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Dengue Virus Type 2 Antagonizes IFN-
␣
but Not IFN-
␥
Antiviral Effect via Down-Regulating Tyk2-STAT Signaling in
the Human Dendritic Cell
1
Ling-Jun Ho,* Li-Feng Hung,* Chun-Yi Weng,
†
Wan-Lin Wu,
†
Ping Chou,
†
Yi-Ling Lin,
‡
Deh-Ming Chang,
†
Tong-Yuan Tai,* and Jenn-Haung Lai
2†
The immunopathogenesis mechanism of dengue virus (DV) infection remains elusive. We previously showed that the target of DV
in humans is dendritic cells (DCs), the primary sentinels of immune system. We also observed that despite the significant amount
of IFN-
␣
induced; DV particles remain massively produced from infected DCs. It suggests that DV may antagonize the antiviral
effect of IFN-
␣
. Recent work in animal studies demonstrated the differential critical roles of antiviral cytokines, namely IFN-
␣
/
IFN-

and IFN-
␥
, in blocking early viral production and in preventing viral-mediated disease, respectively. In this study, we
examined the effects of IFN-
␣
and IFN-
␥
in DV infection of monocyte-derived DCs. We showed that the preinfection treatment
with either IFN-
␣
or IFN-
␥
effectively armed DCs and limited viral production in infected cells. However, after infection, DV
developed mechanisms to counteract the protection from lately added IFN-
␣
, but not IFN-
␥
. Such a selective antagonism on
antiviral effect of IFN-
␣
, but not IFN-
␥
, correlated with down-regulated tyrosine-phosphorylation and DNA-binding activities of
STAT1 and STAT3 transcription factors by DV. Furthermore, subsequent studies into the underlying mechanisms revealed that
DV attenuated IFN-
␣
-induced tyrosine-phosphorylation of Tyk2, an upstream molecule of STAT activation, but had no effect on
expression of both IFN-
␣
receptor 1 and IFN-
␣
receptor 2. Moreover, DV infection by itself could activate STAT1 and STAT3
through IFN-
␣
-dependent and both IFN-
␣
-dependent and IFN-
␣
-independent mechanisms, respectively. These observations pro-
vide very useful messages with physiological significance in investigation of the pathogenesis, the defense mechanisms of human
hosts and the therapeutic considerations in DV infection. The Journal of Immunology, 2005, 174: 8163– 8172.
Dengue viruses (DV)
3
belonging to mosquito-born flavi-
viruses are single-stranded positive-polarity RNA vi-
ruses that cause serious infectious diseases all over the
world especially in Asian and Latin American countries (1, 2).
Infection by DV may result in benign course called dengue fever
(DF) or life-threatening presentations such as dengue hemorrhagic
fever (DHF) and dengue shock syndrome. In the past few decades,
the incidence of DF/DHF has significantly increased. The esti-
mated rate yearly for DF is ⬃50,000,000 –100,000,000 cases and
for DHF is ⬃250,000 –500,000 cases worldwide (3). It is not until
recently that the natural cellular target of initial infection of DV in
humans was identified to be dendritic cells (DCs) (4 – 6). DCs, the
best and professional APCs critical in both innate and adaptive
immune responses, play crucial roles in different species of virus
infection (7, 8). Through binding to DC-specific ICAM-3-grabbing
nonintegrin, DV infect immature DC and replicate inside the cell,
and in the meantime, activate DC to drive cytokine production and
cell maturation (5, 9).
After infection, one major cytokine produced is IFN-
␣
(5). The
type I IFNs, IFN-
␣
and IFN-

, together with the type II IFN,
IFN-
␥
, are crucial in mediating antiviral response through blocking
viral replication or through modulating immune responses to in-
hibit viral spreading (10 –14). We previously showed that, despite
a significant amount of IFN-
␣
produced in culture medium of DV-
infected DCs, the viruses remain actively replicating and produc-
ing viral progenies (5). Such an observation is also reflected in
DV-infected children (15). It may suggest the presence of mech-
anisms antagonizing antiviral effect of IFN-
␣
in DV-infected DCs.
By studying genetically deficient animals, Shresta et al. (16)
showed that both IFN-
␣
/IFN-

and IFN-
␥
receptors play critical
and nonoverlapping roles in resolving primary DV infection in
mice. Meanwhile, recent work performed using immortalized cell
lines suggests that DV nonstructural protein 4B may interfere
IFN-

- and IFN-
␥
-mediated antiviral response through blocking
STAT1 activities (17). As observed in many DNA and RNA vi-
ruses, STAT proteins are indeed one of the major targets for vi-
ruses to block and to counteract the antiviral effects of IFNs (13).
To appreciate whether and how both IFN-
␣
and IFN-
␥
may pos-
sibly mediate their antiviral activities in DV-infected DC and how
DV may interfere with these mechanisms, we used purified mono-
cyte-derived DCs to examine such effects and mechanisms. Our
results revealed that DV effectively suppressed IFN-
␣
-induced but
not IFN-
␥
-induced antiviral effect at least in part through blocking
STAT1 and STAT3 activation as well as reducing tyrosine-phos-
phorylation of Tyk2 tyrosine kinase.
*Division of Gerontology Research, National Health Research Institute,
†
Rheuma-
tology/Immunology and Allergy, Department of Medicine, Tri-Service General Hos-
pital, National Defense Medical Center, and
‡
Institute of Biomedical Sciences, Aca-
demia Sinica, Taipei, Taiwan
Received for publication October 19, 2004. Accepted for publication March 30, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work is supported in part by grants from the National Health Research Insti-
tutes (to L.-J.H. and to J.-H.L., NHRI-EX92-9208SI) and the National Science Coun-
cil (NSC-91-2314-B-016-044), Taipei, Taiwan.
