2O7
and 400 mM Na3VO4). Cells
were pelleted and then solubilized in 100 µl ice-cold lysis buffer
[50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 10
mM iodoacetamide, 10 mM NaF, 10 mM
Na4P2O7,
400 mM Na3VO4, 1 mM
Pefabloc SC, and 100 U/ml aprotinin]. The lysates were clarified
(12,000 g at 4°C for 10 min) and aliquots of the
supernatants were mixed with SDS-PAGE sample buffer, boiled for 2 min,
and subjected to 10% SDS-PAGE. The separated proteins were
electrophoretically transferred onto nitrocellulose (Hybond-C,
Amersham) and probed with the anti-phosphotyrosine mAb, PY20
(Transduction Laboratories, Lexington, Ky.). The blots were incubated
with goat anti-mouse IgG peroxidase (Jackson Immunochemicals) and then
developed by enhanced chemiluminescence (Amersham). Prestained standard
markers (Bio-Rad, Hercules, Calif.) were used for molecular weight
determinations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) that is similar to the carboxyl-terminal hydrophobic
extension of GPI-linked eukaryotic protein precursors (20
, 21
, 23)
. Given that NS1 becomes membrane associated in the absence
of a membrane-spanning domain and would appear from hydropathy plots to
be essentially a hydrophilic species, we hypothesized that GPI-mediated
anchoring may provide an explanation for at least a component of this
membrane association. To examine this hypothesis, the expression of NS1
on the surface of HeLa cell lines stably transfected with NS1
constructs encoding either NS1 alone (NS1) or with the
carboxyl-terminal hydrophobic extension (NS1H) was investigated. This
carboxyl-terminal extension represents the hydrophobic signal-like
domain described above and terminates at a potential serine protease
cleavage site (R-X-R) that is highly conserved among flavivirus
sequences. Using a recombinant baculovirus system, we had reported that
this 26 amino acid extension is sufficient for correct processing of
NS1 (34)
. In initial studies, expression levels were
analyzed by flow cytometry. NS1H-derived NS1 was found to be expressed
at high levels on the cell surface (Fig. 2A
) whereas NS1-derived NS1 was efficiently secreted and
expressed only at very low levels on the cell surface (data not shown).
To determine whether NS1H-derived NS1 was processed to a GPI-anchored
form, HeLa.NS1H cells were incubated with and without PI-PLC, an enzyme
that specifically releases GPI-linked proteins from the cell surface
(31)
. FACS analysis showed that PI-PLC treatment reduced
the median fluorescence intensity of NS1H-derived NS1 on the cell
surface by ~90% (Fig. 2A
). To verify that PI-PLC
treatment did not cause nonspecific degradation of NS1, removal of
cell-surface NS1 by PI-PLC was further analyzed by immunoblotting. As
seen in Fig. 2B
, PI-PLC treatment released intact NS1 from
the cell surface into the supernatant. The higher molecular weight of
the released NS1 reflects differences in the carbohydrate composition
of NS1 species in different cellular compartments: the majority of
intracellular NS1, which is inaccessible to the action of PI-PLC on
intact cells, contains carbohydrate of the high-mannose form at both
linkage sites whereas cell-surface NS1 released by the action of PI-PLC
has transited the Golgi, where one of these moieties is trimmed and
processed to a complex form. These results show that NS1H-derived NS1
is sensitive to PI-PLC digestion and suggest the presence of a GPI
anchor.
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There are two possible explanations for the observation that
NS1H-derived NS1 is released from the cell surface by PI-PLC treatment.
Either it is covalently modified by GPI linkage and anchored in the
plasma membrane by GPI or it becomes tightly associated with another
protein that is itself anchored in the plasma membrane by GPI. To
distinguish these possibilities, metabolic labeling of the HeLa cell
lines with 3H-ethanolamine was performed.
Ethanolamine is a major constituent of the GPI anchor structure and is
incorporated into intracellular GPI anchor precursors, which are then
transferred to newly synthesized protein (35)
.
