of May 7, 2018.This information is current as

Apoptosis and Hemorrhage Development Results in Endothelial Cellβ/αB Inhibitor

κDengue Viral Protease Interaction with NF-

Miaw and Betty A. Wu-HsiehYen, Chin-Wen Lai, Mi-Hua Tao, Yi-Ling Lin, Shi-Chuen Jung-Chen Lin, Shih-Ching Lin, Wen-Yu Chen, Yu-Ting

ol.1302675http://www.jimmunol.org/content/early/2014/06/27/jimmun

published online 27 June 2014J Immunol 

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The Journal of Immunology

Dengue Viral Protease Interaction with NF-kB Inhibitor a/bResults in Endothelial Cell Apoptosis and HemorrhageDevelopment

Jung-Chen Lin,* Shih-Ching Lin,* Wen-Yu Chen,* Yu-Ting Yen,* Chin-Wen Lai,†,‡

Mi-Hua Tao,†,‡ Yi-Ling Lin,‡ Shi-Chuen Miaw,* and Betty A. Wu-Hsieh*

Hemorrhagic manifestations occur frequently accompanying a wide range of dengue disease syndromes. Much work has focused on

the contribution of immune factors to the pathogenesis of hemorrhage, but how dengue virus (DENV) participates in the pathogenic

process has never been explored. Although there is no consensus that apoptosis is the basis of vascular permeability in human

dengue infections, we showed in dengue hemorrhage mouse model that endothelial cell apoptosis is important to hemorrhage de-

velopment in mice. To explore the molecular basis of the contribution of DENV to endothelial cell death, we show in this study that

DENV protease interacts with cellular IkBa and IkBb and cleaves them. By inducing IkBa and IkBb cleavage and IkB kinase

activation, DENV protease activates NF-kB, which results in endothelial cell death. Intradermal inoculation of DENV protease

packaged in adenovirus-associated virus-9 induces endothelial cell death and dermal hemorrhage in mice. Although the H51

activity site is not involved in the interaction between DENV protease and IkB-a/b, the enzymatic activity is critical to the ability

of DENV protease to induce IkBa and IkBb cleavage and trigger hemorrhage development. Moreover, overexpression of IkBa or

IkBb protects endothelial cells from DENV-induced apoptosis. In this study, we show that DENV protease participates in the

pathogenesis of dengue hemorrhage and discover IkBa and IkBb to be the new cellular targets that are cleaved by DENV

protease. The Journal of Immunology, 2014, 193: 000–000.

Dengue virus (DENV), a mosquito-borne flavivirus, causesillness ranging from flu-like dengue fever to severedengue hemorrhagic fever (DHF) and dengue shock

syndrome (DSS) (1). Major features of DHF and DSS includehemorrhage; an increase of vascular permeability with plasmaleakage, pleural, or peritoneal effusion; bleeding manifestations;and thrombocytopenia (2, 3). The most important pathophysio-logical event in DHF and DSS is plasma leakage, which occa-sionally leads to death (4–6).We showed previously in an experimental dengue hemorrhage

mouse model that DENV targets the endothelium and that boththe virus and host factors contribute to endothelial cell death inhemorrhage tissues (7, 8). In addition, administration of pan-caspase inhibitor inhibits hemorrhage development (8). These re-sults showed that endothelial cell apoptosis is key to hemorrhage

development in the mouse model. In recent years, much effort inthe study of dengue is devoted to the discovery of immune fac-tors, and TNF and vascular endothelial growth factor are the lead-ing candidates that contribute to endothelium damage (4). Less isknown about viral component and the molecular mechanism ofhow it participates in this pathogenic process.The DENV genomic RNA is translated during viral replication to

a single polyprotein that is cleaved into three structural and sevennonstructural (NS) proteins by host cell furin proteases and viralNS2B-NS3 (2B-3) protease complex (9). The NS3 protein hasa serine protease domain (activity sites: H51, D75, and S135) thatspans 180 residues in the N terminus (10–12). The cleavage sitesfor DENV protease on the polyprotein have the consensus se-quence of dibasic amino acids (R and K), followed by a smallamino acid (G, A, or S) (13–15). It has been reported recently thatDENV protease cleaves human MITA at LRR/96G through whichit modulates human host innate immune response to infection (16,17). Whether DENV protease is also involved in the pathogenicmechanism of dengue hemorrhage is a question that has neverbeen addressed.NF-kB is a transcription factor involved in the regulated ex-

pression of genes that encode for cytokines, chemokines, anti-apoptotic proteins and proapoptosis proteins (18, 19). The NF-kBcomplex is a heterodimer (p65/p50 subunits) in association witha family of inhibitory proteins, called IkB proteins (18). Upondegradation of the IkB proteins, the NF-kB complex translocate tothe nucleus to activate target genes (18). NF-kB is also shown tobe involved in the induction of apoptosis after DENV infection(20, 21). However, the role of NF-kB pathway in DENV patho-genesis is still not clear.Flaviviral proteases, including that in Langat virus, West Nile

virus, and DENV, are functional in inducing host cell apoptosis(22–25), while activity-abolished NS2B-NS3185 (H51 replacedby A, 2B-Pro-H51A) fails to induce apoptosis in Vero cells (24).

*Graduate Institute of Immunology, National Taiwan University College of Medi-cine, Taipei 100, Taiwan; †Department of Clinical Laboratory Sciences and MedicalBiotechnology, College of Medicine, National Taiwan University, Taipei 100, Tai-wan; and ‡Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan

Received for publication October 9, 2013. Accepted for publication May 21, 2014.

This work was supported by National Research Program for Genomic MedicineGrants 98, 99, and 100-3112-B-002 and National Science Council (Republic ofChina) Grants NSC-98-2745-B-002-003 and NSC-101-2321-B-002-062.

Address correspondence and reprint requests to Dr. Betty A. Wu-Hsieh, GraduateInstitute of Immunology, National Taiwan University College of Medicine, Number 1Jen-Ai Road, Section 1, Taipei 100, Taiwan. E-mail address: bwh@ntu.edu.tw

The online version of this article contains supplemental material.