2
Address correspondence and reprint requests to Dr. Jenn-Haung Lai, Rheumatology/
Immunology and Allergy, Tri-Service General Hospital, Number 325, Section 2, Cheng-
Kung Road, Neihu 114, Taipei, Taiwan. E-mail address: haungben@tpts5.seed.net.tw
3
Abbreviations used in this paper: DV, dengue virus; DC, dendritic cell; DF, dengue
fever; DHF, dengue hemorrhagic fever; MOI, multiplicity of infection; NGC, New
Guinea C; IFNAR, IFN-
␣
receptor; DV2, DV serotype 2.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
Materials and Methods
Culture medium and reagents
The cell culture medium consisted of RPMI 1640 (Invitrogen Life Tech-
nologies) supplemented with 10% FBS, 2 mM glutamine, and 1000 U/ml
penicillin-streptomycin (Invitrogen Life Technologies). Recombinant GM-
CSF and IL-4 were purchased from R&D Systems. Abs against total
STAT1, STAT2, STAT3, STAT5, and STAT6 were purchased from Santa
Cruz Biotechnology. Anti-tyrosine phosphorylated Tyk2 (anti-Tyk2-pY),
anti-tyrosine phosphorylated STAT1 (anti-STAT1-pY), anti-STAT3-pY,
anti-STAT5-pY, and anti-STAT6-pY were purchased from Cell Signaling
Technology. Anti-STAT2-pY was purchased from Upstate Biotechnology.
Both human IFN-
␣
and IFN-
␥
were purchased from R&D Systems. The
fluorescence-labeled anti-IFN-
␣
receptor (IFNAR)1, anti-IFNAR2, CD3,
CD14, CD19, HLA-DR, CD83, CD86, CD1a, CD11c and CD11b mAbs
were purchased from BD Pharmingen. Both anti-IFN-
␣
neutralizing Ab
and mouse IgG control were purchased from R&D Systems. Unless spec-
ified, the rest of the reagents were purchased from Sigma-Aldrich.
Establishment of DCs from human peripheral blood monocytes
DCs were established from positively selected CD14
⫹
monocytes from
80 –100 different healthy donors by using a MACS cell isolation column
following manufacturer’s instructions (Miltenyi Biotech). In brief, buffy
coat (each buffy coat is equivalent to 500 ml of whole blood) from a blood
bank (Taipei, Taiwan) was mixed with Ficoll-Hypaque, after centrifuga-
tion, the layer of mononuclear cells was collected. After lysis of RBC, the
PBMC were obtained. To obtain DCs with high purity, PBMC were incu-
bated with anti-CD14 microbeads at 4 –8°C for 15 min. After wash, the
CD14
⫹
cells were isolated using a MACS cell isolation column (Miltenyi
Biotec). The obtained monocytes were then cultured in the medium con-
taining 800 U/ml GM-CSF and 500 U/ml IL-4 at a cell density of 1 ⫻10
6
cells/ml. The culture medium was changed every other day with 300
lof
fresh medium containing 2400 U GM-CSF and 1500 U IL-4, and the cells
were used for experiments after 5–7 days of culture. The purity of DCs, as
determined by the positive staining of CD1a, was consistently higher than
90% as described in our previous work (18).
Preparation of DV and determination of virus titers
The preparation of DV has been described previously with some modifi-
cations (5, 19). In brief, DV serotype 2 (DV2) New Guinea C (NGC) strain
(American Type Culture Collection) and DV2 PL046 strain, a wild-type
with unknown passage and nonmouse adapted local Taiwanese strain iso-
lated from a patient with DF in 1981, were propagated in C6/36 mosquito
cells in RPMI 1640 containing 5% heat-inactivated FCS and maintained at
28°C in a 5% CO
2
atmosphere for 7 days. The supernatants were collected,
and virus titers were determined and then stored at ⫺70°C until use. To
determine virus titers, the culture supernatants were harvested for plaque-
forming assays. Various virus dilutions were added to 80% confluent baby
hamster kidney (BHK-21) cells and incubated at 37°C for 1 h. After ad-
sorption, cells were washed and overlaid with 1% agarose (SeaPlaque;
FMC BioProducts) containing RPMI 1640 and 1% FCS. After incubation
for 7 days, cells were fixed with 10% formaldehyde and stained with 0.5%
crystal violet. The numbers of plaques were counted and the results were
shown as PFU per milliliter. Aside from Fig. 1D, where the PL046 strain
was the viral strain used, the NGC strain was the only source of DV to
infect cells throughout the studies.
Infection of DC with DV
DCs cultured for 5 days were infected with mock or DV at different mul-
tiplicity of infections (MOIs) or at MOI 5 (in most of the conditions of this
report) for4hat37°C (5). After viral absorption, cells were then washed
and cultured in six-well plates (Costar) with culture medium in the absence
of exogenously added cytokines for various periods of time as indicated in
the figures. For treatment, the cell density was maintained at 1 ⫻10
6
/ml in
culture medium.
Flow cytometric analysis
The determination of single expression or coexpression of both cell surface
and intracellular molecules has been described in our previous report (18).