Incorporation of 3H-ethanolamine into NS1 was
analyzed by immunoprecipitation of cell lysates using a monoclonal
anti-NS1 antibody, followed by SDS-PAGE. Figure 3B
(lane 3) shows that 3H-ethanolamine
was efficiently incorporated into NS1H-derived NS1. Labeling with
[35S]methionine/cysteine demonstrated that
similar amounts of NS1 were synthesized during the labeling period by
both HeLa.NS1 and HeLa.NS1H cell lines (Fig. 3A
), yet
3H-ethanolamine was not incorporated into NS1
expressed in the HeLa.NS1 line (Fig. 3B
, lane 2). The two
constructs differ only in that NS1H is expressed with a 26 amino acid
carboxyl-terminal extension, corresponding to the
NH2 terminus of NS2A (Fig. 1C
). Taken
together, these results show that NS1H-derived NS1 is
post-translationally modified by covalent addition of a GPI anchor and
that the NH2 terminus of NS2A can act as a signal
sequence for GPI linkage of NS1.
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GPI-linked NS1 in dengue-infected cells
After the results with the transfected cell lines, expression of a
GPI-linked form of NS1 in cells infected with dengue virus was
investigated. To determine whether a PI-PLC sensitive form of NS1 was
expressed on the surface of infected cells, dengue-infected HeLa cells
were incubated with and without PI-PLC and analyzed using FACS. PI-PLC
treatment reduced the median fluorescence intensity of NS1 on the
surface of infected cells by ~20% (Fig. 4A
). In contrast, surface expression of the dengue virus E
glycoprotein, which is known to be associated with the plasma membrane
via a transmembrane anchor, was unaffected by PI-PLC treatment (Fig. 4B
). Metabolic labeling of NS1 expressed in infected HeLa
cells with 3H-ethanolamine was attempted, but
incorporation was barely detectable, possibly owing to the fact that
dengue virus replicates to relatively low titers in HeLa cells. Dengue
virus yields from infected C6/36 cells, a mosquito cell line, are
routinely more than 100-fold higher than those from infected HeLa
cells. Therefore, metabolic labeling of NS1 expressed in
dengue-infected C6/36 cells was performed using both
3H-ethanolamine and another labeled component of
the GPI anchor, 3H-inositol. Incorporation of
3H-inositol is highly specific for the presence
of a GPI anchor structure. Both components were incorporated into
cell-associated NS1 (Fig. 4C
), albeit at low levels,
confirming the presence of a GPI moiety.
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Signal transduction induced by anti-NS1 antibody
It is well established that GPI-linked proteins are capable of
acting as signaling molecules in response to binding by specific
antibody (26
, 36)
. Given that NS1 is an important target
of humoral immunity in dengue virus infections, we investigated the
possibility that binding of NS1-specific antibody to GPI-linked NS1 on
the surface of cells could initiate cell signaling events. As tyrosine
phosphorylation is a crucial early step in GPI-mediated signal
transduction leading to cell activation (26)
, the profile
of phosphorylated cellular proteins induced by the interaction of
anti-NS1 antibody with cells expressing NS1 on their surface was
examined. Incubation of HeLa.NS1H cells with a mixture of mAbs specific
for NS1 (1A12.3, 1E2.3 and 5H5.4) resulted in enhanced tyrosine
phosphorylation of a number of cellular proteins (Fig. 5
, lane 5). These species had molecular weights of 56,000, 110,000,
190,000 and a doublet in excess of 200,000. This effect is attributable
to specific binding of GPI-anchored NS1 by antibody since it was not
produced by incubation of HeLa.NS1H cells with an irrelevant
isotype-matched antibody at the same concentration (Fig. 5
, lane 2) nor
by incubation of control cells (transfected with empty vector and
therefore not expressing NS1) or HeLa.NS1 cells (expressing NS1 that is
not GPI anchored) with the anti-NS1 mAb mixture (Fig. 5
, lanes 3 and 4,
respectively). Exactly the same set of proteins was found to be
phosphorylated after incubation of HeLa.NS1H cells with a mAb specific
for another GPI-anchored membrane protein, decay-accelerating factor
(DAF/CD55) (Fig. 5
, lane 7). To assess whether the observed
phosphorylation profile was merely an artifact of the stable
transfectant system, we also examined the response of dengue
virus-infected HeLa cells to incubation with the anti-NS1 mAb mixture.
