Dengue Virus Type 2 Antagonizes IFN-alpha but Not IFN-gamma Antiviral Effect via Down-Regulating Tyk2-STAT Signaling in the Human Dendritic Cell.pdf

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the Human Dendritic CellDown-Regulating Tyk2-STAT Signaling inAntiviral Effect viaγ Not IFN-

butαDengue Virus Type 2 Antagonizes IFN-

and Jenn-Haung LaiPing Chou, Yi-Ling Lin, Deh-Ming Chang, Tong-Yuan TaiLing-Jun Ho, Li-Feng Hung, Chun-Yi Weng, Wan-Lin Wu,

http://www.jimmunol.org/content/174/12/81632005; 174:8163-8172; ; J Immunol

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Materials and MethodsCulture 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/mlpenicillin-streptomycin (Invitrogen Life Technologies). Recombinant GM-CSF and IL-4 were purchased from R&D Systems. Abs against totalSTAT1, STAT2, STAT3, STAT5, and STAT6 were purchased from SantaCruz 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 SignalingTechnology. Anti-STAT2-pY was purchased from Upstate Biotechnology.Both human IFN- and IFN- were purchased from R&D Systems. Thefluorescence-labeled anti-IFN- receptor (IFNAR)1, anti-IFNAR2, CD3,CD14, CD19, HLA-DR, CD83, CD86, CD1a, CD11c and CD11b mAbswere purchased from BD Pharmingen. Both anti-IFN- neutralizing Aband 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 from80–100 different healthy donors by using a MACS cell isolation columnfollowing manufacturer’s instructions (Miltenyi Biotech). In brief, buffycoat (each buffy coat is equivalent to 500 ml of whole blood) from a bloodbank (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, theCD14 cells were isolated using a MACS cell isolation column (MiltenyiBiotec). 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 1 106

cells/ml. The culture medium was changed every other day with 300 l of fresh medium containing 2400 U GM-CSF and 1500 U IL-4, and the cellswere used for experiments after 5–7 days of culture. The purity of DCs, asdetermined by the positive staining of CD1a, was consistently higher than90% 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-typewith unknown passage and nonmouse adapted local Taiwanese strain iso-

lated from a patient with DF in 1981, were propagated in C6/36 mosquitocells in RPMI 1640 containing 5% heat-inactivated FCS and maintained at28°C in a 5% CO2 atmosphere for 7 days. The supernatants were collected,and virus titers were determined and then stored at 70°C until use. Todetermine virus titers, the culture supernatants were harvested for plaque-forming assays. Various virus dilutions were added to 80% confluent babyhamster 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 incubationfor 7 days, cells were fixed with 10% formaldehyde and stained with 0.5%crystal violet. The numbers of plaques were counted and the results wereshown as PFU per milliliter. Aside from Fig. 1 D, where the PL046 strainwas the viral strain used, the NGC strain was the only source of DV toinfect 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 thisreport) for 4 h at 37°C (5). After viral absorption, cells were then washedand cultured in six-well plates (Costar) with culture medium in the absenceof exogenously added cytokines for various periods of time as indicated inthe figures. For treatment, the cell density was maintained at 1 106 /ml inculture medium.

Flow cytometric analysis

The determination of single expression or coexpression of both cell surfaceand 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 30min. 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 mAbconjugated 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 104 events.

Nuclear extract preparation

Nuclear extracts were prepared according to our published work (20).Briefly, the treated cells (1–2 107 cells in average in each treatmentcondition) were left at 4°C in 50 l of buffer A (10 mM HEPES, pH 7.9,10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF, and 3.3 g/mlaprotinin) for 15 min with occasional gentle vortexing. The swollen cellswere centrifuged at 15,000 rpm for 3 min. After removal of the superna-

tants (cytoplasmic extracts), the pelleted nuclei were washed with 50 l of buffer A and subsequently, the cell pellets were resuspended in 30 l of buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mMEDTA, 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 thesupernatants were used as nuclear extracts.

EMSA

The EMSA was performed as detailed in our previous report (20). Theoligonucleotides containing STAT1, STAT3, STAT5, and STAT6 werepurchased and used as DNA probes (Promega). The DNA probes wereradiolabeled with [ -32P]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 MgCl2, 4% glycerol, and 2 g poly(dI-dC). Thereaction mixture was left at room temperature to proceed with bindingreaction for 20 min. If unradiolabeled competitive oligonucleotides wereadded, they were used as 100-fold molar excess and preincubated withnuclear 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 inlysis buffer. After periodic vortexing, the mixture was centrifuged, thesupernatant 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, thenitrocellulose filter was incubated with TBS-T containing 5% nonfat milk (milk buffer) for 2 h, and then blotted with antisera against individualproteins 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 orunpaired Student’s t test was used to determine the difference that wasthought to be significant when p 0.05.

ResultsTreatment 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-mediateddisease 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. 1 A, 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. 1 B). 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. 1 D, 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. 1 D, 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 1 106 /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 1 106 /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 B was 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. C and 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. 2 A, 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 104 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 1 106 /ml (4–7

106 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.




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. 2 B). 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, A and 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. 3 B).

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. 4 A, 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. 4 B and data not shown). Within the deter-

mined time points, the DNA-binding activities of both STAT5 and

STAT6 were not detectable (Fig. 4 B). 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. 5 A) and STAT3 (Fig. 5 B) 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, A and 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. 5C illustrated 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 1 106 /ml (1–2 107 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.




than 6 h before the addition of IFN- (Fig. 5 D). 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. 7 A) and STAT3-containing (Fig. 7 B) specific bands

were identified. Consistently, these STAT1- and STAT3-specific

protein-DNA complexes induced by IFN- were suppressed by

DV infection (Fig. 7, A and 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, A and 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. 3 B and 4 A) 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. 8 A, 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. 8 B). 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

106 /ml (4 –7 106 cells in average in

each treatment condition) were in-

fected with mock or DV (NGC

strain) for 18–24 h and then treatedwith 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 STAT1were 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

106 /ml (4 –7 106 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 15min, respectively, and then the total and tyrosine-phosphorylated STAT1

and STAT3 were determined by Western blotting.




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. 9 A, 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. 9 B, 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).

DiscussionEarlier 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 1 106 /ml (1–2

107 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 leastthree independent experiments with similar results.




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 limitingthe 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 ratherthan 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 1 106 /ml (4 –7 106 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.




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 inIFN-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 IFNsobserved 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.

1 D). In support of our observations, Munoz-Jordan 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, suchobservations 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.

AcknowledgmentsWe 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.




DisclosuresThe authors have no financial conflict of interest.

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