For dual stainings, 20 h after infection, DCs were collected and resus-
pended in 50
l of PBS containing 1% BSA. Then anti-CD1a mAb con-
jugated with PE was added, and the mixture was incubated at 4°C for 30
min. After this, cells were permeabilized by adding 0.25% saponin (Sigma-
Aldrich). After incubation at 4°C for another 20 min, the anti-NS1 mAb
(19) was added. After a wash with cold PBS, the goat anti-mouse mAb
conjugated with FITC was added and incubated for another 30 min. After
wash, the samples were analyzed in a flow cytometer (BD Biosciences).
Each density plot was comprised of at least 10
4
events.
Nuclear extract preparation
Nuclear extracts were prepared according to our published work (20).
Briefly, the treated cells (1–2 ⫻10
7
cells in average in each treatment
condition) were left at 4°C in 50
l of buffer A (10 mM HEPES, pH 7.9,
10 mM KCl, 1.5 mM MgCl
2
, 1 mM DTT, 1 mM PMSF, and 3.3
g/ml
aprotinin) for 15 min with occasional gentle vortexing. The swollen cells
were centrifuged at 15,000 rpm for 3 min. After removal of the superna-
tants (cytoplasmic extracts), the pelleted nuclei were washed with 50
lof
buffer A and subsequently, the cell pellets were resuspended in 30
lof
buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl
2
, 0.2 mM
EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, and 3.3
g/ml aproti-
nin) and incubated at 4°C for 30 min with occasional vigorous vortexing.
Then the mixtures were centrifuged at 15,000 rpm for 20 min, and the
supernatants were used as nuclear extracts.
EMSA
The EMSA was performed as detailed in our previous report (20). The
oligonucleotides containing STAT1, STAT3, STAT5, and STAT6 were
purchased and used as DNA probes (Promega). The DNA probes were
radiolabeled with [
␥
-
32
P]ATP using the T4 kinase according to the man-
ufacturer’s instructions (Promega). For the binding reaction, the radiola-
beled STAT probe was incubated with 5
g of nuclear extracts. The bind-
ing buffer contained 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM
EDTA, 1 mM DTT, 1 mM MgCl
2
, 4% glycerol, and 2
g poly(dI-dC). The
reaction mixture was left at room temperature to proceed with binding
reaction for 20 min. If unradiolabeled competitive oligonucleotides were
added, they were used as 100-fold molar excess and preincubated with
nuclear extracts for 10 min before the addition of the radiolabeled probes.
Western blotting
ECL Western blotting (Amersham) was performed as described (21).
Briefly, after extensive wash, the cells were pelleted and resuspended in
lysis buffer. After periodic vortexing, the mixture was centrifuged, the
supernatant was collected, and the protein concentration was measured.
Equal amounts of whole cellular extracts were analyzed on 10% SDS-
PAGE and transferred to the nitrocellulose filter. For immunoblotting, the
nitrocellulose filter was incubated with TBS-T containing 5% nonfat milk
(milk buffer) for 2 h, and then blotted with antisera against individual
proteins for overnight at 4°C. After washing with milk buffer twice, the
filter was incubated with secondary Ab conjugated to HRP at a concen-
tration of 1/5000 for 30 min. The filter was then incubated with the sub-
strate and exposed to x-ray film.
Statistics
When necessary, the results were expressed as means ⫾SD. A paired or
unpaired Student’s ttest was used to determine the difference that was
thought to be significant when p⬍0.05.
Results
Treatment with IFN-
␣
or IFN-
␥
before or after viral infection
distinguished their antiviral effects in DV infection of DC
Although both IFN-
␣
and IFN-
␥
preserve antiviral activities, mice
that are deficient of IFN-
␣
/IFN-

or IFN-
␥
receptor appear to have
different manifestations in terms of viral load and viral-mediated
disease in DV infection (16). To investigate the effects of antiviral
cytokines in DV infection, IFN-
␣
or IFN-
␥
was added into the
culture medium of DCs; after incubation for 6 h, the medium was
washed and the cells were infected with DV. After viral absorption
for 4 h, the DCs were washed and left for an additional 20 h. Then
the supernatants were collected for viral titer determination by
plaque assays. As shown in Fig. 1A, the treatment of IFN-
␣
or
IFN-
␥
before viral infection effectively reduced viral production in
DCs. Such results were compatible with the observations by Dia-
mond et al. (22) demonstrate the inhibition of DV production by
either IFN-
␣
or IFN-

pretreatment in various human cell lines. To
examine the antiviral effects of these two cytokines in already in-
fected cells, DCs were first infected by DV; after viral absorption
and extensive wash, the cells were incubated with different con-
centrations of IFN-
␣
or IFN-
␥
2 h later and left for an additional
8164 DV BLOCK IFN-
␣
BUT NOT IFN-
␥
EFFECT VIA INHIBITING Tyk2-STAT
18 h incubation. Viral titers in supernatants then were determined.
