research article dengue virus infection causes the activation of...

Research ArticleDengue Virus Infection Causes the Activation ofDistinct NF-120581B Pathways for Inducible Nitric OxideSynthase and TNF-120572 Expression in RAW2647 Cells

Yi-Lin Cheng1 Yee-Shin Lin123 Chia-Ling Chen4 Shu-Wen Wan3 Yi-Dan Ou2

Chia-Yi Yu3 Tsung-Ting Tsai5 Po-Chun Tseng6 and Chiou-Feng Lin57

1 Institute of Basic Medical Science College of Medicine National Cheng Kung University Tainan 701 Taiwan2Department of Microbiology and Immunology College of Medicine National Cheng Kung University Tainan 701 Taiwan3Center of Infectious Diseases and Signaling Research National Cheng Kung University Tainan 701 Taiwan4Translational Research Center Taipei Medical University Taipei 110 Taiwan5Department of Microbiology and Immunology College of Medicine Taipei Medical University Taipei 110 Taiwan6Institute of Clinical Medicine College of Medicine National Cheng Kung University Tainan 701 Taiwan7Graduate Institute of Medical Sciences College of Medicine Taipei Medical University Taipei 110 Taiwan

Correspondence should be addressed to Chiou-Feng Lin cflin2014tmuedutw

Received 29 October 2014 Revised 14 April 2015 Accepted 19 May 2015

Academic Editor Dennis D Taub

Copyright copy 2015 Yi-Lin Cheng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Infection with dengue virus (DENV) causes an increase in proinflammatory responses such as nitric oxide (NO) generation andTNF-120572 expression however the molecular mechanism underlying this inflammatory activation remains undefined although theactivation of the transcription factor NF-120581B is generally involved In addition to TNF-120572 production in DENV-infected murinemacrophage RAW2647 cells inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied byNO generation NF-120581B is known to be activated by DENV infection Pharmacologically inhibiting NF-120581B activation abolishesiNOSNO biosynthesis and TNF-120572 production With inhibition the potential role of NF-120581B in oxidative signaling regulation wasprevented during DENV infection Heat-inactivated DENV failed to cause the identified inflammatory responses Pharmacologicalinhibition of TLR3 partly decreased NF-120581B activation however it effectively abolished inducible iNOSNO biosynthesis but didnot inhibit TNF-120572 production In contrast to TLR3 viral protein NS2B3 also independently contributed to NF-120581B activation toregulate TNF-120572 productionThese results show the distinct pathways for NF-120581B activation caused by DENV infection individuallyfor the regulation of iNOSNO and TNF-120572 expression

1 Introduction

Dengue virus (DENV) which has 4 serotypes is a mosquito-borne enveloped positive-stranded RNA virus in the Fla-viviridae family [1] It is estimated that approximately 390million dengue infections occur per year of which 96millionmanifest apparent symptoms [2] Although most cases ofDENV infection are asymptomatic a variable spectrum ofsymptoms can occur ranging from a mild fever to fatalsevere dengue hemorrhagic fever (DHF) and dengue shocksyndrome (DSS) In DHFDSS cases potentially lethal com-plications including plasma leakage severe hemorrhages

and organ failure can develop [1] Unfortunately there iscurrently no effective vaccine or specific antiviral treatment

Although the mechanisms of DHFDSS have not beenfully clarified it is believed that several proinflammatorymediators are involved in hemorrhage development suchas nitric oxide (NO) and tumor necrosis factor-120572 (TNF-120572)[3 4] In monocytes collected from DENV patients withacute fever inducibleNO synthase (iNOS) themajor enzymecatalyzing NO production is significantly increased [5] Asseen in our previous studies NO can cause endothelial celldamage resulting in vascular leakage which is induced byantibodies against DENV nonstructural protein (NS) 1 [6ndash8]

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 274025 13 pageshttpdxdoiorg1011552015274025

2 Mediators of Inflammation

In addition Yen et al also observed increased macrophageinfiltration and TNF-120572 production in the endothelium ofthe hemorrhagic tissue and endothelial cell apoptosis in thetissues with enhanced expression of iNOS and nitrotyrosinein DENV-infected mice [9] Furthermore TNF-120572 has beenreported to induce hemorrhage by causing endothelial celldeath which is associated with increased endothelial cellpermeability in an experimental dengue hemorrhage mousemodel [9 10] TNF receptor levels have also shown a highlypositive correlation with the severity of DHF in patients [11]However the underlying molecular mechanism of DENV-induced inflammatory activation remains largely unknown

Nuclear factor-120581B (NF-120581B) which is a well-known tran-scription factor participates in the regulation of proinflam-matory mediators including iNOS and TNF-120572 NF-120581B is aheterodimer consisting of p65 and p50 subunits which asso-ciate with the inhibitor of NF-120581B (I120581B) family of inhibitoryproteins Upon I120581B degradation NF-120581B translocates to thenucleus and drives the expression of downstream target genes[12] NF-120581B can be activated by several different signalingpathways including the receptor-mediated signaling of pat-tern recognition receptors such as Toll-like receptors (TLRs)retinoic-acid-inducible gene I melanoma differentiation-associated gene 5 [13] and reactive oxygen species- (ROS-)induced pathways [14] In DENV infection Tsai et al showedthat TLR3 plays a major role in interleukin- (IL-) 8 produc-tion and viral replication compared with other TLRs [15]NF-120581B activation during DENV infection has been reportedsince the 1990s when several studies showed that DENVcan induce NF-120581B activation leading to the induction ofapoptosis in different cell types [16ndash18] however the under-lyingmolecularmechanism remains undefined Recently Linet al clarified that the activation of NF-120581B which leads toendothelium cell apoptosis and hemorrhage developmentoccurs through the interaction of the DENV NS2B-NS3(NS2B3) protease complex with I120581B120572120573 NS2B3 cleavesI120581B120572120573 and activates I120581B kinase [19] However whetherNF-120581B is involved in DENV-induced iNOSNO and TNF-120572production and which pathwaymediates these events are stilla mystery Becausemonocytic cells are the DENV target cellswe usedmurinemononuclear phagocyte RAW2647 cells as amodel [20 21] In this study we aimed to investigate the roleof NF-120581B in DENV-induced iNOSNO and TNF-120572 produc-tion and to unveil the underlying molecular mechanism ofNF-120581B activation

2 Materials and Methods

21 Antibodies and Reagents The reagents and antibodiesused were translation inhibitor cycloheximide transcriptioninhibitor actinomycin D NF-120581B inhibitor caffeic acid phe-nethyl ester (CAPE) antioxidant N-acetyl-cysteine (NAC)Poly(IC) TLR3 inhibitor dimethyl sulfoxide (DMSO)and mouse monoclonal antibody (Ab) specific for 120573-actin(Sigma-Aldrich St Louis MO) Abs against DENV NS1 andenvelope (E) (GeneTex San Antonio TX) Abs against iNOSand NF-120581B (Cell Signaling Technology Beverly MA) Absagainst isotype control IgG (Millipore BillericaMA) donkeyanti-goat IgG conjugated with horseradish peroxidase (HRP)

(Santa Cruz Biotechnology Santa Cruz CA) and goat anti-rabbit IgG conjugated with HRP (Chemicon InternationalTemecula CA) rabbit anti-mouse IgG conjugated with HRP(Abcam Cambridge MA) and Alexa Fluor 488- and AlexaFluor 594-conjugated goat anti-mouse and goat anti-rabbit(Invitrogen Carlsbad CA) Inhibitors were then dissolvedin DMSO prior to dilution with sterile phosphate-bufferedsaline (PBS) for use in experiments Other chemical reagentswere obtained from Sigma-Aldrich All drug treatments oncells were assessed for cytotoxic effects using cytotoxicityassays prior to experiments Noncytotoxic dosages were usedin this study

