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but Not IFN-
Antiviral Effect via Down-Regulating Tyk2-STAT Signaling in the Human Dendritic Cell1
* Division of Gerontology Research, National Health Research Institute, Rheumatology/Immunology and Allergy, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, and Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
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Abstract |
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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 provide very useful messages with physiological significance in investigation of the pathogenesis, the defense mechanisms of human hosts and the therapeutic considerations in DV infection. |
Introduction |
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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, 5, 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 inhibit viral spreading (10, 11, 12, 13, 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 producing viral progenies (5). Such an observation is also reflected in DV-infected children (15). It may suggest the presence of mechanisms 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 viruses, STAT proteins are indeed one of the major targets for viruses to block and to counteract the antiviral effects of IFNs (13). To appreciate whether and how both IFN- and IFN- may possibly mediate their antiviral activities in DV-infected DC and how DV may interfere with these mechanisms, we used purified monocyte-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-phosphorylation of Tyk2 tyrosine kinase.
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Materials and Methods |
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The cell culture medium consisted of RPMI 1640 (Invitrogen Life Technologies) 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 specified, 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 centrifugation, the layer of mononuclear cells was collected. After lysis of RBC, the PBMC were obtained. To obtain DCs with high purity, PBMC were incubated 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 containing 800 U/ml GM-CSF and 500 U/ml IL-4 at a cell density of 1 x 106 cells/ml. The culture medium was changed every other day with 300 µl of 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 modifications (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 isolated 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% CO2 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 adsorption, 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.
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DCs cultured for 5 days were infected with mock or DV at different multiplicity of infections (MOIs) or at MOI 5 (in most of the conditions of this report) for 4 h at 37°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 x 106/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 resuspended in 50 µl of PBS containing 1% BSA. Then anti-CD1a mAb conjugated 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 104 events.
Nuclear extract preparation
Nuclear extracts were prepared according to our published work (20). Briefly, the treated cells (1–2 x 107 cells in average in each treatment condition) were left at 4°C in 50 µl 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 µg/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 supernatants (cytoplasmic extracts), the pelleted nuclei were washed with 50 µl of buffer A and subsequently, the cell pellets were resuspended in 30 µl of buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, and 3.3 µg/ml aprotinin) 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 [-32P]ATP using the T4 kinase according to the manufacturer’s instructions (Promega). For the binding reaction, the radiolabeled STAT probe was incubated with 5 µg of nuclear extracts. The binding 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 µg 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 concentration of 1/5000 for 30 min. The filter was then incubated with the substrate and exposed to x-ray film.
Statistics
When necessary, the results were expressed as means ± SD. A paired or unpaired Student’s t test was used to determine the difference that was thought to be significant when p < 0.05.
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Results |
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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 Diamond 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 infected cells, DCs were first infected by DV; after viral absorption and extensive wash, the cells were incubated with different concentrations of IFN- or IFN- 2 h later and left for an additional 18 h incubation. Viral titers in supernatants then were determined. In contrast to preinfection treatment of cytokines, the antiviral effects 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 observed 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 preinfection 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 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 peripheral 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 expression of CD1a (DCs) and CD14 (monocytes) clearly distinguished 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 affected 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 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, respectively (Fig. 2B). The infectious rate of viruses in DCs correlated quite well with another report (4) and our unpublished observations.
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It has been generally accepted that binding of IFN- to its ligand results in the activation of STAT1, STAT2, and STAT3 (23, 24, 25). In addition, IFN- also activates STAT4, STAT5, and STAT6 in T cells, B cells, and Daudi cells (26, 27, 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 activation of STAT1, IFN- also activates several genes like chemokine 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, 32, 33, 34). Interestingly, 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. 3B). At examined time points, the activation of both STAT5 and STAT6 was not detectable (Fig. 3C). Because after tyrosine-phosphorylation, 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 activities 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. 4B and data not shown). Within the determined 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.
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-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-mediated 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--induced STAT1 (Fig. 5A) and STAT3 (Fig. 5B) tyrosine-phosphorylation was reduced by DV. In contrast, as a side-by-side comparison, 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 than 6 h before the addition of IFN- (Fig. 5D). Subsequent experiments further demonstrated that the IFN--induced phosphorylation of STAT1 and STAT3 was reduced as DV MOI was increased, 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 phosphorylation 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, A and B). Again, the IFN--induced DNA-binding activities of both STAT-1 and STAT-3 seemed to be unsuppressed or mildly increased by DV infection (Fig. 7, A and B).
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-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. 3B and 4A) suggested that the DV-induced activation of STAT3 might be independent of IFN-. To explore this possibility, 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 infection induced activation of STAT1 in a IFN--dependent manner. However, both IFN--dependent and IFN--independent mechanisms were responsible for the activation of STAT3 in DV infection.
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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 receptors, 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 tyrosine 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 activation of Tyk2 tyrosine phosphorylation in DV-infected cells but not in mock-infected cells (data not shown).
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Discussion |
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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 productivity remains unsuppressed (5). As natural cellular targets for DV, it is likely that DCs may respond inappropriately to IFN- or ignore 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 activation was unsuppressed or mildly enhanced by DV. Therefore, our studies suggest that although both preserved antiviral activities, IFN- and IFN- might play different roles at least in different stages or different tissues of DV infection.