In contrast to preinfection treatment of cytokines, the antiviral ef-
fects of IFN-
␣
, but not IFN-
␥
, were greatly reduced when the
cytokine was added after DV infection (Fig. 1B). To be more close
to physiological viral loads, DV at MOI 1 were used to infect DCs,
and the protective effects of IFN-
␣
and IFN-
␥
were examined. As
shown in Fig. 1C, the protection of IFN-
␣
was only observed when
it was added before DV infection of the cells. Consistently, IFN-
␥
reduced DV production no matter if it was added before or after
DV infection of DCs. In considering that the observed different
response to IFNs may be due to the examination of viral NGC
strain, a high mouse brain-passaged virus known to be attenuated
for human beings, similar experiments were conducted to examine
the DV2 PL046 strain isolated locally in Taiwan instead. We ob-
served that although high doses of postinfection-added IFN-
␣
could inhibit virus production, at the comparable dosages, there
was 1.5–2.5 log attenuation of suppressive intensity compared
with the effects of preinfection-added IFN-
␣
(Fig. 1D,upper
panel). In contrast, the antiviral effect of IFN-
␥
was consistently
observed at comparable intensity when it was added in both pre-
infection and postinfection conditions (Fig. 1D,lower panel). In
addition, we observed that the PL046 strain appeared to be less
able than the NGC strain to antagonize the antiviral effect of
IFN-
␣
. The difference of IFN-
␣
and IFN-
␥
antiviral effects in DCs
FIGURE 1. Effects of preinfection treatment or
postinfection treatment of IFN-
␣
or IFN-
␥
on DV pro-
duction in DCs. Human DCs at 1 ⫻10
6
/ml were treated
with various doses of IFN-
␣
or IFN-
␥
for 6 h. The cells
were washed and infected with DV (NGC strain) at MOI
5. A, After viral absorption for 4 h, the cells were then
incubated for an additional 20 h and the supernatants
were collected for viral titer determination by plaque
assays. B, Two hours after viral absorption for 4 h, DCs
at 1 ⫻10
6
/ml were treated with various doses of IFN-
␣
or IFN-
␥
and left for an additional 18 h. The superna-
tants were collected for virus titer measurements (as the
scheme shows). The percentage of inhibition in both A
and Bwas calculated as follows: (virus titer in the ab-
sence of cytokine treatment ⫺virus titer in the presence
of cytokine treatment)/virus titer in the absence of cy-
tokine treatment. Cand D, Similar experiments were
performed, except DV NGC strain (C) and DV PL046
strain (D) at MOI 1 were used to infect DCs instead. The
data shown are representative of three to six indepen-
dent experiments using different donor DCs with similar
results.
8165The Journal of Immunology
infected by these two DV2 strains was not exactly clear. Both the
virulence and the different growth curve in culture of these viral
strains may contribute in part to the difference. Altogether, these
results suggest that, after infection, DV may develop a defense
mechanism in infected cells to fight against the antiviral effect of
IFN-
␣
, but not IFN-
␥
.
Flow cytometric analysis of DV-infected DCs
To determine the phenotype of DCs established from human pe-
ripheral blood monocytes, the cells were stained with various cell
surface markers and analyzed with a flow cytometer. As shown in
Fig. 2A, compared with monocytes, immature DCs expressed
higher levels of CD83, CD11c, and CD11b. Meanwhile, the ex-
pression of CD1a (DCs) and CD14 (monocytes) clearly distin-
guished these two cell populations. Both monocytes and DCs did
not express CD3 and CD19, markers for T and B lymphocytes,
respectively. Because the effects on IFN-
␣
and IFN-
␥
may be af-
fected to certain extent by the infectious rate of viruses, the DV-
infected DCs expressing both cell surface CD1a and intracellular
viral NS1 proteins were studied and analyzed by flow cytometric
FIGURE 2. Flow cytometric anal-
ysis of immature DC phenotypes and
the percentages of DCs infected by
DV. Human DCs were stained with
various cell surface markers as indi-
cated and analyzed by a flow cytom-
eter as described A.B, DCs after in-
fection by mock or DV (NGC strain)
at MOI 1, 5, or 10 were stained with
both NS-1 and CD1a or isotype-
matched control mAb and analyzed
by a flow cytometer as described in
Materials and Methods. Each density
plot or histogram is generated using
at least 10
4
events. The representative
data from at least three different do-
nor DCs are shown. Ctl indicates the
staining using isotype-matched con-
trol mAb.
FIGURE 3. Tyrosine-phosphorylation of STATs in
DV-infected DCs. Human DCs at 1 ⫻10
6
/ml (4 –7 ⫻
10
6
cells in average in each treatment condition) in-
fected by mock or DV (NGC strain) at MOI 5 at various
time points were collected and lysed, and the whole ly-
sates were prepared. Aside from the 3-h time point that
was within the period of viral absorption, the rest of the
time points shown all included the first 4 h for viral
absorption. Western blotting was performed to deter-
mine the levels of total (-t) and tyrosine-phosphorylated
(-p) STAT1 and STAT2 (A), STAT3 (B), STAT5 and
STAT6 (C) as described in Materials and Methods.As
a positive control, IFN-
␣
(1,000 U/ml) was added 5 min
before cell collection. The data shown are representative
of three to six independent experiments using different
donor DCs with similar results.
8166 DV BLOCK IFN-
␣
BUT NOT IFN-
␥
EFFECT VIA INHIBITING Tyk2-STAT
analysis. We showed that the percentages of DCs infected by DV
were 25, 52, and 56% in the presence of MOI 1, 5, and 10, re-
spectively (Fig. 2B). The infectious rate of viruses in DCs corre-
lated quite well with another report (4) and our unpublished
observations.
DV infection induced STAT activities
It has been generally accepted that binding of IFN-
␣
to its ligand
results in the activation of STAT1, STAT2, and STAT3 (23–25).