Abbreviations used in this article: AAV9, adenovirus-associated virus-9; ANK,ankyrin; 2B-3, NS2B-NS3; 2B-Pro, NS2B-NS3185; 2B-Pro-H51A, activity-abolishedNS2B-NS3185 (H51 replaced by A); CVB3, Coxsackie B3 virus; DENV, dengue virus;DHF, dengue hemorrhagic fever; DSS, dengue shock syndrome; HMEC-1, humanmicrovascular endothelial cell-1; IKK, IkB kinase; IRES, internal ribosome entry site;MOI, multiplicity of infection; NS, nonstructural; PI, propidium iodide; TU, transduc-tion unit.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302675

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Human microvascular endothelial cell-1 (HMEC-1) infected byDENV or transfected with DENV NS2B-NS3185 (2B-Pro) un-dergo apoptosis (8, 25). Caspase-3 activation was observed intransfected cells (25). These reports indicate that DENV protease–induced endothelial apoptosis is activity dependent and involvesthe apoptotic pathway. In this study, we aimed to investigate themolecular mechanism of how DENV protease induces apoptosis.In this study, we discovered that DENV protease interacted with

both IkBa and IkBb and cleaved them. The interaction resulted inNF-kB p65 and p50 nuclear translocation and activation of ex-trinsic apoptotic pathway. While endothelial cells transduced withDENV protease became apoptotic, overexpressing either IkBa orIkBb protected them from cell death. Interestingly, DENV pro-tease targeted to endothelium by adenovirus-associated virus-9(AAV9) displaying SLRSPPS (AAV9-SLR) in the capsid (26)induced endothelial cell death and hemorrhage development inmice. The ability of DENV protease to induce IkB-a/b cleavageand the associated events leading to cell death was protease ac-tivity dependent. To our knowledge, our results showed for thefirst time that dengue viral protease is involved in hemorrhagedevelopment in mice and that IkBa and IkBb can be added to thelist of cellular proteins that are cleaved by DENV protease.

Materials and MethodsEthics statement

All experiments involving animals were carried out in strict accordancewith the recommendations in the Guidebook for the Care and Use ofLaboratory Animals, 3rd Ed., 2007, published by The Chinese TaipeiSociety of Laboratory Animal Sciences. The experiment protocol wasapproved by the Ethics of Animal Experiments Committee of the NationalTaiwan University College of Medicine (permit number 20120041)

Mice

C57BL/6 mice were obtained from National Laboratory Animal Center,National Applied Research Laboratories, Taiwan, and bred at the Labo-ratory Animal Center of the National Taiwan University. Mice were housedin sterilized cages, bedding, filter cage tops and fed with sterilized food andwater. Mice at 6 wk of age were used in all experiments.

Yeast-two-hybrid assay

Plasmids pGilda, pJG4-5 and pSH18-34 as well as EGY48 yeast wereobtained from Origene Technologies in the DupLEX-AYeast Two HybridSystem kit. DENV 2B-3 fragment was cloned into pGilda (bait) plasmidand mouse cDNA library into pJG4-5 plasmid. Yeast EGY48 were cotrans-formed with pGilda-2B-3, pJG4-5-library and reporter pSH18-34. Trans-formants were selected in 5-bromo-4-chloro-3-indolyl b-D-galactoside–containing synthetic defined media without His, Leu, Trp, and Ura inquadruple dropout plates. The potential positive transformants were col-onies that turned blue. DNA isolated from blue colonies was used totransform Escherichia coli (KC8), and the library plasmid was recovered.The recovered library plasmids were amplified by PCR and sequenced.

Vector construction and transfection

DENV components 2B-3, 2B-Pro, 2B-Pro-H51A mutant, Pro, and 2B withFlag-tag on the C terminus were cloned into pRV vectors (at restriction sitesBglII and XhoI) with GFP under the control of internal ribosomal entry site(IRES). Human IkBa and IkBb were cloned into pRV vectors with DsRedunder the control of IRES. The plasmids were transiently transfected into293T cells using Maestrofectin transfection reagent (Omics Bio). HumanIkBa and IkBb were cloned into pWPI vectors with DsRed under thecontrol of IRES. DENV 2B-Pro, 2B-Pro-H51A, Pro, and 2B with Flag-tagon the C terminus were cloned into pWPI vectors with GFP under thecontrol of IRES.

Lentivirus production and transduction

To generate lentivirus, pWPI carrying different DENV viral components,IkBa or IkBb, pSPAX2-1 (CMV-Gag polymerase), and pMD2.G (CMV-envelope plasmid) were mixed in Maestrofectin transfection reagent. Themixture was added to 293T cells, which had been plated the prior day ina 10-cm culture dish. Supernatants were collected at 48 or 72 h after

transfection, concentrated with buffer containing PEG-6000 (Sigma-Aldrich) and NaCl and resuspended in 150 ml PBS. To transduce cells,lentivirus preparations (8.83 3 104 transduction unit [TU]) were diluted inM200 medium (Invitrogen) with polybrene at 8 mg/ml. The mixture wasadded to HMEC-1 cultured in 6-well plate. The plates were centrifuged at1100 3 g in room temperature for 55 min. The supernatant was removed,and the infected cells were washed by PBS and replenished with M200complete medium (M200 medium plus 2% of low serum growth supple-ment (Invitrogen)). To determine the titer of purified lentivirus, HMEC-1cells (2 3 104) cultured in 12-well plates were transduced with 10 mlresuspended virus. The percentage of GFP+ cells was determined by flowcytometry at 48 h postinfection. The TU was calculated by the formula:TU/ml = percentage of GFP+ cells 3 (2 3 104)/0.01 ml.

Abs

Rabbit anti-IkBa, rabbit anti-IkBb, rabbit anti-IkB kinase (IKK)a, andmouse anti-IKKb Abs were obtained from Santa Cruz Biotechnology.Rabbit anti–caspase-3, -8, and -9 Abs were from Cell Signaling Technology.Mouse anti-Flaviviruses NS3 Ab was from Yao-Hong Biotechology. Mouseanti-Flag, mouse HRP-conjugated anti-Flag, and mouse anti-tubulin Abswere purchased from Sigma-Aldrich. Rabbit anti-GAPDH Ab was fromGeneTex. Secondary HRP-conjugated anti-mouse Ig and HRP-conjugatedanti-rabbit Ig Abs were from Cell Signaling Technology and GeneTex, re-spectively. HRP-conjugated anti-mouse L chain and HRP-conjugated anti-rabbit L chain Abs were from Millipore.

Reagents and inhibitors

Pan-caspase inhibitor Boc-Asp(OMe)-fluoromethyl ketone (Sigma-Aldrich),control peptide Z-F-A-FMK (BD Pharmingen), caspase-3 inhibitor Z-D(OMe)-E(OMe)-V-D(OMe)-FMK (R&D), caspase-8 inhibitor Z-I(OMe)-E(OMe)-T-D(OMe)-FMK(R&D), caspase-9 inhibitor Z-L(OMe)-E(OMe)-H-D(OMe)-FMK (R&D Systems), and BAY 11-7082 (Sigma-Aldrich)were dissolved in absolute DMSO. Lymphotoxin (staurosporine) wasobtained from Sigma-Aldrich. The IKK-a/b Kinase (Human) Assay/InhibitorScreening Assay Kit was from Abnova.

Immunoprecipitation and Western blotting

Transfected 293T cells or transduced HMEC-1 cells with or without DENVinfection were lysed with radioimmunoprecipitation assay buffer containingprotease inhibitor mixture (Sigma-Aldrich). Anti-IkBa, anti-IkBb, or anti-Flag Ab (2 mg) was added to cell lysate. The mixture was left at 4˚C for 1–4 h. Protein A/G–conjugated agarose beads were added to the cell lysate,and the mixture was rotated at 4˚C overnight. Samples were washed withcold PBS, resuspended in 23 sample buffer (Bio-Rad), and subjected to12–15% SDS-PAGE, followed by transfer to polyvinylidene difluoridemembranes (Millipore). The membranes were blotted with primary Absand secondary HRP-conjugated Abs, followed by ECL (Millipore).