22 Cell Culture and Virus Culture RAW2647 mouse macro-phage was purchased from American Type Culture Collec-tion (ATCC number TIB-71) and were grown on plastic inDulbeccorsquos modified Eaglersquos medium (DMEM) with 10fetal bovine serum (FBS) (Sigma-Aldrich St Louis MO)100 units of penicillin and 100 120583gmL of streptomycin at37∘C under 5 CO

2 Cells were used at 3 to 5 passages

Baby hamster kidney (BHK) cells and C636 cells werecultured inDMEM(Invitrogen Life Technologies) containingFBS DENV2 PL046 strain was maintained in C636 cellsMonolayers of C636 cells were incubated with DENV at amultiplicity of infection (MOI) of 001 and incubated at 28∘Cin 5 CO

2for 5 days The virus supernatant was further

filtered with 022120583mfilter and then stored at minus80∘C until useVirus titer was determined by plaque assay using the BHKcell line

23 DENV Infection Cells were resuspended at a concentra-tion of 5 times 105 cellsmL in appropriate medium with DENVand incubated for 90min at 37∘CThen the cells were washedonce with DMEM medium resuspended at a concentrationof 5 times 105 cellsmL and incubated at 37∘C with 5 CO

2 The

viral supernatants were checked using plaque assays

24 Plaque Assay BHK-21 cells were plated into 12-wellplates (2 times 105 cellswell) and cultured in DMEM underCO2-enriched conditions After adsorption with a serially

diluted virus solution for 1 h the solution was replaced withfresh DMEM containing 2 FBS and 05 methyl cellulose(Sigma-Aldrich) Five days after infection the medium wasremoved and the cells were fixed and stained with crystalviolet solution containing of 1 crystal violet 064 NaCland 2 formalin

25 Western Blotting Harvested cells were lysed in a buffercontaining 1 Triton X-100 50mM Tris (pH 75) 10mMEDTA 002 NaN

3 and a protease inhibitor cocktail (Roche

Boehringer Mannheim Diagnostics Mannheim Germany)After a freeze-thaw cycle cell lysates were centrifuged at10000timesg at 4∘C for 20min The lysates were boiled in asample buffer for 5min Proteins were then subjected toSDS-PAGE and transferred to PVDF membranes (MilliporeBillerica MA USA) using a semidry electroblotting systemAfter blocking with 5 skim milk in PBS the membraneswere incubated overnight with a 1 1000 dilution of primaryantibodies at 4∘C The membranes were then washed with

Mediators of Inflammation 3

005 PBS-Tween 20 and incubated with a 1 5000 dilutionof HRP-conjugated secondary antibody at room tempera-ture for 1 h After washing the membranes were soakedin ECL solution (PerkinElmer Life and Analytical SciencesInc Boston MA USA) for 1min and exposed to an X-ray film (BioMax Eastman Kodak Rochester NY USA)The relative signal intensity was quantified using ImageJsoftware (version 141o W Rasband National Institutes ofHealth Bethesda MD USA) The changes in the ratio ofproteins compared with the normalized value of untreatedcells (indicated protein120573-actin) are also determined Oneset of representative data obtained from three independentexperiments is shown and the data shown as the mean plusmn SDvalues from three independent experiments

26 TNF-120572 Expression After treatment we used a commer-cial enzyme-linked immunosorbent assay (ELISA) kit (RampDMN USA) to detect the concentration of murine TNF-120572 incell-conditioned culture medium according to the manufac-turerrsquos instructions

27 Detection of NO Production Production of NO wasassessed as the accumulation of nitrite (NO

2

minus) in themediumusing a colorimetric reaction with the Griess reagent Brieflysamples (cell culture supernatants or murine ascites) weremixed with an equal (1 1) volume of Griess reagent (01 N-(1-naphthyl)ethylenediamine dihydrochloride 1 sulfanil-amide and 25 H

3PO4) The absorbance was measured at

540 nm using a 96-well microplate reader (Spectra MAX340PC Molecular Devices) data were analyzed using Soft-max Pro software Sodium nitrite was dissolved in double-distilled water and then used as standards (from 1 to 50 120583M)

28 Luciferase Reporter Assay To analyze NF-120581B promoteractivity by a luciferase reporter assay transient transfec-tion was performed using the TurboFect Cell TransfectionReagent (Thermo PA USA) In short cells were cotrans-fected with 02 120583g of an NF-120581B-promoter-driven firefly luci-ferase reporter and 001 120583g of a Renilla luciferase-expressingplasmid (pRL-TK Promega) Twenty-four hours after thetransfection the cells were infected with DENV for 24 hlysed and then harvested for luciferase and Renillameasure-ment using a luciferase assay system (Dual-Glo Promega)For each lysate the firefly luciferase activity was normalizedto theRenilla luciferase activity to assess transfection efficien-cies

29 Immunostaining To detect expression of NF-120581B andDENV E protein cells were fixed with 4 paraformaldehydepermeabilized with 05 Triton X-100 and washed twicewith ice-cold PBS Cells were stained with anti-NF-120581B andDENV E Abs and then with Alexa 488-conjugated goatanti-mouse IgG and Alexa 594-conjugated goat anti-rabbitIgG DAPI (5 120583gmL) was used for nuclear staining Cellswere visualized under a laser-scanning immunofluorescencemicroscope (IX71 Olympus PA USA)

210 Transfection Transient transfection was performedusing TurboFect Cell Transfection Reagent (Thermo PA

USA) according to the manufacturerrsquos instructions for opti-mization and usage The plasmid expressing Flag-taggedwild-type (WT) NS2B3 and a protease-dead mutant NS2B3which has a single point mutation changing serine residue135 of NS3 to alanine (S135A) and its control pCR31 weregenerated previously [22] After transfection the cells werecultured for 24 h before the experiments

211 Intracellular ROS Assay Intracellular oxidative stress wasmeasured by dichlorodihydrofluorescein diacetate oxidationCells were plated at 1 times 105well in 96-well plates culturedovernight and washed twice with Hankrsquos Buffered Salt Solu-tion (HBSS) before experiments Cells were exposed to 20120583M5-(and-6)-chloromethyl-2101584071015840-dichlorodihydrofluorescein dia-cetate acetyl ester (CM-H

2DCFDA) (Invitrogen Life Technol-

ogies Carlsbad CAUSA) for 1 h Fluorescencewas read imme-diately at wavelengths of 485 nm for excitation and 530 nm foremission on a fluorescence plate reader (Fluoroskan AscentThermo Electron Corporation Milford MA USA)

212 Statistical Analysis Data obtained from three indepen-dent experiments are presented as the mean plusmn standarddeviation (SD) Statistical analysis of data analyses wereperformed using Prism version 5 (GraphPad Software SanDiego CA) Two sets of the datawere analyzed by anunpairedStudentrsquos 119905 test Three or more sets of data were analyzed byone-wayANOVAwith Tukeyrsquosmultiple-comparison posttestStatistical significance was set at 119875 lt 005

3 Results

31 DENV Infection Transcriptionally and TranslationallyUpregulates iNOSNO Biosynthesis and TNF-120572 ProductionWe first examined whether murine mononuclear phagocyteRAW2647 cells could be efficiently infected by DENV sero-type 2 (DENV2) strain PL046 in vitro Usingwestern blottingand plaque assays we confirmed the expression of viral NS1and showed that viral titers were significantly increased at24 h after infection with an MOI of 50 (Figure 1(a)) Theseresults demonstrate that DENV infects RAW2647 cells invitro To evaluate the expression of TNF-120572 NO and iNOSwe determined the multiple MOIs of DENV infection andthe kinetics of response to DENV by ELISA Griess reagentand western blotting respectively The results showed thata relative increased response of TNF-120572 production andiNOS expression in the multiple MOIs of DENV infection(Figure 1(b)) According to the results we selected a highMOI of DENV infection for this study and all of these valuesincreased in a time-dependent manner (Figure 1(c)) Todeterminewhether the upregulation of TNF-120572 and iNOSNOoccurred through new protein synthesis RAW2647 cellswere infected with DENV in the presence or absence of thetranscription inhibitor cycloheximide (CHX) and the trans-lation inhibitor actinomycin D (ACD) for 24 h Treatmentwith CHX and ACD significantly suppressed the expressionof TNF-120572 NO and iNOS (Figure 1(d))These results demon-strate that RAW2647 cells infected by DENV induce TNF-120572production and iNOSNO biosynthesis through new proteinsynthesis