In addition, IFN-
␣
also activates STAT4, STAT5, and STAT6 in
T cells, B cells, and Daudi cells (26 –28). Interestingly, the IFN-
␣
-induced activation of both STAT3 and STAT5, but not both
STAT1 and STAT3, is sensitive to a Syk/ZAP70-specific kinase
inhibitor (29). It suggests that the activation of STATs by IFN-
␣
is
mediated through diverging signaling pathways. With regard to the
IFN-
␥
-mediated signaling pathway, aside from the well-known ac-
tivation of STAT1, IFN-
␥
also activates several genes like che-
mokine genes through STAT1-independent signaling pathway
(30). In this context, STAT proteins such as STAT3 and STAT5
are shown to be activated after IFN-
␥
stimulation (31–34). Inter-
estingly, although STAT6 is not directly activated by IFN-
␥
, both
IFN-
␣
and IFN-
␥
can block IL-4-induced STAT6 activation (35).
Because STAT proteins are one of the major targets for both
RNA and DNA viruses to counteract the antiviral effect of IFNs
(13), we first determined the activation status of STAT proteins in
DV-infected DCs. The Western blotting assays were performed to
examine the tyrosine-phosphorylation of STAT proteins. As
shown in Fig. 3, Aand B, DV infection was able to induce the
tyrosine-phosphorylation of STAT1, STAT2, and STAT3 albeit
the kinetics of activation was different. Notably, the activation of
STAT3 could be clearly demonstrated 3 h after infection (Fig. 3B).
At examined time points, the activation of both STAT5 and
STAT6 was not detectable (Fig. 3C). Because after tyrosine-phos-
phorylation, STATs form homo- or heterodimers and translocate
from the cytoplasm to the nucleus, where they bind specific DNA
sequences and activate transcription of many genes. Therefore, the
EMSA analysis was performed to examine the DNA-binding ac-
tivities of STATs in DV-infected DCs. As shown in Fig. 4A,DV
infection potently induced DNA-binding activities of STAT3 that
was detectable as early as 3 h after infection compatible to the
kinetics of its tyrosine-phosphorylation status. Consistently, the
DNA-binding activity of STAT1 could only be detected 18 –24 h
after infection (Fig. 4Band data not shown). Within the deter-
mined time points, the DNA-binding activities of both STAT5 and
STAT6 were not detectable (Fig. 4B). These results suggest that
DV infection might by itself or via secondarily secreted mediators
induce the activation of STAT proteins in human DCs.
DV infection blocked IFN-
␣
-induced but not IFN-
␥
-induced
STAT activation
Knowing both that DV infection by itself was able to activate
STAT proteins and that STAT proteins are critical in IFN-medi-
ated antiviral activities, we were interested to know how STATs
might be regulated by IFN-
␣
or IFN-
␥
in the presence of DV
infection. DCs were infected with mock or DV for 18 –24 h and
treated with IFN-
␣
or IFN-
␥
, and then the total cell lysates were
collected to determine tyrosine-phosphorylated STAT1 and
STAT3 by Western blotting. As shown in Fig. 5, the IFN-
␣
-in-
duced STAT1 (Fig. 5A) and STAT3 (Fig. 5B) tyrosine-phospho-
rylation was reduced by DV. In contrast, as a side-by-side com-
parison, the IFN-
␥
-induced STAT1 and STAT3 activation was not
only unsuppressed but also mildly enhanced by DV infection (Fig.
5, Aand B). Because the dosage of IFN-
␥
(50 U/ml) used in these
experiments was relatively lower than that of IFN-
␣
(1,000 U/ml),
we also examined the effects of DV on different doses of IFN-
␥
-
induced STAT1 activation. Fig. 5Cillustrated an additive effect
between DV-induced and different doses of IFN-
␥
-induced STAT1
activation. Consistently, the suppression by DV infection was still
observed when DCs were treated with lower concentrations (both
200 and 500 U/ml) of IFN-
␣
(data not shown). In addition, the
viral-mediated suppression of IFN-
␣
-induced STAT1 activation
was only observed when DCs were preinfected by DV for longer
FIGURE 4. DV induced DNA-binding activities of
STATs in DCs. To determine the DNA-binding activi-
ties of STATs, human DCs 1 ⫻10
6
/ml (1–2 ⫻10
7
cells
in average in each treatment condition) were infected
with mock or DV (NGC strain) for various periods of
time, and the cells were collected and nuclear extracts
were prepared. The DNA-binding activities of STAT3
(A) or STAT1, STAT5, and STAT6 (B) in nuclear ex-
tracts of different treatment conditions were analyzed by
EMSA as described in Materials and Methods. The fold
induction was presented as a comparison with the signal
intensity in mock-infected control. The results were ob-
tained from at least three to six different donors DC with
similar results.
8167The Journal of Immunology
than 6 h before the addition of IFN-
␣
(Fig. 5D). Subsequent ex-
periments further demonstrated that the IFN-
␣
-induced phosphor-
ylation of STAT1 and STAT3 was reduced as DV MOI was in-
creased, whereas IFN-
␥
-induced phosphorylation of STAT1 and
STAT3 was unsuppressed or mildly increased under similar DV
MOIs infection (Fig. 6). To further correlate the tyrosine phos-
phorylation status of STATs with their DNA-binding activities, the
nuclear extracts of the treated cells were prepared and analyzed
with EMSA. As shown in Fig. 7, by competition with the excess
wild-type or mutant nonradiolabeled oligonucleotides, both
STAT1- (Fig. 7A) and STAT3-containing (Fig. 7B) specific bands
were identified. Consistently, these STAT1- and STAT3-specific
protein-DNA complexes induced by IFN-
␣
were suppressed by
DV infection (Fig. 7, Aand B). Again, the IFN-
␥
-induced DNA-
binding activities of both STAT-1 and STAT-3 seemed to be un-
suppressed or mildly increased by DV infection (Fig. 7, Aand B).