Anti-NS1 specific mAb, but not control antibody, induced tyrosine
phosphorylation of the same proteins, although to a lesser degree (Fig. 5
, lanes 6 and 1, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A comparison of NS1 expression in transfected and infected cells
suggests that not all NS1 in infected cells is processed to a
GPI-anchored form. This may be explained by the fact that internal
cleavage events within NS2A occur during processing of the viral
polyprotein (34)
, which in turn may determine whether or
not NS1 acquires an appropriate carboxyl-terminal signal sequence for
GPI anchor addition. Alternatively, in the absence of appropriate NS2A
cleavage, the hydrophobic domain in the NH2
terminus of NS2A may act as an internally positioned GPI anchor
addition signal. An internally positioned signal sequence has
previously been shown experimentally to be capable of signaling GPI
anchor addition, although inefficiently when compared with a signal
sequence in the usual carboxyl-terminal position (37)
. To
our knowledge there is no biological precedent for utilization of an
internal GPI anchor addition signal sequence. The present study does
not define the exact mechanism and site of GPI linkage to NS1, but
these are subjects of current investigation. Requirements for the amino
acids at and around the GPI acceptor site (the 
site) have been
proposed (23
, 38)
, and examination of the predicted amino
acid sequence encoded at the 3' end of the NS1 gene and in the 5'
portion of the NS2A gene reveals several potential sites for GPI
linkage.
The function of a GPI-anchored form of NS1 in the virus life cycle is
not known. Recent evidence from a study seeking to clarify the role of
NS1 in viral RNA replication suggests that GPI-linkage is not essential
for replication of the closely related yellow fever (YF) virus in
mammalian cell culture (16)
. In that study, a YF virus
infectious clone containing a lethal deletion in NS1 was successfully
replicated in mammalian cells by trans-complementation with
NS1 expressed in the absence of downstream NS2A sequences (and
therefore in the absence of a GPI anchor addition signal sequence).
However, the failure of dengue virus NS1 to trans-complement
a similarly deleted dengue virus infectious clone was recently
reported, raising the possibility of fundamental differences between
these two flaviviruses (39)
. As part of the virus life
cycle, mosquito-borne flaviviruses including dengue and YF must also
replicate in insect cells. Mechanisms of flavivirus replication in
mosquito and mammalian cells are yet to be fully defined, but NS1
maturation is strikingly different in the two cell types. Whereas NS1
is both membrane associated and efficiently secreted from mammalian
cells, it remains membrane associated in insect cells
(40)
. A unique role may be possible for a GPI-anchored
form of NS1 in viral replication in insect cells.
It is also possible that a GPI-anchored form of NS1 serves to exert a
functional effect only in vivo. NS1 is a major target of
humoral immunity in dengue virus infection (41
42
43
44)
, and
our results show that binding of specific anti-NS1 antibody to
cell-surface NS1 in both transfected and infected cells initiates
signal transduction, leading to protein tyrosine phosphorylation.
Signal transduction induced by antibodies to GPI-linked proteins is
well established (45
46
47)
and appears to be a general
property of GPI-anchored proteins that depends on the presence of the
GPI moiety (24
, 25)
. GPI anchors lack an intracellular
domain for signal transduction and the transmembrane components of the
GPI signal transduction pathway have remained elusive. However,
association of GPI anchors with members of the src homology
kinase family (such as lck, fyn, and
lyn) and phosphorylation of intracellular substrates in
response to antibody binding have been shown for GPI-anchored proteins
expressed by eukaryotic cells (45
, 48)
. Identification of
a similar subset of phosphorylated proteins in response to antibody
binding of both NS1 and CD55 (Fig. 5)
suggests the involvement of a
common signal transduction pathway for these two GPI-linked proteins.