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Figure 1 DENV infection induces TNF-120572 production and iNOSNObiosynthesis through newprotein synthesis (a) RAW2647 cells infectedwithDENV serotype 2 PL046were assessed for DENVNS1 expression by western blot analysis and for viral replication by a plaque assay lowast119875 lt005 and lowastlowast119875 lt 001 compared with untreated cells (b c and d) ELISA Griessrsquo reagent and western blot analysis quantified the expressionof TNF-120572 and iNOSNO in DENV 2-infected cells for the differentMOIs the changes over time and in the presence of cycloheximide (CHX)and actinomycin D (ACD) DMSOwas used as the negative control lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with untreated cells119875 lt 005 compared with DENV ns not significant For western blot results one set of representative data obtained from three independentexperiments is shownThe relative ratio to 120573-actin based on densitometer quantification and analysis using ImageJ software is shown For allexperiments the quantitative data shown represent mean plusmn SD values of three independent experiments

32DENV InfectionCausesNF-120581BActivation Wenext inves-tigated the effects of DENV infection on NF-120581B activationA luciferase reporter assay showed a significant increaseof NF-120581B activation at the high MOIs of DENV infec-tion (Figure 2(a)) To explore the activation of NF-120581B weused immunocytochemical staining to detect the nucleartranslocation of NF-120581B in the context of DENV infection In

the infection group the p65 subunit of NF-120581B translocatedinto the nucleus in E protein-positive cells (Figure 2(b)) at6 h after infection Furthermore a luciferase reporter assayshowed that inhibitingNF-120581BwithCAPE an inhibitor ofNF-120581B blocked DENV-induced NF-120581B activation (Figure 2(c))These results demonstrate that DENV infection activates NF-120581B in RAW2647 cells


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Figure 2 DENV infection promotes NF-120581B nuclear translocation and activation (a) NF-120581B reporter assay quantified activation of NF-120581B inDENV 2-infected cells for the indicated MOIs 6 h after infection (b) Immunostaining followed by immunofluorescence microscopy revealsthe expression and intracellular localization of theNF-120581B p65 (green) andDENVEprotein (red) 6 h after infectionwithDENV2DAPI (blue)was used for nuclear staining Images are representative of three independent experiments (c) NF-120581B reporter assay quantified activation ofNF-120581B in DENV 2-infected cells pretreated with CAPE DMSO was used for the negative control lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 comparedwith untreated cells 119875 lt 005 compared with DENV ns not significant The quantitative data shown represent the mean plusmn SD values ofthree independent experiments

33 DENV Infection Induces the Expression of TNF-120572 iNOSand NO in an NF-120581B-Regulated Manner To further deter-mine the essential role of NF-120581B in DENV infection weexamined the effect of NF-120581B inhibition on TNF-120572 NOand iNOS expression After treatment with CAPE DENV-induced TNF-120572 production (Figure 3(a)) NO generation(Figure 3(b)) and iNOS expression (Figure 3(c)) were dra-matically downregulatedThese results confirmed thatNF-120581B

plays a critical role in DENV-induced TNF-120572 NO and iNOSexpression

34 DENV-InducedROS IndependentNF-120581BActivation TNF-120572 Production and iNOSNo Biosynthesis One of the possi-ble mechanisms modulating NF-120581B activation is the ROS-mediated pathway [14] We examined the possibility of ROSinvolvement in DENV-induced NF-120581B activation followed


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Figure 3 NF-120581B activation determines TNF-120572 NO and iNOS expression duringDENV infection ELISA (a) Griessrsquo reagent (b) andwesternblot analysis (c) quantified expression of TNF-120572 and iNOSNO in DENV 2-infected cells pretreated with CAPE DMSO was used for thenegative control lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with untreated cells 119875 lt 005 compared with DENV ns not significant Forwestern blot results one set of representative data obtained from three independent experiments is shownThe relative ratio to 120573-actin basedon densitometer quantification and analysis using ImageJ software is shown For all experiments the quantitative data shown represent themean plusmn SD values of three independent experiments

by TNF-120572 NO and iNOS induction DCFDA stainingfollowed by flow cytometry was used to detect changesin the ROS level in a time-dependent manner Howeverthere was no significant difference after DENV infectionat the indicated time points (Figure 4(a)) Inhibiting ROSusing NAC did not affect DENV-induced NF-120581B activation(Figure 4(b)) NAC also had no influence on TNF-120572 andiNOS expression (Figures 4(c) and 4(e)) However inhibitionof ROS reduced NO generation significantly during DENVinfection (Figure 4(d))These results indicate that ROS is notresponsible for the induction of NF-120581B activation followed

by TNF-120572 and iNOS upregulation but plays some role in theconversion of iNOS to NO during DENV infection

35 Heat-Inactivated DENV Induces NF-120581B Activation TNF-120572 Production and iNOSNO Biosynthesis Inefficiently Toinvestigate whether DENV-induced NF-120581B activation fol-lowed by TNF-120572 production and iNOSNO biosynthesisoccurs in a protein-mediated manner RAW2647 cells wereinoculated with MOI of 50 of heat-inactivated DENV (HI-DENV)The results revealed that HI-DENVwas less efficientat inducing NF-120581B activity than live-DENV (Figure 5(a))


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Figure 4 ROS is independent of NF-120581B activation followed by TNF-120572 and iNOS expression during DENV infection (a) CM-H2

DCFDA-based staining followed by flow cytometric analysis determinedROS generation inRAW2647 cells infectedwithDENV2 at the indicated timepoints The mean fluorescence intensity (MFI) of each stain is shown as the mean plusmn SD of three individual experiments ns not significantIn the presence of the ROS-scavenger NAC NF-120581B reporter assay (b) ELISA (c) Griess reagent (d) and western blot analysis (e) quantifiedthe activation of NF-120581B and the expression of TNF-120572 and iNOSNO in DENV 2-infected cells DMSO was used for the negative controllowastlowast

119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with untreated cells 119875 lt 005 compared with DENV ns not significant For western blot resultsone set of representative data obtained from three independent experiments is shown The relative ratio to 120573-actin based on densitometerquantification and analysis using ImageJ software is shown For all experiments the quantitative data shown represent the mean plusmn SD valuesof three independent experiments

Similarly the levels of TNF-120572 (Figure 5(b)) NO (Figure 5(c))and iNOS expression (Figure 5(d)) were lower in the HI-DENV group compared with the live DENV group Theseexperiments show that DENV activates NF-120581B followed byTNF-120572 and iNOSNO biosynthesis through viral proteins

36 TLR3 Contributes to NF-120581B Activation of iNOSNOBiosynthesis but Not TNF-120572 Production According to Tsai etal TLR3 plays a major role in cellular activation comparedwith other TLRs during DENV infection [15] TLR3 is a well-defined receptor for viral RNA which can induce NF-120581B