DV activated STATs through IFN-
␣
-dependent and IFN-
␣
-
independent signaling pathways
Although STATs can be activated by IFN-
␣
, a cytokine produced
from DV-infected DCs, the early kinetics of activation of STAT3
(Fig. 3Band 4A) suggested that the DV-induced activation of
STAT3 might be independent of IFN-
␣
. To explore this possibil-
ity, we examined the blocking effects of anti-IFN-
␣
neutralizing
Ab in DV-infected DCs. As shown in Fig. 8A, the pretreatment
with anti-IFN-
␣
neutralizing Ab successfully blocked the IFN-
␣
-
induced STAT3 tyrosine-phosphorylation. However, the anti-
IFN-
␣
neutralizing Ab failed to block the DV-induced STAT3
tyrosine-phosphorylation (Fig. 8B). Under the same conditions, the
DV-induced STAT1 tyrosine-phosphorylation was totally blocked
by anti-IFN-
␣
neutralizing Ab (Fig. 8C). It suggested that DV
FIGURE 5. DV inhibited STAT1
and STAT3 tyrosine-phosphorylation
induced by IFN-
␣
but not that in-
duced by IFN-
␥
. Human DCs at 1 ⫻
10
6
/ml (4 –7 ⫻10
6
cells in average in
each treatment condition) were in-
fected with mock or DV (NGC
strain) for 18 –24 h and then treated
with IFN-
␣
(1,000 U/ml) or IFN-
␥
(50 U/ml) for 5 or 15 min, respec-
tively, and then the total cell lysates
were prepared and the tyrosine-phos-
phorylated STAT1 (A) or STAT3 (B)
was determined by Western blotting.
Meanwhile, the total amounts of
STAT1 and STAT3 were deter-
mined. C, Similarly, the DV effects
on various concentrations of IFN-
␥
-
induced STAT1 activation were ex-
amined. D, DCs were infected by
mock or DV for 4, 6, 9, or 24 h before
the addition of IFN-
␣
. The tyrosine-
phosphorylated and total STAT1
were determined by Western blot-
ting. A–D, The fold induction was
presented as a comparison with the
signal intensity in mock-infected
control. The results were obtained
from at least three to six different do-
nors DCs with similar results.
FIGURE 6. Dose-dependent regulation of IFN-
␣
- or IFN-
␥
-induced
STAT1 and STAT3 tyrosine-phosphorylation by DV. Human DCs at 1 ⫻
10
6
/ml (4–7 ⫻10
6
cells in average in each treatment condition) were
infected by mock or DV (NGC strain) at various doses for 18 –24 h and
then stimulated with IFN-
␣
(1,000 U/ml) or IFN-
␥
(50 U/ml) for 5 or 15
min, respectively, and then the total and tyrosine-phosphorylated STAT1
and STAT3 were determined by Western blotting.
8168 DV BLOCK IFN-
␣
BUT NOT IFN-
␥
EFFECT VIA INHIBITING Tyk2-STAT
infection induced activation of STAT1 in a IFN-
␣
-dependent manner.
However, both IFN-
␣
-dependent and IFN-
␣
-independent mecha-
nisms were responsible for the activation of STAT3 in DV infection.
DV blocked antiviral effect of IFN-
␣
through targeting Tyk2 and
had no effect on IFNAR expression
According to Chee et al. (36), one of the mechanisms for herpes
simplex virus to block STAT activation is mediated through down-
regulation of IFNAR expression. To investigate whether this
mechanism was also operating in DV infection, the expression of
IFNAR1 and IFNAR2 after mock or DV infection was determined
by a flow cytometer. As shown in Fig. 9A, compared with mock
infection, DV infection did not affect the level of expression of
both IFNAR1 and IFNAR2. It then became relatively clear that the
target of DV might be the molecule transmitting signals between
IFNARs and STAT molecules. IFN-
␣
, after binding to its recep-
tors, immediately activates two tyrosine kinases, namely Jak1 and
Tyk2. Somewhat different from IFN-
␣
, IFN-
␥
, after binding to
IFN-
␥
receptors, activates both Jak1 and Jak2 but not Tyk2 ty-
rosine kinase. The inhibition of IFN-
␣
-induced but not IFN-
␥
-
induced STAT activities by DV suggested that Tyk2 might be the
one targeted by DV. The IFN-
␣
-induced tyrosine-phosphorylation
of Tyk2 was examined in DCs preinfected or not by DV. As shown
in Fig. 9B, DV greatly suppressed IFN-
␣
-induced Tyk2 tyrosine-
phosphorylation. Under such conditions, IFN-
␥
did not induce
Tyk2 tyrosine-phosphorylation. In these experiments, we also
noted that the longer exposure of the film revealed the weak acti-
vation of Tyk2 tyrosine phosphorylation in DV-infected cells but
not in mock-infected cells (data not shown).
Discussion
Earlier observations in DV-infected children reveal high plasma
concentrations of IFN-
␣
that coexist with elevated viral titers (37,
38). Our previous work also demonstrated that despite significant
amount of IFN-
␣
produced in DV-infected DCs, the viral produc-
tivity remains unsuppressed (5). As natural cellular targets for DV,
it is likely that DCs may respond inappropriately to IFN-
␣
or ig-
nore the signal from IFN-
␣
that results in viral overproduction. In
the present study, we showed that preinfection of DCs by DV
counteracted the antiviral protection of IFN-
␣
but not IFN-
␥
.In
addition, although IFN-
␣
potently induced STAT1 and STAT3
activation, such an effect was greatly attenuated by DV. The down-
regulation of IFN-
␣
-induced STAT activities by DV appeared to
involve the inhibition of Tyk2 tyrosine kinase activation (these
sequential events were summarized in Fig. 10). In contrast to IFN-
␣
-mediated effects, the IFN-
␥
-induced STAT1 and STAT3 acti-
vation was unsuppressed or mildly enhanced by DV. Therefore,
our studies suggest that although both preserved antiviral activi-
ties, IFN-
␣
and IFN-
␥
might play different roles at least in different
stages or different tissues of DV infection.