The demonstration of signal transduction induced by specific anti-NS1
antibody provides the first suggestion of pathophysiologically relevant
counterparts to previous experimental observations of antibody-induced
signal transduction by cell-encoded GPI-anchored proteins
(45
46
47)
. Signal transduction by NS1 is likely to promote
cellular activation, which in turn may increase production and release
of virus progeny (49)
. This may have relevance to viral
transmission as Aedes mosquitoes, which transmit dengue, are
surprisingly resistant to infection by ingested dengue virus and high
titers of virus in human blood are essential for mosquito transmission
to be sustained (50)
. In addition, virus-encoded
nonstructural proteins are known to be phosphorylated by cellular
enzymes (51
, 52)
. Given that phosphorylation status may
affect protein function (53)
, it is possible that
GPI-mediated signaling may be exploited by the virus as a means of
modifying its own replication.
Whatever the explanation for use of the cellular GPI linkage pathway by
dengue virus to produce a GPI-anchored form of NS1, this finding may
have implications for understanding the pathogenesis of DHF/DSS.
DHF/DSS is a severe, life-threatening form of illness characterized by
disordered hemostasis and vascular leak. The predominant risk factor
predisposing individuals to DHF/DSS is precirculating anti-dengue
antibody, which may enhance viral replication. However, the molecular
mechanisms linking enhanced viral replication to disease pathogenesis
remain unknown (2)
. Shock is caused by endothelial cell
dysfunction rather than cell death (3)
and does not
coincide with the peak of viral replication, but instead occurs as
fever subsides and at a time when circulating dengue-specific antibody
is detected (54
, 55)
. The finding that anti-NS1 antibody
induces signal transduction by NS1 expressed as a GPI-anchored species
on the surface of infected cells suggests a possible mechanism that may
contribute to the pathogenesis of DHF/DSS. Release of vasoactive
cytokines from infected monocytes/macrophages, which is believed to
underlie the endothelial dysfunction in DHF/DSS (4)
, may
be augmented in response to NS1-mediated signaling and cellular
activation. In addition, it is possible that GPI-linked NS1 may
transfer from infected monocytes/macrophages to the surface of
endothelial cells, where antibody-induced signal transduction may
directly initiate endothelial cell activation (29
, 30)
.
The concept of cellular activation resulting from a surface-expressed,
GPI-anchored viral antigen interacting with the specific antibodies it
induces is a new paradigm in viral pathogenesis, and further studies
are indicated to elucidate its role in human disease.
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ACKNOWLEDGMENTS |
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Received for publication September 7, 1999.
Revision received December 1, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-site: constraints for the polypeptide binding site of the putative transamidase. Prot. Eng. 11,1155-1161This article has been cited by other articles:
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Y.-W. Lin, K.-J. Wang, H.-Y. Lei, Y.-S. Lin, T.-M. Yeh, H.-S. Liu, C.-C. Liu, and S.-H. Chen Virus Replication and Cytokine Production in Dengue Virus-Infected Human B Lymphocytes J. Virol., October 25, 2002; 76(23): 12242 - 12249. [Abstract] [Full Text] [PDF]
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C.-F. Lin, H.-Y. Lei, A.-L. Shiau, H.-S. Liu, T.-M. Yeh, S.-H. Chen, C.-C. Liu, S.-C. Chiu, and Y.-S. Lin Endothelial Cell Apoptosis Induced by Antibodies Against Dengue Virus Nonstructural Protein 1 Via Production of Nitric Oxide J. Immunol., July 15, 2002; 169(2): 657 - 664. [Abstract] [Full Text] [PDF]
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