Measurement of IkBa and IkBb stability

To assay for the stability of endogenous IkBa and IkBb, transfected293T cells were treated with or without cycloheximide (20 mg/ml) at 48 hafter transfection. Cell lysates were then subjected to Western blot anal-ysis. The density of the protein bands was analyzed by ImageJ software.The half life was determined from the slopes of ordinary least squaresregression where the relative level of IkBa or IkBb was 0.5.

Detecting the cleavage forms of IkBa and IkBb

293T cells were cultured in 6-well plate and transfected with increasingamounts of plasmids encoding viral components and fixed amounts of IkBaor IkBb. Proteasome inhibitor (MG132; 5 mg/ml) (Sigma-Aldrich) andIKK1/2 inhibitor VII (IKK inhibitor VII; 0.5 mM) (Merck Millipore) wereadded at 36 h after transfection. Cells were cultured in the presence of theinhibitor for an additional 24 h. Cleavage forms of IkBa and IkBb in celllysates were detected by anti–C-terminal IkBa (Santa Cruz Biotechnol-ogy) and anti–C-terminal IkBb (Santa Cruz Biotechnology) Abs, respec-tively.

IKK activity assay

IKKa/b assay/Inhibitor Screening Kit (Abnova) was used to measure IKKactivity. Briefly, 293T cell were transfected with pRV vectors carryingdifferent viral components. Cells were harvested at 24, 36, and 48 h aftertransfection and resuspended in PBS. Cells were lysed through three220˚C/37˚C freeze/thaw cycles. After centrifugation, cell lysates wereharvested and applied to wells precoated with a substrate corresponding torecombinant IkBa with two serine residues that can be phosphorylated by

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IKKa and IKKb. Primary anti-phosphorylated IkBa Ab was added andleft at room temperature for 30 min, followed by incubation with sec-ondary HRP-conjugated anti-mouse IgG Ab at room temperature for 30min. Tetramethylbenzidine was used for chemiluminescence develop-ment. Absorbance was measured in a spectrophotometric iMark microplatereader (Bio-Rad) at 450 nm wavelength.

Characterization of NF-kB activation

The cytoplasmic and nuclear protein extracts from transfected 293T cells,transduced HMEC-1 or DENV-infected HMEC-1 were fractionated byreagents provided in NE-PER Nuclear and Cytoplasmic Extraction Kit(Thermo Scientific Pierce). The protein extracts were subjected to Westernblot analysis. Anti-p50 (Santa Cruz Biotechnology), anti-p65 Abs (SantaCruz Biotechnology), anti-tubulin (Sigma-Aldrich) and anti-Lamin A(Abcam) Abs were used.

Staining for cell death

DENV-2–infected (multiplicity of infection [MOI] = 10) or transducedHMEC-1 was trypsinized at different time points and resuspended in 50 mlallophycocyanin-conjugated Annexin V in Annexin V Binding Buffer (BDPharmingen) and incubated at room temperature for 30 min in the dark.Cells were resuspended in the same buffer with propidium iodide (PI;Sigma-Aldrich) and acquired by FACSCanto (BD Biosciences) and ana-lyzed by FlowJo software (Tree Star).

Targeted transfer of 2B-Pro and 2B-Pro-H51A to mice by AAV9

2B-Pro-IRES-GFP and 2B-Pro-H51A-IRES-GFP gene fragments werecloned into the pXX-UF1-CB-lacZ (B1268) plasmid by replacing the lacZfragment. An empty pXX-UF1-CB plasmid was also generated by deletingthe lacZ gene fromB1268. To generate the endothelial cell-targeting AAV9-SLR vector, 293T cells were transfected with three plasmids: p5E18-VD2/9-SfiI1959-SLRSPPS (capsid plasmid) (26), helper plasmid and the emptyB1268 plasmid, or B1268 plasmid carrying 2B-Pro or 2B-Pro-H51A. Thepurification and quantification of AAV9 were performed as describedpreviously (27). Six-week-old C57BL/6 mice were inoculated intrader-mally with 2 3 1011 vector genome (vg) of AAV9-SLR carrying emptyplasmid, 2B-Pro or 2B-Pro-H51A as described previously (7). Mice weresacrificed on day 2 after inoculation. Skin was exposed to observe hem-orrhage development, then excised, snap-frozen, and embedded in optimalcutting temperature medium and kept at 80˚C.

Immunofluorescence staining

Frozen skin was cryosectioned (Cryotome SME; Shandon) at a thicknessof 5 mm. Sections were stained with TUNEL reagents in the In Situ CellDeath Detection kit (Roche). Alexa Fluor 488–conjugated anti-DYKDDDDK(anti-Flag; Cell Signaling Technology) and allophycocyanin-conjugated anti-CD31 (BD Pharmingen) Abs were added and left at 4˚C overnight. Hoechst33258 (Sigma-Aldrich) was used to stain nuclei. Sections were imaged byconfocal microscope (Zeiss) and analyzed using LSM Image Browser soft-ware (Zeiss).

Stable expression of IkBa or IkBb in HMEC-1 cells

HMEC-1 cells were transduced with IkBa or IkBb and cultured for 48 h.GFP-positive IkBa or IkBb-overexpressing HMEC-1 cells were sorted byfluorescence-activated cell sorter (FACSAria; BD Biosciences) and ex-panded in M200 complete medium containing 2% of low serum growthsupplement.

DENV infection of HMEC-1 cells

HMEC-1 and mock-transduced and stable IkB-a/b–overexpressing-HMEC-1 cells were infected with DENV-2 16681 at an MOI of 10. Thecells were gently shaken every 10 min, harvested after incubation, andsubjected to analysis.

Statistical analysis

A two-tailed Student t test was used to compare the difference betweengroups. Data are presented as mean 6 SD.

ResultsCellular IkBa and IkBb interact with DENV protease

Using yeast-two-hybrid system with DENV 2B-3 as a bait, wescreened mouse cDNA library and identified IkBb as a cellularprotein candidate that interacts with 2B-3. To confirm this inter-

action in mammalian cells and to expand the observation to IkBa,we cloned DENV 2B-3, 2B-Pro, 2B-Pro-H51A mutant, NS3185(Pro), and NS2B (2B) components as well as cellular IkBb intoretroviral vectors. The vectors carrying DENV components hadFlag-tag on the C terminus. Immunoprecipitation of 2B-3, 2B-Pro,and Pro, but not 2B, coprecipitated both IkBa (Fig. 1A, 1B) andIkBb (Fig. 1C, 1D). Reciprocally, immunoprecipitation of IkBaor IkBb coprecipitated 2B-3, 2B-Pro, and Pro but not 2B(Supplemental Fig. 1A, 1B), showing that protease-containingDENV components directly interact with IkBa and IkBb. In-terestingly, enzyme activity-dead H51A mutation did not affectthe interactions of 2B-Pro with IkBa (Supplemental Fig. 1C,1D) and IkBb (Supplemental Fig. 1E, 1F), indicating that pro-tease H51 activity site is not involved in its physical interactionwith IkB proteins. We then generated pWPI constructs carryingcellular IkBa and IkBb to evaluate whether the physical inter-action between NS3 and IkB proteins occurs in DENV infection.HMEC-1 cells were transduced with lentiviral vectors express-ing either IkBa or IkBb and infected with DENV. Resultsshowed that immunoprecipitation of either IkBa or IkBbcoprecipitated viral NS3 in endothelial cells infected withDENV (Fig. 1E, 1F). Taken together, these experiments dem-onstrated that DENV protease physically interacts with IkBaand IkBb during infection and that this interaction does notinvolve its H51 activity site.