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Figure 5 Heat-inactivated DENVdoes not efficiently cause NF-120581B activation TNF-120572 production or iNOSNO biosynthesis NF-120581B reporterassay (a) ELISA (b) Griessrsquo reagent (c) and western blot analysis (d) quantified the activation of NF-120581B and the expression of TNF-120572 andiNOSNO in DENV 2-infected and heat-inactivated DENV 2-treated RAW2647 cells lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 comparedwith untreated cells 119875 lt 005 comparedwithDENV For western blot results one set of representative data obtained from three independentexperiments is shownThe relative ratio to 120573-actin based on densitometer quantification and analysis using ImageJ software is shown For allexperiments the quantitative data shown represent the mean plusmn SD values of three independent experiments

activation during viral infection [13] We further confirmedthe involvement of TLR3 in NF-120581B activation and inflamma-tory response First we used the TLR3 agonist polyinosinic-polycytidylic acid poly (Poly(IC)) to determine whetherTLR3 signaling can activate NF-120581B The results revealedthat Poly(IC) activated luciferase activity of NF-120581B and thatthis activity was inhibited by CAPE (Figure 6(a)) Moreoverpharmacological inhibition of TLR3 suppressed NF-120581B acti-vation (Figure 6(b)) Surprisingly there was no difference inTNF-120572 between the presence and absence of TLR3 inhibitorduring DENV infection (Figure 6(c)) In contrast DENV-induced NO (Figure 6(d)) and iNOS (Figure 6(e)) biosyn-thesis was significantly attenuated by TLR3 inhibition Theseresults demonstrate that TLR3-activated NF-120581B is requiredfor iNOSNObiosynthesis but not TNF-120572 expression duringDENV infection

37 DENV Protease NS2B3 also Promotes NF-120581B Activationto Induce TNF-120572 Production A recent study showed thatthe DENV protease NS2B3 activates NF-120581B by cleaving I120581B-120572 and I120581B-120573 [19] Therefore to investigate whether NS2B3-activated NF-120581B also participates in the induction of TNF-120572NO and iNOS we overexpressed a wild-type form of NS2B3(NS2B3-WT) and a protease-dead mutant NS2B3 which hasa single point mutation changing serine residue 135 of NS3to alanine (S135A) that abolished protease activity Westernblotting confirmed that the overexpression ofNS2B3-WTandNS2B3-S135A worked efficiently in RAW2647 cells Westernblotting detected the proform and cleavage form of NS2B3in NS2B3-WT cells but only one band in NS2B3-S135Acells demonstrating the loss of NS2B3 autocleavage ability(Figure 7(a))NF-120581Bactivitywas enhanced by overexpressingNS2B3-WT but decreased by overexpressing NS2B3-S135A


DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+

Poly(IC) (120583gmL)CAPE (120583M)

lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+

TLR3 inhibitor (120583M)

(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)

Figure 6 DENV infection induces TLR3-regulated NO and iNOS expression but not TNF-120572 expression following NF-120581B activation (a)In the absence and presence of CAPE an NF-120581B reporter assay was performed to measure NF-120581B activation in Poly(IC)-treated RAW2647cells NF-120581B reporter assay (b) ELISA (c) Griess reagent (d) and western blot analysis (e) quantified the activation of NF-120581B and theexpression of TNF-120572 and iNOSNO in DENV 2-infected RAW2647 cells pretreated with TLR3 inhibitor lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 compared with untreated cells 119875 lt 005 compared with Poly(IC) or DENV ns not significant For western blot resultsone set of representative data obtained from three independent experiments is shown The relative ratio to 120573-actin based on densitometerquantification and analysis using ImageJ software is shown For all experiments the quantitative data shown represent the mean plusmn SD valuesof three independent experiments


WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)

Figure 7 DENV protease NS2B3 also induces NF-120581B activation followed specifically by TNF-120572 expression (a)Western blot analysis showingthe expression of NS2B3 in Flag-tagged wild-type (WT) and a mutated NS2B3S135A (S135A)-transfected RAW2647 cells NF-120581B reporterassay (b) ELISA (c) Griess reagent (d) and western blot analysis (e) quantified the activation of NF-120581B and the expression of TNF-120572 andiNOSNO in transfected RAW2647 cells lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with the mock 119875 lt 005 compared with WT NS2B3 nsnot significant For western blot results one set of representative data obtained from three independent experiments is shown The relativeratio to 120573-actin based on densitometer quantification and analysis using ImageJ software is shown For all experiments the quantitative datashown represent the mean plusmn SD values of three independent experiments

(Figure 7(b)) Distinct from TLR3 signaling NS2B3-S135Asuppressed TNF-120572 expression (Figure 7(c)) compared withthe NS2B3-WT sample In addition neither NS2B3-WT norNS2B3-S135A induced iNOSNO biosynthesis (Figures 7(d)and 7(e)) These results reveal that forced expression ofDENVproteaseNS2B3 inducesNF-120581B activation followed byTNF-120572 expression but not iNOSNO biosynthesis

4 Discussion

In this study we identified the distinct pathways of NF-120581Bactivation for regulating iNOSNO and TNF-120572 expressionduring DENV infection in RAW2647 cells As summarizedin Figure 8 the viral protein NS2B3 causes NF-120581B activationspecifically for TNF-120572 expression and alternatively TLR3

mediatesNF-120581B activation for iNOSNObiosynthesis ROS isnot involved in NF-120581B activation as demonstrated in DENV-infected RAW2647 cells While this is not consistent withprevious studies that have shown crosstalk between ROSand NF-120581B activation [14] the presence of ROS remainsimportant for the generation of nitrite (NO

2

minus) We speculatethat ROS may directly modulate iNOS activity or indirectlyregulate nitrite generation It may have a limitation onour findings that these results are demonstrated in murinemacrophages In the future the findings are suggested to befurther confirmed in human primary macrophages

It has generally been suggested that TLRs are required fortriggering NF-120581B-mediated expression of downstream targetgenes of proinflammatory mediators during the inductionof inflammatory responses in DENV infection In addition


DENV

NS2B3 TLR3

ROS-independent

iNOSNO

Transcriptiontranslation

ROS-regulated

NF-120581B NF-120581B

TNF-120572

Figure 8 A hypothetical model of the distinct pathways of DENV-inducedNF-120581B activation followed byTNF-120572 and iNOSNOexpres-sion

to TLR3 we confirmed an alternative activation of NF-120581B caused by viral protein NS2B3 These results show thediverse effects of molecular regulation on NF-120581B duringDENV infection In activated macrophages the activationof NF-120581B usually induces inflammatory cytokine TNF-120572and iNOSNO generation by controlling the transcriptionalactivation of the genes [23] Even under NF-120581B activationthe different upstream signaling pathways and coactivatorsmay contribute considerably to inducing specific genes inresponse to different stimuli A previous study reported NF-120581B-mediated IL-8 expression [24] The positive transcriptionelongation factor b DENV core and nonstructural protein5 may also synthetically regulate IL-8 mRNA transacti-vation [25 26] For DENV-induced expression of TNF-120572and iNOS in macrophages other factors involved in thetranscriptional activation of TNF-120572 and iNOS need furtherinvestigation

During DENV infection the activation of NF-120581B hasbeen widely reported to activate apoptosis in neuroblastomahepatoma and endothelial cells [16 19 27] accompaniedby chemokine production in endothelial cells [17] IL-8production in monocytes epithelial cells and endothelialcells [24] MHC production in hepatoma cells [28] and typeI IFN response in lung epithelial cells [29] However themolecular mechanisms underlying the activation of NF-120581Bcaused by DENV infection or viral proteins remain largelyundefined DENV nonstructural protein 1 can increase NF-120581B activity in hepatoma cells [30] Consistent with a studydemonstrating that NS2B3 is required for DENV-inducedNF-120581B activation in endothelial cells [19] we showed anNS2B3-mediated NF-120581B activation of TNF-120572 expressionin DENV-activated RAW2647 cells Interestingly DENV-infected RAW2647 cells also underwent apoptosis 48 hafter infection (data not shown) For NS2B3-mediated NF-120581B activation in endothelial cells [19] the endogenous NF-120581Binhibitors I120581B120572 and I120581B120573 are cleaved by the protease activity

of NS2B3 In our study protease-dead mutant NS2B3 S315A-transfected RAW2647 cells lost the ability to trigger NF-120581B activation and TNF-120572 expression compared to wild-typeNS2B3TheNF-120581B-promoting effect of downregulating I120581B120572and I120581B120573 may need to be confirmed in DENV-infectedmacrophages