FIGURE 7. DV infection inhibited IFN-
␣
-induced but not IFN-
␥
-induced STAT1 and STAT3 DNA-binding activities. Human DCs 1 ⫻10
6
/ml (1–2 ⫻
10
7
cells in average in each treatment condition) were infected with mock or DV (NGC strain) for 18 –24 h, after wash, the cells were then treated or not
with IFN-
␣
(1,000 U/ml) or IFN-
␥
(50 U/ml) for 5 or 15 min, respectively. The nuclear extracts were then prepared and analyzed with EMSA for
determination for STAT1 (A) or STAT3 (B) DNA-binding activities. For competition assays, the nonradiolabeled wild-type (wt) or mutant (mt) probes were
added into the reaction mixtures 10 min before adding the radiolabeled probes. The bands were indicated as nonspecific (NS) because they could either
be competed by both wild-type and mutant probes or totally unaffected by both probes. The specific STAT1-containing (A) or STAT-3-containing (B)
DNA-binding complex was indicated because it could only be competed by the wild-type but not the mutant probe. The data are representative of at least
three independent experiments with similar results.
8169The Journal of Immunology
In fact, the differential critical roles of both IFN-
␣
and IFN-
␥
in
DV infection are suggested in animal studies using mice deficient
of either IFN-
␣
/IFN-

or IFN-
␥
or both receptors (16, 39). In the
absence of IFN-
␣
/IFN-

receptors, after DV infection, viral par-
ticles are detectable in various organs such as serum, liver, spleen,
lymph nodes, brain, and spinal cords (16). In contrast, viral parti-
cles are not detectable in these organs in IFN-
␥
receptor-deficient
mice. These experiments clearly suggest that the IFN-
␣
/IFN-

re-
ceptor-mediated protection is crucial in controlling initial DV pro-
duction and subsequent viral spreading. To the contrary, the IFN-
␥
receptor-mediated protection appears to be more important in con-
trolling viral-induced diseases, but it is less important in limiting
the early expansion of viral load (16). Nevertheless, in the absence
of IFN-
␣
/IFN-

receptor signaling, IFN-
␥
receptor signaling can
also mediate viral clearance (16).
It is currently not clear why DV selectively inhibited IFN-
␣
- but
not IFN-
␥
-mediated signaling events. One of the possibilities may
be due to the fact that IFN-
␣
, but not IFN-
␥
, is produced by virus-
infected cells (40). Depending on the environment, virus may or
may not have a chance to encounter IFN-
␥
. Therefore, the revo-
lutionary process does not arm virus to fight against IFN-
␥
.In
addition, in an example of hepatitis C virus infection, there is ev-
idence suggesting that long-term exposure of IFN-
␥
reduces the
protection from antiviral effect of IFN-
␣
in virus-infected cells
(41). The preservation of IFN-
␥
response may, in the long run,
provide an additional protection on virus from IFN-
␣
-mediated
effects. Furthermore, such a selection and differential roles of
IFN-
␣
and IFN-
␥
in DV-mediated immunopathologies as dis-
cussed above also indicates that virus cares more about itself rather
than the subsequent pathologies after infection.
Our studies also suggest the critical role of STAT proteins in the
pathogenesis of DV infection that attenuated the antiviral effect of
IFN-
␣
. There are many ways for viruses to escape from the pro-
tection by IFNs induced in viral-infected hosts. Examples such as
FIGURE 9. DV attenuated IFN-
␣
-induced tyrosine-
phsphorylation of Tyk2 but had no effect on IFNAR1
and IFNAR2 expression. A, DCs infected by mock or
DV (NGC strain) for 24 h were collected, and the ex-
pression of both IFN receptors (IFNAR1 and IFNAR2)
were determined by flow cytometry as described in Ma-
terials and Methods. The dashed line represented the
staining with isotype-matched control Ab. B, Human
DCs were infected with mock or DV (NGC strain) for
24 h and then treated with IFN-
␣
(1000 U/ml) or IFN-
␥
(50 U/ml) for 5 or 15 min, respectively, and then the
total cell lysates were prepared and the tyrosine-phos-
phorylated Tyk2 was determined by Western blotting.
The total amounts of Tyk2 were also determined. Here,
longer exposure of the film could reveal the tyrosine-
phosphorylated Tyk2 band in DV-infected but not in
mock-infected cells (data not shown). The results were
obtained from at least three to six different donors DC
with similar results.
FIGURE 8. DV induced both IFN-
␣
-dependent and IFN-
␣
-independent STAT activation. Human DCs 1 ⫻10
6
/ml (4 –7 ⫻10
6
cells in average in each
treatment condition) were pretreated in the presence or absence of anti-IFN-
␣
neutralizing Ab for 2 h and then stimulated or not with IFN-
␣
at 1000 IU/ml
for 5 min. A, The tyrosine-phosphorylated STAT3 was determined by Western blotting. B, Human DCs were pretreated or not with anti-IFN-
␣
neutralizing
Ab or control IgG (Ctl Ig) and then infected with mock or DV (NGC strain) for 18 –24 h. The tyrosine-phosphorylated STAT3 was determined. C, Similarly,
the tyrosine-phosphorylated STAT1 was determined. The results were obtained from at least three to six different donors DCs with identical results.