DENV protease reduces the stability of IkBa and IkBb

We first examined the stability of IkBa and IkBb in 2B-Pro–transfected cells to determine the effect of DENV protease on IkBproteins. Interestingly, 2B-Pro transfection significantly reducedthe stability of endogenous IkBa (t1/2 = 27.7 6 3.7 min in 2B-Pro–transfected versus t1/2 = 43.46 10.1 min in mock transfected,p, 0.05; Fig. 2A, top and bottom) and that of IkBb (t1/2 = 26.562.5 min in 2B-protransfected versus t1/2 = 51.1 6 15.1 minin mock transfected, p , 0.05; Fig. 2A, middle and bottom) incyclohexamide-treated cells. The stability of IkBa and IkBb incells transfected with activity-dead H51A mutant was comparableto mock controls (Fig. 2B). These data revealed that the specificinteraction between DENV protease and IkB proteins reduces thestability of the latter.

DENV protease cleaves IkBa and IkBb and activates IKK

To examine whether IkBa and IkBb are the potential 2B-3 targets,we added MG132 and IKK inhibitor VII to prevent IkB proteindegradation by 26S proteasome and phosphorylation by IKK, re-spectively. We coexpressed 2B-Pro and IkBa or IkBb in 293Tcells and found that 2B-Pro dose-dependently cleaved IkBaand IkBb (Fig. 2C). The sizes of the cleaved forms of IkBa andIkBb were ∼30 and 33 kDa, respectively. Coexpressions of2B-Pro-H51 and IkBa or IkBb did not result in IkBs cleavage(Fig. 2C). Kinase assay also showed that although transfections of2B-Pro and H51A mutant did not affect IKK a/b expression, theformer but not the latter induced IKK activation (Fig. 2D). Theseresults together indicate that DENV protease interacts with bothIkBa and IkBb and induces the cleavage of these cellular proteinsthrough its enzymatic activity and also activates IKK.

DENV protease induces NF-kB activation

NF-kB activation is tightly controlled by a series of inhibitoryproteins including IkBa and IkBb. Hence, we tested the effect ofthe interaction between DENV protease and IkBa/IkBb on NF-kB activity. Results showed that expression of DENV proteaseincluding that in 2B-3, 2B-Pro, and Pro, but not NS2B, resulted inincreased translocation of p50 and p65 to the nucleus and de-

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creased cytosolic p50 and p65 (Fig. 3A, 3B). Expression of H51Amutant failed to induce p50 and p65 translocation (Fig. 3C, 3D).These data demonstrate that DENV protease activates NF-kB byreducing the stability of IkB proteins.

DENV protease induces endothelial cell apoptosis through itsenzymatic activity

Endothelial cells are one of the targets of DENV and their inter-action with DENV plays a critical role in hemorrhage development(8). We studied the effect of DENV protease on endothelial cellsin vitro. Lentiviral vectors carrying DENV 2B-Pro, 2B-Pro-H51Aand 2B with Flag-tag on the C terminus and GFP under IREScontrol were constructed. Immunofluorescence staining revealedthat DENV 2B-Pro but not 2B induced HMEC-1 apoptosis (Fig.4A), and H51A mutant failed to induce apoptosis (Fig. 4B). Thisdemonstrates that DENV protease induces endothelial cell apo-ptosis through its enzymatic activity.

DENV protease induces hemorrhage in vivo

We further investigated the effects of DENV protease in vivo.Recent report showed that AAV9-SLR specifically targets humanumbilical cord vein endothelial cells in situ (26). Thus, we usedthe AAV9-SLR transduction system to deliver DENV protease tomice (Fig. 4C). Empty AAV9-SLR or AAV9-SLR carrying 2B-Proor 2B-Pro-H51A was injected intradermally to mice at four siteson the upper back. Interestingly, mice transduced with AAV9-SLRexpressing 2B-Pro developed hemorrhage along the blood vesselson day 2 after inoculation, whereas those transduced with H51Amutant or empty AAV9-SLR had no hemorrhagic manifestation(Fig. 4D). Further analysis of the skin biopsies showed that al-though 2B-Pro and 2B-Pro-H51A mutant colocalized with CD31+

endothelium, TUNEL+ cells were found only in 2B-Pro–trans-duced mice where the nuclei of most blood vessel endothelial cellsstained positive (Fig. 4E). These results showed that DENV pro-tease induces endothelial cell death and hemorrhage in mice and

the effect is dependent on protease activity. It is interesting to notethat other cells in the vicinity, possibly infiltrating macrophages,were also apoptotic (Fig. 4E). It is possible that TNF released byapoptotic macrophages also contributes to protease-induced hem-orrhage development (7, 8).

DENV protease induces endothelial cell death throughextrinsic apoptosis pathway

Viral protein–transduced endothelial cells were analyzed for cas-pase cleavage. Cleaved caspase-3 and -8 were detected in 2B-Pro-and Pro- but not 2B-Pro-H51A- or 2B–transduced HMEC-1 cells,whereas no caspase-9 cleavage was observed in cells transducedwith any of them (Fig. 5A–C). Although treatment with caspaseinhibitors did not change the transduction efficiency (SupplementalFig. 2A), treatment with pan-caspase or caspase-3 or -8 inhibitorsignificantly reduced 2B-Pro–induced HMEC-1 apoptosis (Fig. 5D),indicating that DENV protease induces HMEC-1 apoptosis byactivation of the extrinsic apoptotic pathway.