An increase in TNF-120572may contribute to the pathogenesisof dengue diseases by modulating cell death and survivalinflammation and endothelial dysfunction [31] AlthoughTNF-120572 may not contribute to viral replication directly [32]overproduction of TNF-120572 may further cause NF-120581B activa-tion which has important consequences for NF-120581B-drivenrelease of inflammatory cytokines during DENV infection[33] According to our findings NS2B3 may be a targetfor inhibiting DENV-induced TNF-120572 overproduction Inaddition to NS2B3 we also showed a TLR3-mediated NF-120581B activation of iNOSNO biosynthesis The induction ofiNOSNObiosynthesismay contribute not only to inflamma-tory response but also to initiation of NO-mediated antiviralactivity [34] Regarding the canonical pathway of TLR-3-mediated antiviral response it is speculated that a TLR3-mediated iNOSNO induction may be increased for anantiviral purpose in DENV-infected macrophages

In conclusion this work identifies two different pathwaysfor NF-120581B activation in DENV-infected RAW2647 macro-phages through the viral proteinNS2B3 and through the hostpattern recognition receptor TLR3 The viral protein NS2B3-regulated production of TNF-120572 may represent an inflam-matory response with a potential immunopathogenic rolein DENV infection In contrast TLR3-regulated iNOSNObiosynthesis may be a host response to confer antiviral activ-ity Obviously both TNF-120572 andNOmaymodulate inflamma-tory activation in DENV-infected macrophages

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the grants from the Ministryof Science and Technology Taiwan (MOST102-2628-B-006-005-MY3) and Taipei Medical University Taiwan (TMU103-AE1-B03)

References

[1] C P Simmons J J Farrar N van Vinh Chau and B WillsldquoDenguerdquo The New England Journal of Medicine vol 366 no15 pp 1423ndash1432 2012

[2] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013

[3] B E E Martina P Koraka and A DM E Osterhaus ldquoDenguevirus pathogenesis an integrated viewrdquo Clinical MicrobiologyReviews vol 22 no 4 pp 564ndash581 2009


[4] M T Fernandez-Mestre K Gendzekhadze P Rivas-Veten-court and Z Layrisse ldquoTNF-120572-308A allele a possible severityrisk factor of hemorrhagic manifestation in dengue feverpatientsrdquo Tissue Antigens vol 64 no 4 pp 469ndash472 2004

[5] P C F Neves-Souza E L Azeredo S M O Zagne et alldquoInducible nitric oxide synthase (iNOS) expression in mono-cytes during acute Dengue Fever in patients and during in vitroinfectionrdquo BMC Infectious Diseases vol 5 article 64 2005

[6] C-F Lin H-Y Lei A-L Shiau et al ldquoEndothelial cell apoptosisinduced by antibodies against dengue virus nonstructuralprotein 1 via production of nitric oxiderdquo Journal of Immunologyvol 169 no 2 pp 657ndash664 2002

[7] C-F Lin H-Y Lei A-L Shiau et al ldquoAntibodies from denguepatient sera cross-react with endothelial cells and induce dam-agerdquo Journal of Medical Virology vol 69 no 1 pp 82ndash90 2003

[8] C-F Lin S-W Wan H-J Cheng H-Y Lei and Y-S LinldquoAutoimmune pathogenesis in dengue virus infectionrdquo ViralImmunology vol 19 no 2 pp 127ndash132 2006

[9] Y-T Yen H-C Chen Y-D Lin C-C Shieh and B A Wu-Hsieh ldquoEnhancement by tumor necrosis factor alpha of denguevirus-induced endothelial cell production of reactive nitrogenand oxygen species is key to hemorrhage developmentrdquo Journalof Virology vol 82 no 24 pp 12312ndash12324 2008

[10] H-C Chen F M Hofman J T Kung Y-D Lin and B A Wu-Hsieh ldquoBoth virus and tumor necrosis factor alpha are criticalfor endothelium damage in a mouse model of dengue virus-induced hemorrhagerdquo Journal of Virology vol 81 no 11 pp5518ndash5526 2007

[11] D B Bethell K Flobbe C X T Phuong et al ldquoPathophysio-logic and prognostic role of cytokines in dengue hemorrhagicfeverrdquoThe Journal of Infectious Diseases vol 177 no 3 pp 778ndash782 1998

[12] Q Li and I M Verma ldquoNF-120581B regulation in the immune sys-temrdquo Nature Reviews Immunology vol 2 no 10 pp 725ndash7342002

[13] A G Bowie and L Unterholzner ldquoViral evasion and subversionof pattern-recognition receptor signallingrdquo Nature ReviewsImmunology vol 8 no 12 pp 911ndash922 2008

[14] M J Morgan and Z-G Liu ldquoCrosstalk of reactive oxygenspecies and NF-kappaB signalingrdquo Cell Research vol 21 no 1pp 103ndash115 2011

[15] Y-T Tsai S-Y Chang C-N Lee and C-L Kao ldquoHumanTLR3 recognizes dengue virus and modulates viral replica-tion in vitrordquo Cellular Microbiology vol 11 no 4 pp 604ndash6152009

[16] P Marianneau A Cardona L Edelman V Deubel and PDespres ldquoDengue virus replication in human hepatoma cellsactivates NF-120581b which in turn induces apoptotic cell deathrdquoJournal of Virology vol 71 no 4 pp 3244ndash3249 1997

[17] P Avirutnan P Malasit B Seliger S Bhakdi and M Hus-mann ldquoDengue virus infection of human endothelial cellsleads to chemokine production complement activation andapoptosisrdquoThe Journal of Immunology vol 161 no 11 pp 6338ndash6346 1998

[18] J-T Jan B-H Chen S-H Ma et al ldquoPotential dengue virus-triggered apoptotic pathway in human neuroblastoma cellsarachidonic acid superoxide anion andNF-120581B are sequentiallyinvolvedrdquo Journal of Virology vol 74 no 18 pp 8680ndash86912000

[19] J Lin S Lin W Chen et al ldquoDengue viral protease inter-action with NF-120581B inhibitor alphabeta results in endothelialcell apoptosis and hemorrhage developmentrdquo The Journal ofImmunology vol 193 no 3 pp 1258ndash1267 2014

[20] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoThe Journal of Infectious Diseases vol 189 no 8 pp 1411ndash14182004

[21] S-J L Wu G Grouard-Vogel W Sun et al ldquoHuman skinLangerhans cells are targets of dengue virus infectionrdquo NatureMedicine vol 6 no 7 pp 816ndash820 2000

[22] C-Y Yu T-H Chang J-J Liang et al ldquoDengue virus targets theadaptor protein MITA to subvert host innate immunityrdquo PLoSPathogens vol 8 no 6 Article ID e1002780 2012

[23] T Lawrence ldquoThenuclear factorNF-kappaB pathway in inflam-mationrdquo Cold Spring Harbor perspectives in biology vol 1 no 6Article ID a001651 2009

[24] I Bosch K Xhaja L Estevez et al ldquoIncreased produc-tion of interleukin-8 in primary human monocytes and inhuman epithelial and endothelial cell lines after dengue viruschallengerdquo Journal of Virology vol 76 no 11 pp 5588ndash55972002

[25] L-L Li S-T Hu S-H Wang H-H Lee Y-T Wang andY-H Ping ldquoPositive transcription elongation factor b (P-TEFb) contributes to dengue virus-stimulated induction ofinterleukin-8 (IL-8)rdquo Cellular Microbiology vol 12 no 11 pp1589ndash1603 2010