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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 particles are detectable in various organs such as serum, liver, spleen, lymph nodes, brain, and spinal cords (16). In contrast, viral particles are not detectable in these organs in IFN- receptor-deficient mice. These experiments clearly suggest that the IFN-/IFN- receptor-mediated protection is crucial in controlling initial DV production and subsequent viral spreading. To the contrary, the IFN- receptor-mediated protection appears to be more important in controlling 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 revolutionary process does not arm virus to fight against IFN-. In addition, in an example of hepatitis C virus infection, there is evidence 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 discussed 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 protection by IFNs induced in viral-infected hosts. Examples such as the reduction of IFN synthesis (42) and the inhibition of IFN receptor 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, 44, 45, 46, 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 infection by microbial pathogens and viruses (48, 49). Under such conditions, 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 activation were at this moment unclear. To our surprise, the inhibition of Tyk2 tyrosine kinase was not unique in DV infection because similar mechanism was also observed in human papilloma virus infection 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 possibly 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 culture-passaged virus could reflect attenuated vs wild-type phenotypes. Nevertheless, although not as strong as NGC strain infection, 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, Muñoz-Jordán 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 hepatoma 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 infection. 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.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work is supported in part by grants from the National Health Research Institutes (to L.-J.H. and to J.-H.L., NHRI-EX92-9208SI) and the National Science Council (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{at}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.
Received for publication October 19, 2004. Accepted for publication March 30, 2005.
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  J. A. Souza-Neto, S. Sim, and G. Dimopoulos An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense PNAS, October 20, 2009; 106(42): 17841 - 17846. [Abstract] [Full Text] [PDF]
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I. Umareddy, K. F. Tang, S. G. Vasudevan, S. Devi, M. L. Hibberd, and F. Gu Dengue virus regulates type I interferon signalling in a strain-dependent manner in human cell lines J. Gen. Virol., December 1, 2008; 89(12): 3052 - 3062. [Abstract] [Full Text] [PDF]
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 W. Dejnirattisai, T. Duangchinda, C.-L. S. Lin, S. Vasanawathana, M. Jones, M. Jacobs, P. Malasit, X.-n. Xu, G. Screaton, and J. Mongkolsapaya A Complex Interplay among Virus, Dendritic Cells, T Cells, and Cytokines in Dengue Virus Infections J. Immunol., November 1, 2008; 181(9): 5865 - 5874. [Abstract] [Full Text] [PDF]
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 Z. D. Nightingale, C. Patkar, and A. L. Rothman Viral replication and paracrine effects result in distinct, functional responses of dendritic cells following infection with dengue 2 virus J. Leukoc. Biol., October 1, 2008; 84(4): 1028 - 1038. [Abstract] [Full Text] [PDF]
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R. V. Warke, A. Becerra, A. Zawadzka, D. J. Schmidt, K. J. Martin, K. Giaya, J. H. Dinsmore, M. Woda, G. Hendricks, T. Levine, et al. Efficient dengue virus (DENV) infection of human muscle satellite cells upregulates type I interferon response genes and differentially modulates MHC I expression on bystander and DENV-infected cells J. Gen. Virol., July 1, 2008; 89(7): 1605 - 1615. [Abstract] [Full Text] [PDF]
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R. E. Randall and S. Goodbourn Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures J. Gen. Virol., January 1, 2008; 89(1): 1 - 47. [Abstract] [Full Text] [PDF]
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 R. V. Warke, K. J. Martin, K. Giaya, S. K. Shaw, A. L. Rothman, and I. Bosch TRAIL Is a Novel Antiviral Protein against Dengue Virus J. Virol., January 1, 2008; 82(1): 555 - 564. [Abstract] [Full Text] [PDF]
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G. S. Park, K. L. Morris, R. G. Hallett, M. E. Bloom, and S. M. Best Identification of Residues Critical for the Interferon Antagonist Function of Langat Virus NS5 Reveals a Role for the RNA-Dependent RNA Polymerase Domain J. Virol., July 1, 2007; 81(13): 6936 - 6946. [Abstract] [Full Text] [PDF]
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 C. A. Sariol, J. L. Munoz-Jordan, K. Abel, L. C. Rosado, P. Pantoja, L. Giavedoni, I. V. Rodriguez, L. J. White, M. Martinez, T. Arana, et al. Transcriptional Activation of Interferon-Stimulated Genes but Not of Cytokine Genes after Primary Infection of Rhesus Macaques with Dengue Virus Type 1 Clin. Vaccine Immunol., June 1, 2007; 14(6): 756 - 766. [Abstract] [Full Text] [PDF]
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J. J. H. Chu and P. L. Yang c-Src protein kinase inhibitors block assembly and maturation of dengue virus PNAS, February 27, 2007; 104(9): 3520 - 3525. [Abstract] [Full Text] [PDF]
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T. Chareonsirisuthigul, S. Kalayanarooj, and S. Ubol Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells J. Gen. Virol., February 1, 2007; 88(2): 365 - 375. [Abstract] [Full Text] [PDF]
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K. Clyde, J. L. Kyle, and E. Harris Recent Advances in Deciphering Viral and Host Determinants of Dengue Virus Replication and Pathogenesis J. Virol., December 1, 2006; 80(23): 11418 - 11431. [Full Text] [PDF]
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R.-J. Lin, B.-L. Chang, H.-P. Yu, C.-L. Liao, and Y.-L. Lin Blocking of Interferon-Induced Jak-Stat Signaling by Japanese Encephalitis Virus NS5 through a Protein Tyrosine Phosphatase-Mediated Mechanism. J. Virol., June 1, 2006; 80(12): 5908 - 5918. [Abstract] [Full Text] [PDF]
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