DENV protease-induced endothelial cell death isNF-kB-dependent

Bay 11-7082 has been shown to inactivate NF-kB through irre-versible inhibition of IkBa phosphorylation (28). 2B-Pro and2B-Pro-H51A–transduced HMEC-1 cells were treated with Bay11-7082. Although treatment with Bay11-7082 did not affecttransduction efficiency or viral protein expression (SupplementalFig. 2B, 2C), it dose-dependently inhibited NS2B-protease–inducedp50 and p65 nuclear translocation (Supplemental Fig. 3). Impor-tantly, Bay11-7082 treatment blocked 2B-Pro–induced HMEC-1apoptosis (Fig. 5E), demonstrating that NF-kB activation is criti-cal to DENV protease–induced endothelial cell apoptosis. In ad-dition, Bay11-7082 treatment dose-dependently reduced DENVprotease–induced caspase-3 and -8 cleavages in HMEC-1 cells(Fig. 5F). Inversely, although blocking the activity of caspase-3 or-8 did not affect viral protein expression, it also did not inhibit

FIGURE 1. DENV protease interacts with cellular IkBa and IkBb. (A) 293T cells were transfected with empty pRV vector (mock) or pRV vector

carrying 2B-3, 2B-Pro, Pro, or 2B. The viral components as well as endogenous IkBa were detected by indicated Abs. (B) Cell lysates were immuno-

precipitated with anti-Flag Ab and blotted with anti-IkBa and anti-Flag Abs. (C) 293T cells were cotransfected with pRV vectors carrying the same viral

components as described in (A) and pRV vector carrying IkBb. Cell lysates were blotted with indicated Abs. (D) Immunoprecipitations were performed as

described in (B), except that anti-IkBb instead of anti-IkBa Ab was used. (E) HMEC-1 cells were stably transduced with IkBa (left panel) or IkBb (right

panel) by lentivirus and infected with or without DENV (MOI = 10). Cell lysates were collected 36 h later and blotted with indicated Abs. (F) Cell lysates

were immunoprecipitated with anti-IkBa (left panel) or anti-IkBb (right panel) Ab and blotted with indicated Abs. Either rabbit IgG (rIgG) or mouse IgG

(mIgG) Ab was used as immunoprecipitation control. Tubulin was used as a loading control. Data shown in (A)–(F) are representative of three independent

experiments. Because 2B-3 and 2B-Pro were enzymatically active and contained some autocleavage sites (14, 47, 48), multiple bands were observed on the

Western blot. Arrows point to uncleaved 2B-3 and 2B-Pro and white arrowheads to cleaved fragments.

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NS2B-protease-induced p50 and p65 nuclear translocation (Fig. 5G,5H, Supplemental Fig. 2D). These results together indicate that NF-kB activation precedes caspases-8 and -3 cleavages in DENVprotease-induced apoptosis.

Overexpressing IkBa or IkBb protects HMEC-1 fromDENV-induced apoptosis

BecauseDENVprotease induces HMEC-1 apoptosis by reducing thestability of IkB proteins, we investigated whether overexpressingIkBa or IkBb protects HMEC-1 cells from DENV-induced apo-ptosis. HMEC-1 cells infected by DENV expressed NS3 and thecells underwent apoptosis (Supplemental Fig. 4A–C). Bay11-7082treatment dose-dependently blocked DENV-induced p50 and p65nuclear translocation and reduced virus-induced apoptosis, dem-

onstrating that DENV-induced cell death is dependent on NF-kBactivation (Fig. 6A, 6B). HMEC-1 cells stably transduced withIkBa and IkBb were sorted. Although overexpressing IkBa orIkBb did not affect virus NS3 expression or the percentage of ap-optotic cells (Fig. 6C, 6D, upper panel), overexpressing IkBa– orIkBb–protected cells from DENV-induced apoptosis (Fig. 6D,lower panel). These results showed that IkBa or IkBb over-expression protects HMEC-1 cells from DENV-induced, NF-kB–dependent apoptosis.

DiscussionMild hemorrhagic manifestations occur quite frequently accom-panying a wide range of dengue disease syndromes. The etiologyof these hemorrhagic phenomena is not well understood. We

FIGURE 2. DENV protease reduces the stability of IkBa and IkBb by cleaving them and activating IKK. (A and B) 293T cells were transfected with

empty pRV vector (mock) and pRV vector carrying 2B-Pro or 2B-Pro-H51A. At 48 h after transfection, 293T cells were treated with or without cyclo-

heximide (CHX; 20 mg/ml) for indicated periods of time. Cell lysates were blotted with anti-Flag, anti-IkBa (top panels), or anti-IkBb (middle panels) and

anti-tubulin Abs and quantified by ImageJ software. The relative levels of IkBa and IkBb were calculated by dividing the density of IkBa or IkBb by the

density of tubulin at the same time point. The relative levels of IkBa and IkBb at time 0 were set as 1 (bottom panels). Data presented are means 6 SD

obtained from five independent experiments. *p , 0.05, **p , 0.01 by comparing either 2B-Pro (A) or 2B-Pro-H51A (B) to mock. (C) 293T cells were

transfected with IkBa (0.8 mg) or IkBb (0.5 mg) along with increasing quantities of plasmid encoding Flag-tagged 2B-Pro or 2B-Pro-H51A (1, 3, and 6 mg)

(upper panels) and 2B-Pro or 2B-Pro-H51A (1.5, 3.5, and 6.5 mg) (lower panels). At 36 h after transfection, MG132 and IKK inhibitor VII were added.

Viral protein was detected with anti-Flag Ab. The cleavage forms of IkBa and IkBb were detected by anti-C terminus-IkBa and -IkBb Abs, respectively.

(D) 293T cells were transfected with pRV vector (mock) and pRV vectors containing 2B-Pro or 2B3-Pro-H51A and left in culture for 24, 36, or 48 h before

harvest. Cell lysates collected at 24 and 48 h were applied to wells coated with IkBa. Phosphorylated IkBa was measured by anti-phosphorylated IkBa Ab

(upper panels). Data presented are the mean6 SD of data obtained from three independent experiments with three replicates each. Cell lysates were blotted

with anti-IKKa or anti-IKKb Ab (lower panels). Tubulin was used as loading control. Arrows point to uncleaved 2B-Pro fragment and white arrowheads to

cleaved fragments. *p , 0.05, **p , 0.01.

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demonstrated in mouse hemorrhage model that DENV infectingendothelial cells and infiltration of TNF-producing macrophagesare both important to hemorrhage development. How DENVparticipates in the pathogenic mechanism has never been studied,and neither has it been reported how a viral component can be

involved in this pathogenic process. We found that DENV proteaseinduces endothelial cell apoptosis through interaction with IkBaand IkBb and causes hemorrhage development when delivered tomice. DENV protease cleaves IkBa and IkBb after binding tothem and activates IKK, thus triggering NF-kB activation, which

FIGURE 3. DENV protease induces NF-kB activation. 293T cells were transfected with empty pRV vector (mock), pRV vector carrying 2B-3, 2B-Pro,

Pro, or 2B (A and B) or pRV vector carrying 2B-3, 2B-Pro or 2B-Pro-H51A (C and D). (A, left and C, left) At 48 h after transfection, cell lysates were

blotted with indicated Abs. Tubulin was used as a loading control. (A, right panel, and C, right panel) Cell lysates were separated into cytoplasmic and

nuclear fractions and blotted with anti-p50, anti-p65, anti-lamin A (nuclear marker), and anti-tubulin (cytoplasmic marker) Abs. Arrows point to uncleaved

2B-3 and 2B-Pro and white arrowheads to cleaved fragments. (B and D) The p50 and p65 bands were scanned and quantified by ImageJ software. The

relative density was calculated by dividing the nuclear density of p50 and p65 by the total cytosol plus nuclear density of p50 and p65, respectively. Data

presented are the means 6 SD of data obtained from three independent experiments. *p , 0.05, **p , 0.01 by comparing experimental group to mock

control or by comparing the two groups linked by a horizontal line.