[26] C LMedin KA Fitzgerald andA L Rothman ldquoDengue virusnonstructural protein NS5 induces interleukin-8 transcriptionand secretionrdquo Journal of Virology vol 79 no 17 pp 11053ndash11061 2005

[27] P Despres M Flamand P-E Ceccaldi and V Deubel ldquoHumanisolates of dengue type 1 virus induce apoptosis in mouseneuroblastoma cellsrdquo Journal of Virology vol 70 no 6 pp4090ndash4096 1996

[28] S Othman N A Rahman and R Yusof ldquoInduction of MHCClass I HLA-A2 promoter by dengue virus occurs at the NF120581Bbinding domains of the Class I Regulatory Complexrdquo VirusResearch vol 163 no 1 pp 238ndash245 2012

[29] T-H Chang C-L Liao and Y-L Lin ldquoFlavivirus inducesinterferon-beta gene expression through a pathway involvingRIG-I-dependent IRF-3 and PI3K-dependent NF-120581B activa-tionrdquoMicrobes and Infection vol 8 no 1 pp 157ndash171 2006

[30] B M Silva L P Sousa A C Gomes-Ruiz et al ldquoThe denguevirus nonstructural protein 1 (NS1) increases NF-kappaB tran-scriptional activity inHepG2 cellsrdquoArchives of Virology vol 156no 7 pp 1275ndash1279 2011

[31] I Kurane ldquoDengue hemorrhagic fever with special empha-sis on immunopathogenesisrdquo Comparative Immunology Micro-biology and Infectious Diseases vol 30 no 5-6 pp 329ndash3402007

[32] S Wati P Li C J Burrell and J M Carr ldquoDengue virus (DV)replication in monocyte-derived macrophages is not affectedby tumor necrosis factor alpha (TNF-120572) and DV infectioninduces altered responsiveness to TNF-120572 stimulationrdquo Journalof Virology vol 81 no 18 pp 10161ndash10171 2007


[33] S Wati S M Rawlinson R A Ivanov et al ldquoTumour necrosisfactor alpha (TNF-120572) stimulation of cells with establisheddengue virus type 2 infection induces cell death that is accompa-nied by a reduced ability of TNF-120572 to activate nuclear factor 120581band reduced sphingosine kinase-1 activityrdquo Journal of GeneralVirology vol 92 no 4 pp 807ndash818 2011

[34] T Akaike and H Maeda ldquoNitric oxide and virus infectionrdquoImmunology vol 101 no 3 pp 300ndash308 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


In addition Yen et al also observed increased macrophageinfiltration and TNF-120572 production in the endothelium ofthe hemorrhagic tissue and endothelial cell apoptosis in thetissues with enhanced expression of iNOS and nitrotyrosinein DENV-infected mice [9] Furthermore TNF-120572 has beenreported to induce hemorrhage by causing endothelial celldeath which is associated with increased endothelial cellpermeability in an experimental dengue hemorrhage mousemodel [9 10] TNF receptor levels have also shown a highlypositive correlation with the severity of DHF in patients [11]However the underlying molecular mechanism of DENV-induced inflammatory activation remains largely unknown

Nuclear factor-120581B (NF-120581B) which is a well-known tran-scription factor participates in the regulation of proinflam-matory mediators including iNOS and TNF-120572 NF-120581B is aheterodimer consisting of p65 and p50 subunits which asso-ciate with the inhibitor of NF-120581B (I120581B) family of inhibitoryproteins Upon I120581B degradation NF-120581B translocates to thenucleus and drives the expression of downstream target genes[12] NF-120581B can be activated by several different signalingpathways including the receptor-mediated signaling of pat-tern recognition receptors such as Toll-like receptors (TLRs)retinoic-acid-inducible gene I melanoma differentiation-associated gene 5 [13] and reactive oxygen species- (ROS-)induced pathways [14] In DENV infection Tsai et al showedthat TLR3 plays a major role in interleukin- (IL-) 8 produc-tion and viral replication compared with other TLRs [15]NF-120581B activation during DENV infection has been reportedsince the 1990s when several studies showed that DENVcan induce NF-120581B activation leading to the induction ofapoptosis in different cell types [16ndash18] however the under-lyingmolecularmechanism remains undefined Recently Linet al clarified that the activation of NF-120581B which leads toendothelium cell apoptosis and hemorrhage developmentoccurs through the interaction of the DENV NS2B-NS3(NS2B3) protease complex with I120581B120572120573 NS2B3 cleavesI120581B120572120573 and activates I120581B kinase [19] However whetherNF-120581B is involved in DENV-induced iNOSNO and TNF-120572production and which pathwaymediates these events are stilla mystery Becausemonocytic cells are the DENV target cellswe usedmurinemononuclear phagocyte RAW2647 cells as amodel [20 21] In this study we aimed to investigate the roleof NF-120581B in DENV-induced iNOSNO and TNF-120572 produc-tion and to unveil the underlying molecular mechanism ofNF-120581B activation

2 Materials and Methods

21 Antibodies and Reagents The reagents and antibodiesused were translation inhibitor cycloheximide transcriptioninhibitor actinomycin D NF-120581B inhibitor caffeic acid phe-nethyl ester (CAPE) antioxidant N-acetyl-cysteine (NAC)Poly(IC) TLR3 inhibitor dimethyl sulfoxide (DMSO)and mouse monoclonal antibody (Ab) specific for 120573-actin(Sigma-Aldrich St Louis MO) Abs against DENV NS1 andenvelope (E) (GeneTex San Antonio TX) Abs against iNOSand NF-120581B (Cell Signaling Technology Beverly MA) Absagainst isotype control IgG (Millipore BillericaMA) donkeyanti-goat IgG conjugated with horseradish peroxidase (HRP)

(Santa Cruz Biotechnology Santa Cruz CA) and goat anti-rabbit IgG conjugated with HRP (Chemicon InternationalTemecula CA) rabbit anti-mouse IgG conjugated with HRP(Abcam Cambridge MA) and Alexa Fluor 488- and AlexaFluor 594-conjugated goat anti-mouse and goat anti-rabbit(Invitrogen Carlsbad CA) Inhibitors were then dissolvedin DMSO prior to dilution with sterile phosphate-bufferedsaline (PBS) for use in experiments Other chemical reagentswere obtained from Sigma-Aldrich All drug treatments oncells were assessed for cytotoxic effects using cytotoxicityassays prior to experiments Noncytotoxic dosages were usedin this study

22 Cell Culture and Virus Culture RAW2647 mouse macro-phage was purchased from American Type Culture Collec-tion (ATCC number TIB-71) and were grown on plastic inDulbeccorsquos modified Eaglersquos medium (DMEM) with 10fetal bovine serum (FBS) (Sigma-Aldrich St Louis MO)100 units of penicillin and 100 120583gmL of streptomycin at37∘C under 5 CO

2 Cells were used at 3 to 5 passages

Baby hamster kidney (BHK) cells and C636 cells werecultured inDMEM(Invitrogen Life Technologies) containingFBS DENV2 PL046 strain was maintained in C636 cellsMonolayers of C636 cells were incubated with DENV at amultiplicity of infection (MOI) of 001 and incubated at 28∘Cin 5 CO

2for 5 days The virus supernatant was further

filtered with 022120583mfilter and then stored at minus80∘C until useVirus titer was determined by plaque assay using the BHKcell line

23 DENV Infection Cells were resuspended at a concentra-tion of 5 times 105 cellsmL in appropriate medium with DENVand incubated for 90min at 37∘CThen the cells were washedonce with DMEM medium resuspended at a concentrationof 5 times 105 cellsmL and incubated at 37∘C with 5 CO