8170 DV BLOCK IFN-
␣
BUT NOT IFN-
␥
EFFECT VIA INHIBITING Tyk2-STAT
the reduction of IFN synthesis (42) and the inhibition of IFN re-
ceptor expression (36) have been reported. In addition, several
mechanisms that include degradation of STAT proteins, down-
regulation of STAT phosphorylation, or STAT methylation as well
as inhibition of STAT binding to promoters of target genes have
been observed in different viral species infection (43– 47). The
reason to target STAT proteins by viruses may be explained in part
by the studies in STAT-deficient animals or humans. In STAT1-
deficient mice, the response to either IFN-
␣
or IFN-
␥
is totally
abolished, and the animals appear to be highly sensitive to infec-
tion by microbial pathogens and viruses (48, 49). Under such con-
ditions, the responses to other cytokines that can also activate
STAT1 are preserved in these mice. In support, human beings with
deficiency of STAT1 died of viral diseases, and the established cell
lines from these victims are unresponsive to antiviral protection of
IFN-
␣
(50). These studies support the significance of STAT1 in
IFN-mediated antiviral effects.
The mechanisms for DV to inhibit Tyk2 tyrosine kinase activa-
tion were at this moment unclear. To our surprise, the inhibition of
Tyk2 tyrosine kinase was not unique in DV infection because sim-
ilar mechanism was also observed in human papilloma virus in-
fection and Japanese encephalitis virus infection (51, 52). Through
the produced E6 protein, human papilloma virus is able to suppress
the activation of IFN-
␣
-induced but not IFN-
␥
-induced Tyk2-
STAT signaling pathway in human epithelial-like cell lines (51).
Additional experiments may be aimed to explore the mechanisms
underlying the suppression of Tyk2 tyrosine kinase activation by
DV infection as well as the viral proteins responsible for this
effect.
Because DV2 NGC strain has been attenuated by serial passages
intracerebrally in mice, the observed anti-IFN-
␣
effect may possi-
bly be questioned about whether the results can be reproducibly
seen using other DV2 viral strains to infect cells. In this regard,
there is also a possibility that the differential sensitivities to IFNs
observed between a mouse brain-passaged virus and a tissue cul-
ture-passaged virus could reflect attenuated vs wild-type pheno-
types. Nevertheless, although not as strong as NGC strain infec-
tion, a certain extent of antagonism of IFN-
␣
-mediated antiviral
effect could also be observed in DV2 PL046 strain infection (Fig.
1D). In support of our observations, Mun˜oz-Jorda´n et al. (17),
using an infectious DV2 cDNA clone to infect different tissue
cells, demonstrated that DV infection antagonizes IFN-

-induced
antiviral activities. In addition, Diamond et al. (22) showed that
when IFN-

is added 4 and 24 h after DV2 16681 strain infection,
the antiviral activities of IFN-

is greatly reduced. Under similar
conditions, IFN-

remains preserving strong antiviral activities
when it is added 4 or 24 h before infection. Furthermore, such
observations are not only demonstrated using DV2 16681 strain to
infect different tissue cells like human foreskin fibroblasts and hep-
atoma cells but also shown using low-passage DV2 viral isolates to
infect cells (22). Therefore, the observed antagonism of IFN-
␣
antiviral effect by DV2 NGC strain infection examined mainly in
this report might also be applied to other DV2 viral strain infec-
tion. We are currently testing whether other DV serotypes (DV1,
DV3, and DV4) and other strains of DV2 infection may also share
similar effects to antagonize IFN-
␣
antiviral activities. Finally, to
be more close to physiological conditions, the study using DV with
only limited passage in tissue cultures becomes very critical and
mandatory in the future.
Acknowledgments
We thank Dr. C. L. Kao for the kind gifts provided.
FIGURE 10. Cartoon of the regulation of IFN-
␣
-STAT and IFN-
␥
-STAT signaling pathways by DV infection. DV infection of DCs led to synthesis of
viral proteins and induced activation of transcription factors that resulted in the induction of IFN-
␣
synthesis and secretion. Meantime, DV infection also
caused activation of STAT3 with unclear significance. The activation of these transcription factors and STAT3 was not necessarily by the newly synthesized
viral proteins as indicated with a question mark (?). The secreted IFN-
␣
then bind IFNARs and induce the activation of Tyk2 and Jak1 as well as the
downstream molecules that include STAT1, STAT2, IFN regulatory factor-9 (IRF-9), and STAT3. Through blocking Tyk2 activation as well as its
downstream molecules, DV viral proteins were able to attenuate IFN-
␣
-induced activation and secretion of IFN-
␣
-responsive genes and proteins. In the
example of IFN-
␥
, after binding to its receptors, activation of STAT1 and STAT3 can be observed. Such an effect was unsuppressed or mildly increased
by DV infection. The down-stream signaling events after activation of STAT3 may involve at least the activation of NF-
B transcription factors that were
not shown in this cartoon. ⫹, Stimulatory; ⫺, inhibitory; dashed lines represent suppressed signaling.
8171The Journal of Immunology
Disclosures
The authors have no financial conflict of interest.
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8172 DV BLOCK IFN-
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BUT NOT IFN-
␥
EFFECT VIA INHIBITING Tyk2-STAT