FIGURE 4. DENV protease induces endothelial cell death and hemorrhage development. (A and B) HMEC-1 cells were transduced with lentivirus

carrying empty pWPI (mock) or pWPI vector carrying 2B-Pro or 2B (A) and 2B-Pro or 2B-Pro-H51A (B). Transduced cells were stained with Annexin V/PI

at 48 h later and analyzed by flow cytometry. Dot plot and bar graph show percentage of dead cells in total GFP+ cell population. Data shown are the mean6SD from three independent experiments. **p , 0.01. (C) Gene fragments encoding 2B-Pro or 2B-Pro-H51A with Flag-tag on the C terminus were cloned

into the pXX-UF1-CB-lacZ (B1268) plasmid by replacing the lacZ fragment. Expression of the viral protein was detected by anti-Flag Ab in transfected

293T cell lysates. GADPH was used as an internal control. Arrows point to uncleaved 2B-Pro fragment and white arrowheads to cleaved fragments. (D and

E) Mice were inoculated intradermally with 2 3 1011 vg AAV9-SLR carrying the empty AAV9-SLR, 2B-Pro, or 2B-Pro-H51A at four diagonal sites on the

upper back. The skin was exposed and photographed on day 2 postinfection (D). Skin within the boundary of the lines connecting the four injection sites

was excised and snap frozen in optimal cutting temperature medium (E). Cryosections were stained with Alexa Fluor 488–conjugated rabbit anti-Flag Ab

(green), allophycocyanin-conjugated rat anti-mouse CD31 Ab (yellow), TUNEL reagents (red), and Hoechst 33258 (blue) stain. White lines outline the

endothelium of blood vessel.

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leads to caspase-mediated endothelial cell apoptosis. Furthermore,overexpression of either IkBa or IkBb protects endothelial cellsfrom DENV-induced apoptosis. To our knowledge, our resultsdemonstrate for the first time that DENV protease induces hem-orrhage in mice, likely through regulating molecules in the NF-kBpathway that controls cell death.In the mouse model, we demonstrated that DENV targets en-

dothelium and endothelial cell death is critical to hemorrhagedevelopment (8). In this study, we used the modified AAV9-SLRsystem to deliver DENV protease to mouse endothelium in vivo(26). This strategy successfully targets DENV protease to mouse

CD31+ vascular endothelial cells and causes hemorrhage to de-velop along the blood vessel. Interestingly, although activity-deadDENV protease mutant was also successfully targeted to endo-thelial cells, it does not induce cell death and no hemorrhage wasobserved. Our results revealed that SLRSPPS-displaying AAV9 isefficient in delivering genes to endothelium in mice, and impor-tantly, that DENV protease, which causes endothelial cell death,is the key viral component that is involved in dengue hemor-rhage. On the basis of our findings, we provide a new molecularpathogenic mechanism of viral hemorrhage and we demonstratehow viral protease contributes to the pathogenic process in mouse

FIGURE 5. DENV protease induces endothelial cell death through NF-kB–dependent extrinsic apoptosis pathway. (A–C) HMEC-1 cells were transduced

with empty pWPI vector (mock), pWPI vector carrying 2B-Pro, 2B-Pro-H51A, Pro, or 2B. The lysates were blotted with indicated Abs. HMEC-1 treated

with lymphotoxin (1:7500) was used as a positive control for caspase activation (Pos Ctrl). (D) HMEC-1 cells were transduced with 2B-Pro-H51A or 2B-

Pro and treated with 4 mM control peptide (Z-FA-FMK), pan-caspase (Boc-D-FMK), caspase-3 (Z-DEVD-FMK), -8 (Z-IETD-FMK), or -9 (Z-LEHD-

FMK) inhibitor. Transduced cells were stained with Annexin V/PI at 48 h. The percentage of dead cells in GFP+ cell population was shown in bar graph.

The mean6 SD are data obtained from three independent experiments. **p, 0.01. (E and F) HMEC-1 cells were transduced with 2B-Pro-H51A or 2B-Pro

before treatment without or with different concentrations of Bay 11-7082 (Bay). (E) Tranduced cells were stained with Annexin V/PI at 36 h and analyzed

by flow cytometry. Bar graph shows the percentage of dead cells in total GFP+ cell population. Values in bar graphs are the means6 SD obtained from three

independent experiments. *p , 0.05. (F) At 48 h after transduction, cell lysates were blotted with indicated Abs (upper panel). Bar graphs show densi-

tometric quantification of cleaved caspase-3 and -8 against GAPDH (lower panel). Data shown are pooled from three separate experiments, one determinant

in each experiment. *p, 0.05, **p, 0.01. Arrows point to uncleaved 2B-Pro fragment and white arrowheads to cleaved fragments in (A), (B), (C), and (F).

(G and H) HMEC-1 cells were transduced with 2B-Pro-H51A or 2B-Pro and treated without or with different concentrations of Z-DEVD-FMK (G) or Z-

IETD-FMK (H). At 48 h after transduction, the lysates were separated into cytoplasmic and nuclear fractions and blotted with anti-p50, anti-p65, anti-lamin

A (nuclear marker), and anti-tubulin (cytoplasmic marker) Abs.

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model. However, whether the phenomena observed in our mousemodel can relate to human dengue remains to be demonstrated.Early in our study employing yeast-two-hybrid system, we

identified IkBb as one of the cellular proteins that interacts withDENV 2B-3. Coimmunoprecipitation/Western blot assay furtherrevealed that DENV protease not only physically interacts withIkBb but also IkBa. In the same way, the N-terminal protease(Npro) of classical swine fever virus, another member of the fla-viviridae that causes hemorrhage in pigs, was shown to interactwith IkBa (29). In the study of Flavivirus NS3 and NS5 proteininteraction networks, IkBa is found to be one of the NS3 inter-acting proteins (30). These studies independently support ourfinding that DENV protease interacts with cellular IkB-a/b. IkBproteins are characterized by the presence of many ankyrin (ANK)repeats that mediate protein–protein interaction (31–34). Throughthe ANK repeats, IkBb interacts with the Rel homology region ofNF-kB (34). DENV NS3 is recently shown to bind to the 19 ANKrepeat-containing ANK repeat domain protein 50 (35). Thus, wepostulate that DENV protease binds to IkB-a/b through the ANKrepeats. Work is under way in our laboratory to test this hypoth-esis. The question of whether interaction with cellular IkB-a/b isa shared attribute among the NS3 proteases of other flavivirusesremains to be addressed.DENV protease is known to cleave viral polyprotein at specific

junctions that contain two basic amino acids (KR, RR, RK, andQR), followed by a small amino acid (G, A, or S) (13–15). In-terestingly, both IkBa and IkBb contain the putative cleavagesequence (PR62GS in IkBa; ERR127G in IkBb). Our resultsshowed that expression of DENV protease reduces the stability ofendogenous IkBa and IkBb in the host cells. We further discov-