2 The

viral supernatants were checked using plaque assays

24 Plaque Assay BHK-21 cells were plated into 12-wellplates (2 times 105 cellswell) and cultured in DMEM underCO2-enriched conditions After adsorption with a serially

diluted virus solution for 1 h the solution was replaced withfresh DMEM containing 2 FBS and 05 methyl cellulose(Sigma-Aldrich) Five days after infection the medium wasremoved and the cells were fixed and stained with crystalviolet solution containing of 1 crystal violet 064 NaCland 2 formalin

25 Western Blotting Harvested cells were lysed in a buffercontaining 1 Triton X-100 50mM Tris (pH 75) 10mMEDTA 002 NaN

3 and a protease inhibitor cocktail (Roche

Boehringer Mannheim Diagnostics Mannheim Germany)After a freeze-thaw cycle cell lysates were centrifuged at10000timesg at 4∘C for 20min The lysates were boiled in asample buffer for 5min Proteins were then subjected toSDS-PAGE and transferred to PVDF membranes (MilliporeBillerica MA USA) using a semidry electroblotting systemAfter blocking with 5 skim milk in PBS the membraneswere incubated overnight with a 1 1000 dilution of primaryantibodies at 4∘C The membranes were then washed with





2












3 Results



Nitr

ite(fo

ld in

crea

se)

iNOS

30

25

20

15

10

05

0

3000

2000

1000

0

Ratio

12

09

06

03

0

DENV (MOI)Time (h)

012

0112

112

512

1012

5012

iNOS

3000

2000

1000

0

TNF-120572

(pg

mL)

120573-actin

012

0112

112

512

1012

5012

DENV (MOI)Time (h)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

NS1

Vira

l tite

r (pf

um

L)

150

120

90

60

30

0

120573-actin1008060402

0

Ratio

(NS1

120573-a

ctin

)

DENV(MOI)

50minus

lowastlowast

DENV(MOI)

50minus

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

0506

5012

5024


lowast

lowast

iNOS

ns

Nitr

ite(fo

ld in

crea

se)

30

25

20

15

10

05

0

ns

ns1600

1200

800

400

0

120573-actin

Ratio

12

09

06

03

0

(iNO

S120573

-act

in)

lowast

DENV (MOI)

DMSO

50 50

005

50025

50minus

minus

minus

minus

minus

minus

minus

minus

minus minus +

minus minus

minus


TNF-120572

(pg

mL)

120573-actin

(iNO

S120573

-act

in)

(a)

(b)

(c) (d)

TNF-120572

(pg

mL)

lowastlowastlowast

lowastlowast

lowast





DENV (MOI) 0 01 1 5 10 50

(fold

incr

ease

)

4

3

2

1

0

NF-120581

B ac

tivity

lowastlowastlowast

lowastlowastlowast

(a)

Moc

kD

ENV

50120583m

(b)

(fold

incr

ease

)

DENV (MOI)

DMSO

50 5025

50

8

6

4

2

0

ns

minus

minus

minus

minus

minus minus

minus

+

lowast

CAPE (120583M)

NF-120581

B ac

tivity

(c)






3000

2000

1000

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(a)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

lowastlowastlowast

(b)

iNOS

10

08

06

04

02

0

Ratio

ns

120573-actin

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(iNO

S120573

-act

in)

50 5025

50minus

minus

minus

minus

minus minus

minus

+

DENV (MOI)

DMSOCAPE (120583M)

lowastlowast

(c)






DCF

(MFI

)

1086420


503

506

5012

501

5024

ns

minus

(a)

ns

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

(b)

2500

2000

1500

1000

500

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(c)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

lowastlowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(d)

iNOS

ns

120573-actin

16

12

08

04

0

Ratio

(iNO

S120573

-act

in)

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

(e)







10

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

lowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(a)

2500

2000

1500

1000

500

0

DENV (MOI)HI-DENV

50

lowastlowastlowast

TNF-120572

(pg

mL)

minus

minus

minus

minus +

(b)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

lowastlowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(c)

iNOS

15

12

09

06

03

0

Ratio

120573-actin

(iNO

S120573

-act

in)

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

lowast

(d)





DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+


lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)




WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2












3 Results



Nitr

ite(fo

ld in

crea

se)

iNOS

30

25

20

15

10

05

0

3000

2000

1000

0

Ratio

12

09

06

03

0

DENV (MOI)Time (h)

012

0112

112

512

1012

5012

iNOS

3000

2000

1000

0

TNF-120572

(pg

mL)

120573-actin

012

0112

112

512

1012

5012

DENV (MOI)Time (h)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

NS1

Vira

l tite

r (pf

um

L)

150

120

90

60

30

0

120573-actin1008060402

0

Ratio

(NS1

120573-a

ctin

)

DENV(MOI)

50minus

lowastlowast

DENV(MOI)

50minus

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

0506

5012

5024


lowast

lowast

iNOS

ns

Nitr

ite(fo

ld in

crea

se)

30

25

20

15

10

05

0

ns

ns1600

1200

800

400

0

120573-actin

Ratio

12

09

06

03

0

(iNO

S120573

-act

in)

lowast

DENV (MOI)

DMSO

50 50

005

50025

50minus

minus

minus

minus

minus

minus

minus

minus

minus minus +

minus minus

minus


TNF-120572

(pg

mL)

120573-actin

(iNO

S120573

-act

in)

(a)

(b)

(c) (d)

TNF-120572

(pg

mL)

lowastlowastlowast

lowastlowast

lowast





DENV (MOI) 0 01 1 5 10 50

(fold

incr

ease

)

4

3

2

1

0

NF-120581

B ac

tivity

lowastlowastlowast

lowastlowastlowast

(a)

Moc

kD

ENV

50120583m

(b)

(fold

incr

ease

)

DENV (MOI)

DMSO

50 5025

50

8

6

4

2

0

ns

minus

minus

minus

minus

minus minus

minus

+

lowast

CAPE (120583M)

NF-120581

B ac

tivity

(c)






3000

2000

1000

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(a)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

lowastlowastlowast

(b)

iNOS

10

08

06

04

02

0

Ratio

ns

120573-actin

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(iNO

S120573

-act

in)

50 5025

50minus

minus

minus

minus

minus minus

minus

+

DENV (MOI)

DMSOCAPE (120583M)

lowastlowast

(c)






DCF

(MFI

)

1086420


503

506

5012

501

5024

ns

minus

(a)

ns

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

(b)

2500

2000

1500

1000

500

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(c)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

lowastlowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(d)

iNOS

ns

120573-actin

16

12

08

04

0

Ratio

(iNO

S120573

-act

in)

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

(e)







10

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

lowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(a)

2500

2000

1500

1000

500

0

DENV (MOI)HI-DENV

50

lowastlowastlowast

TNF-120572

(pg

mL)

minus

minus

minus

minus +

(b)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

lowastlowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(c)

iNOS

15

12

09

06

03

0

Ratio

120573-actin

(iNO

S120573

-act

in)

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

lowast

(d)





DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+


lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)




WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nitr

ite(fo

ld in

crea

se)

iNOS

30

25

20

15

10

05

0

3000

2000

1000

0

Ratio

12

09

06

03

0

DENV (MOI)Time (h)

012

0112

112

512

1012

5012

iNOS

3000

2000

1000

0

TNF-120572

(pg

mL)

120573-actin

012

0112

112

512

1012

5012

DENV (MOI)Time (h)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

NS1

Vira

l tite

r (pf

um

L)

150

120

90

60

30

0

120573-actin1008060402

0

Ratio

(NS1

120573-a

ctin

)

DENV(MOI)

50minus

lowastlowast

DENV(MOI)

50minus

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

0506

5012

5024


lowast

lowast

iNOS

ns

Nitr

ite(fo

ld in

crea

se)

30

25

20

15

10

05

0

ns

ns1600

1200

800

400

0

120573-actin

Ratio

12

09

06

03

0

(iNO

S120573

-act

in)

lowast

DENV (MOI)