ered that DENV protease cleaves both IkBa (37 kDa) and IkBb(43 kDa) into respective 30- and 33-kDa fragments, thus matchingthe size prediction based on the putative cleavage sites. Thus,we speculate that PR62GS and ERR127G are the DENV proteasecleavage sites on IkBa and IkBb, respectively. However, we couldnot rule out the possibility that IkBa and IkBb are cleavedby a cellular protease, which may potentially be regulated by theactivity of DENV protease.During Coxsackie B3 virus (CVB3) infection, IkBa is trans-

located to the nucleus and recombinant protease (3Cpro) cleavescellular IkBa (36). In cells transfected with the cleaved IkBafragment, the TNF-induced NF-kB p65 fragment enters the nu-cleus. The cleaved IkBa fragment functions to retain p65 in thenucleus, inhibits NF-kB transactivation, and increases host cellapoptosis (36). Thus, through interaction with IkBa, CVB3 3Cpro

increases host cell death (36). We found that DENV protease, likeCVB3 3Cpro, interacts with IkB proteins but works through a dif-ferent mechanism to induce cell death. DENV protease cleavesIkBa and IkBb after binding to them and also activates IKK.Through both of these modes of action, DENV protease activatesNF-kB and induces host cell death.NF-kB activation normally leads to the production of anti-

apoptotic proteins, and cells are protected from apoptosis (18, 19).However, in DENV infection, that NF-kB plays a proapoptoticrole has been reported in neuroblastoma (20) and hepatoma celllines (21). In this study, we showed that DENV protease over-expression induces NF-kB activation and that NF-kB activationprecedes caspase-8 and -3 activation but not vice versa. These datasuggest that product(s) of the NF-kB signaling pathway triggersthe extrinsic apoptotic pathway and cell death. Host factors, in-

FIGURE 6. Suppression of NF-kB activity reduces DENV-induced endothelial cell death. (A and B) HMEC-1 cells were treated without or with Bay 11-

7082 before infection with DENV (MOI = 10) at 36 h after treatment. (A, left panel) Cell lysates were blotted with anti-NS3 and anti-tubulin Abs. Tubulin

was used as a loading control. (A, right panel) Cell lysates were separated into cytoplasmic and nuclear fractions and blotted with anti-p50, anti-p65, anti-

lamin A (nuclear marker), and anti-tubulin (cytoplasmic marker) Abs. (B) The cells were stained with Annexin V/PI and analyzed by flow cytometry. The

dot plots (left panel) and bar graph (right panel) show the percentage of dead cells. (C and D) HMEC-1 cells were transduced with empty pWPI vector or

pWPI vector carrying IkBa or IkBb. Cells stably expressing GFP, IkBa, and IkBb were sorted. Sorted cells were infected with or without DENV (MOI =

10). (C) Cell lysates were blotted with anti-NS3, anti-IkBa, anti-IkBb, and anti-tubulin Abs. Arrows point to uncleaved 2B-3 fragment and white

arrowheads to cleaved fragments in (A) and (C). (D) The percentage of dead cells was determined by flow cytometry at 36 h postinfection. Untransduced

HMEC-1 cells were used as a baseline control (Ctrl). The bar graphs show the means 6 SD percentage of dead cells obtained from three independent

experiments. *p , 0.05, **p , 0.01.

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cluding TNF, reactive nitrogen, and oxygen species, are importantto DENV-induced endothelial cell death and hemorrhage devel-opment in DENV infection (4, 7, 8). Our unpublished observationsshowed that intradermal delivery of 2B-Pro but not 2B-Pro-H51Ato mice induced infiltration of TNF-producing macrophages(J.-C. Lin and B.A. Wu-Hsieh, data not shown) which maycontribute to protease-induced hemorrhage development. It is pos-sible that NF-kB-mediated endothelial cell apoptosis trigged byDENV protease is through enhancing macrophage recruitment andalso possibly through enhancing the sensitivity of endothelial cellsto TNF by expressing TNF receptors or by inducing reactive nitrogenor oxygen species or both. These possibilities are currently being tested.Using activity-dead DENV 2B-Pro-H51A, we found that pro-

tease activity site is not physically involved in the interaction be-tween DENV protease and IkB proteins, but protease activity isrequired for the cleavage of them, activation of IKK, and theevents that follows. Furthermore, DENV protease activity is crit-ical to its ability to induce hemorrhage development. It is recentlyshown that DENV protease cleaves human MITA to inhibit typeI IFN response through its protease activity (16, 17). To ourknowledge, these reports are the first ones to show that DENVprotease interacts with and degrades cellular protein. Our reportidentifies IkB-a/b as the new cellular targets that are cleaved byDENV protease and demonstrates that DENV protease and IkB-a/b interaction has pathogenic consequences.Vascular leakage and shock are the major causes of death in

patients with DHF/DSS. The endothelium forms the primaryfluid barrier of the circulation system. Therefore, it is important tounderstand the relationship between DENV-induced endothelialcell dysfunction and vascular leakage. It is reported in Ebola virusand Lassa virus infections that dysfunction of endothelial cellscan affect vascular permeability and leakage (37–39). In vitrostudies showed that DENV productively infects primary HUVECand microvascular endothelial cell line HEMC-1 and causes celldeath, which is enhanced by TNF (8). Several reports claimedthat DENVAgs are found in endothelial cells in autopsied tissuesof patients who died of DHF/DSS (40–44). However, in thesestudies, neither the viral negative strand RNA nor NS3 Agswas demonstrated. Neither was the identity of the endothelialcells established by their specific markers. Therefore, it remainsunclear whether DENV productively infects endothelium inhuman cases.In our dengue hemorrhage mouse model, we showed that

introdermal inoculation of DENV results in hemorrhage devel-opment (8). Both DENV nucleic acid and Ag are detected in theendothelium within one day after inoculation. Temporal studyshowed that there is macrophage infiltration into the vicinity ofendothelium, increased TNF production, and endothelial cell ap-optosis in the hemorrhage tissues by day 2. Administration ofcaspase inhibitor inhibits hemorrhage development. On the basisof these published results and our current findings, we proposea working model that depicts the molecular pathogenic processof dengue hemorrhage involving both viral and immune factors(Fig. 7): 1) DENV infects the endothelium after inoculationthrough mosquito bite or by intradermal injection (Fig. 7A, 7B) (7,8). 2) DENV protease is expressed in the endothelial cells, and itinteracts with cellular IkBa and IkBb (Fig. 7C, 7D). 3) DENVprotease reduces the stability of IkBa and IkBb by enzymaticactivity–dependent cleavage (Fig. 7E) as well as by inducing IKKactivation (Fig. 7F). 4) P50 and p65 are translocated to the nucleusas a result of IkBa and IkBb cleavage and NF-kB is activated(Fig. 7G). 5) NF-kB activation triggers the extrinsic apoptoticpathway directly or by its product(s) and induces endothelial celldeath (Fig. 7H–M). 6) Hemorrhage develops as a result of endo-

thelial cell death. In the presence of TNF, endothelial cell death isenhanced and more severe hemorrhage develops (Fig. 7N, 7O) (7).The vascular endothelium is considered to be the ultimate

battlefield where damage will lead to severe consequences (4, 45,46). On the basis of the study in our mouse model, in this study,we propose that DENV protease is the key viral component thatparticipates in the complex pathogenic process of hemorrhagedevelopment. By interacting with IkBa and IkBb and activatingNF-kB pathway, DENV protease directly triggers the cellularmachinery that can cause endothelial cell death or indirectly bymaking them more susceptible to the detrimental effect of hostfactors.