DMSO

50 50

005

50025

50minus

minus

minus

minus

minus

minus

minus

minus

minus minus +

minus minus

minus


TNF-120572

(pg

mL)

120573-actin

(iNO

S120573

-act

in)

(a)

(b)

(c) (d)

TNF-120572

(pg

mL)

lowastlowastlowast

lowastlowast

lowast





DENV (MOI) 0 01 1 5 10 50

(fold

incr

ease

)

4

3

2

1

0

NF-120581

B ac

tivity

lowastlowastlowast

lowastlowastlowast

(a)

Moc

kD

ENV

50120583m

(b)

(fold

incr

ease

)

DENV (MOI)

DMSO

50 5025

50

8

6

4

2

0

ns

minus

minus

minus

minus

minus minus

minus

+

lowast

CAPE (120583M)

NF-120581

B ac

tivity

(c)






3000

2000

1000

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(a)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

lowastlowastlowast

(b)

iNOS

10

08

06

04

02

0

Ratio

ns

120573-actin

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(iNO

S120573

-act

in)

50 5025

50minus

minus

minus

minus

minus minus

minus

+

DENV (MOI)

DMSOCAPE (120583M)

lowastlowast

(c)






DCF

(MFI

)

1086420


503

506

5012

501

5024

ns

minus

(a)

ns

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

(b)

2500

2000

1500

1000

500

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(c)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

lowastlowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(d)

iNOS

ns

120573-actin

16

12

08

04

0

Ratio

(iNO

S120573

-act

in)

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

(e)







10

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

lowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(a)

2500

2000

1500

1000

500

0

DENV (MOI)HI-DENV

50

lowastlowastlowast

TNF-120572

(pg

mL)

minus

minus

minus

minus +

(b)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

lowastlowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(c)

iNOS

15

12

09

06

03

0

Ratio

120573-actin

(iNO

S120573

-act

in)

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

lowast

(d)





DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+


lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)




WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DENV (MOI) 0 01 1 5 10 50

(fold

incr

ease

)

4

3

2

1

0

NF-120581

B ac

tivity

lowastlowastlowast

lowastlowastlowast

(a)

Moc

kD

ENV

50120583m

(b)

(fold

incr

ease

)

DENV (MOI)

DMSO

50 5025

50

8

6

4

2

0

ns

minus

minus

minus

minus

minus minus

minus

+

lowast

CAPE (120583M)

NF-120581

B ac

tivity

(c)






3000

2000

1000

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(a)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

lowastlowastlowast

(b)

iNOS

10

08

06

04

02

0

Ratio

ns

120573-actin

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(iNO

S120573

-act

in)

50 5025

50minus

minus

minus

minus

minus minus

minus

+

DENV (MOI)

DMSOCAPE (120583M)

lowastlowast

(c)






DCF

(MFI

)

1086420


503

506

5012

501

5024

ns

minus

(a)

ns

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

(b)

2500

2000

1500

1000

500

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(c)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

lowastlowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(d)

iNOS

ns

120573-actin

16

12

08

04

0

Ratio

(iNO

S120573

-act

in)

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

(e)







10

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

lowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(a)

2500

2000

1500

1000

500

0

DENV (MOI)HI-DENV

50

lowastlowastlowast

TNF-120572

(pg

mL)

minus

minus

minus

minus +

(b)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

lowastlowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(c)

iNOS

15

12

09

06

03

0

Ratio

120573-actin

(iNO

S120573

-act

in)

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

lowast

(d)





DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+


lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)




WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















3000

2000

1000

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(a)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

lowastlowastlowast

(b)

iNOS

10

08

06

04

02

0

Ratio

ns

120573-actin

DENV (MOI)

DMSO

50 5025

50minus

minus

minus

minus

minus minus

minus

+

CAPE (120583M)

(iNO

S120573

-act

in)

50 5025

50minus

minus

minus

minus

minus minus

minus

+

DENV (MOI)

DMSOCAPE (120583M)

lowastlowast

(c)






DCF

(MFI

)

1086420


503

506

5012

501

5024

ns

minus

(a)

ns

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

(b)

2500

2000

1500

1000

500

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(c)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

lowastlowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(d)

iNOS

ns

120573-actin

16

12

08

04

0

Ratio

(iNO

S120573

-act

in)

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

(e)







10

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

lowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(a)

2500

2000

1500

1000

500

0

DENV (MOI)HI-DENV

50

lowastlowastlowast

TNF-120572

(pg

mL)

minus

minus

minus

minus +

(b)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

lowastlowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(c)

iNOS

15

12

09

06

03

0

Ratio

120573-actin

(iNO

S120573

-act

in)

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

lowast

(d)





DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+


lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)




WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DCF

(MFI

)

1086420


503

506

5012

501

5024

ns

minus

(a)

ns

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

(b)

2500

2000

1500

1000

500

0

ns

TNF-120572

(pg

mL)

lowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(c)

Nitr

ite(fo

ld in

crea

se)

8

6

4

2

0

ns

lowastlowastlowast

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

(d)

iNOS

ns

120573-actin

16

12

08

04

0

Ratio

(iNO

S120573

-act

in)

DENV (MOI)NAC (mM)

DMSO

50 505

50minus

minus

minus

minus

minus minus

minus

+

lowastlowast

(e)







10

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

lowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(a)

2500

2000

1500

1000

500

0

DENV (MOI)HI-DENV

50

lowastlowastlowast

TNF-120572

(pg

mL)

minus

minus

minus

minus +

(b)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

lowastlowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(c)

iNOS

15

12

09

06

03

0

Ratio

120573-actin

(iNO

S120573

-act

in)

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

lowast

(d)





DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+


lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)




WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10

8

6

4

2

0

(fold

incr

ease

)N

F-120581

B ac

tivity

lowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(a)

2500

2000

1500

1000

500

0

DENV (MOI)HI-DENV

50

lowastlowastlowast

TNF-120572

(pg

mL)

minus

minus

minus

minus +

(b)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

lowastlowast

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

(c)

iNOS

15

12

09

06

03

0

Ratio

120573-actin

(iNO

S120573

-act

in)

DENV (MOI)HI-DENV

50 minus

minus

minus

minus +

lowast

(d)





DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+


lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)




WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DMSO

(fold

incr

ease

)

20

15

10

05

0

ns

50 5025

50minus

minus

minus

minus

minus minus

minus

+


lowast

NF-120581

B ac

tivity

(a)

6

5

4

3

2

1

0

ns

(fold

incr

ease

)N

F-120581

B ac

tivity

lowastlowast

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(b)

ns

ns

lowastlowastlowast

2500

2000

1500

1000

500

0

TNF-120572

(pg

mL)

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


(c)

Nitr

ite(fo

ld in

crea

se)

25

20

15

10

05

0

ns

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

(d)

iNOS

20

15

10

05

0

ns

120573-actin

DENV (MOI)

DMSO

50 5050

50minus

minus

minus

minus

minus minus

minus

+


lowastlowast

Ratio

(iNO

S120573

-act

in)

(e)




WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT

S135

A

NS2B3

(MW)

Flag

7255433426

95

17

130

120573-actin

(a)

5

4

3

2

1

0

(fold

incr

ease

)

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+N

F-120581

B ac

tivity

lowastlowast

(b)

500

400

300

200

100

0

MockNS2B3-WT

NS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

+

lowastlowast

TNF-120572

(pg

mL)

(c)

Nitr

ite(fo

ld in

crea

se)

5

4

3

2

1

0

ns

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(d)

iNOS

Ratio

12

09

06

03

0

120573-actin

(iNO

S120573

-act

in)

lowastlowastlowast

NS2B3-WTNS2B3-S135A

minus

minus

minus minus

minus

minus

+

+

50DENV (MOI)

(e)



4 Discussion



2




DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DENV

NS2B3 TLR3

ROS-independent

iNOSNO


ROS-regulated

NF-120581B NF-120581B

TNF-120572









Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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