AcknowledgmentsWe thank Dr. O.J. Muller (Department of Internal Medicine III, University

Hospital Heidelberg, Heidelberg, Germany) for the gift of the p-5E18-VD-

2/9-SfiI1759 vector. We thank the Cell Sorting Core Facility at the National

Taiwan University College of Medicine for cell sorting service.

FIGURE 7. A proposed model for the mechanism of DENV protease-

induced endothelial cell apoptosis and hemorrhage development. (a)

DENV is introduced to the host intradermally by mosquito bite (in nature)

or through injection (experimentally). (b) DENV infects endothelial cells

after entering the host. (c) DENV protease is expressed in the infected

endothelial cells. (d) DENV protease interacts with cellular IkBa and

IkBb in the cell cytoplasm. (e) IkBa and IkBb are cleaved into smaller

fragments by DENV protease. (f) DENV protease also activates IKK. IKK

phosphorylates IkBa and IkBb by which facilitates their degradation. (g)

IkBa and IkBb cleavage/degradation enables p50 and p65 translocation

into the nucleus, thereby activates NF-kB. (h–l) NF-kB activation triggers

extrinsic apoptotic pathway involving caspase-8 and -3 cleavages directly

or through production of molecule(s) that renders endothelial cells sus-

ceptible to host factors. (m–o) Endothelial cells become apoptotic re-

sulting in hemorrhage development. In the presence of TNF, endothelial

cell death is enhanced, and more severe hemorrhage develops.

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DisclosuresThe authors have no financial conflicts of interest.

References1. Kurane, I. 2003. [Dengue virus]. Nippon Rinsho 61(Suppl. 3): 486–490.2. Bhamarapravati, N. 1989. Hemostatic defects in dengue hemorrhagic fever. Rev.

Infect. Dis. 11(Suppl. 4): S826–S829.3. Gubler, D. J. 1998. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev.

11: 480–496.4. Basu, A., and U. C. Chaturvedi. 2008. Vascular endothelium: the battlefield of

dengue viruses. FEMS Immunol. Med. Microbiol. 53: 287–299.5. Martina, B. E., P. Koraka, and A. D. Osterhaus. 2009. Dengue virus pathogen-

esis: an integrated view. Clin. Microbiol. Rev. 22: 564–581.6. Guzman, M. G., and G. Kourı. 2002. Dengue: an update. Lancet Infect. Dis. 2: 33–42.7. Chen, H. C., F. M. Hofman, J. T. Kung, Y. D. Lin, and B. A. Wu-Hsieh. 2007.

Both virus and tumor necrosis factor a are critical for endothelium damage ina mouse model of dengue virus-induced hemorrhage. J. Virol. 81: 5518–5526.

8. Yen, Y. T., H.-C. Chen, Y. D. Lin, C. C. Shieh, and B. A. Wu-Hsieh. 2008.Enhancement by tumor necrosis factor a of dengue virus-induced endothelialcell production of reactive nitrogen and oxygen species is key to hemorrhagedevelopment. J. Virol. 82: 12312–12324.

9. Lindenbach, B. D., and C. M. Rice. 2003. Molecular biology of flaviviruses. Adv.Virus Res. 59: 23–61.

10. Bazan, J. F., and R. J. Fletterick. 1989. Detection of a trypsin-like serine proteasedomain in flaviviruses and pestiviruses. Virology 171: 637–639.

11. Gorbalenya, A. E., A. P. Donchenko, E. V. Koonin, and V. M. Blinov. 1989.N-terminal domains of putative helicases of flavi- and pestiviruses may be serineproteases. Nucleic Acids Res. 17: 3889–3897.

12. Brinkworth, R. I., D. P. Fairlie, D. Leung, and P. R. Young. 1999. Homologymodel of the dengue 2 virus NS3 protease: putative interactions with bothsubstrate and NS2B cofactor. J. Gen. Virol. 80: 1167–1177.

13. Preugschat, F., C. W. Yao, and J. H. Strauss. 1990. In vitro processing of denguevirus type 2 nonstructural proteins NS2A, NS2B, and NS3. J. Virol. 64: 4364–4374.

14. Yusof, R., S. Clum, M. Wetzel, H. M. Murthy, and R. Padmanabhan. 2000.Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactorNS2B dependence for cleavage of substrates with dibasic amino acids in vitro. J.Biol. Chem. 275: 9963–9969.

15. Zhang, L., P. M. Mohan, and R. Padmanabhan. 1992. Processing and localizationof Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5. J. Virol.66: 7549–7554.

16. Aguirre, S., A. M. Maestre, S. Pagni, J. R. Patel, T. Savage, D. Gutman,K. Maringer, D. Bernal-Rubio, R. S. Shabman, V. Simon, et al. 2012. DENVinhibits type I IFN production in infected cells by cleaving human STING. PLoSPathog. 8: e1002934.

17. Yu, C. Y., T. H. Chang, J. J. Liang, R. L. Chiang, Y. L. Lee, C. L. Liao, andY. L. Lin. 2012. Dengue virus targets the adaptor protein MITA to subvert hostinnate immunity. PLoS Pathog. 8: e1002780.

18. Li, Q., and I. M. Verma. 2002. NF-kB regulation in the immune system. Nat.Rev. Immunol. 2: 725–734.

19. Radhakrishnan, S. K., and S. Kamalakaran. 2006. Pro-apoptotic role of NF-kB:implications for cancer therapy. Biochim. Biophys. Acta 1766: 53–62.

20. Jan, J. T., B. H. Chen, S. H. Ma, C. I. Liu, H. P. Tsai, H. C. Wu, S. Y. Jiang,K. D. Yang, and M. F. Shaio. 2000. Potential dengue virus-triggered apoptoticpathway in human neuroblastoma cells: arachidonic acid, superoxide anion, andNF-kB are sequentially involved. J. Virol. 74: 8680–8691.

21. Marianneau, P., A. Cardona, L. Edelman, V. Deubel, and P. Despres. 1997.Dengue virus replication in human hepatoma cells activates NF-kB which in turninduces apoptotic cell death. J. Virol. 71: 3244–3249.

22. Prikhod’ko, G. G., E. A. Prikhod’ko, A. G. Pletnev, and J. I. Cohen. 2002.Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis. J. Virol.76: 5701–5710.

23. Ramanathan, M. P., J. A. Chambers, P. Pankhong, M. Chattergoon,W. Attatippaholkun, K. Dang, N. Shah, and D. B. Weiner. 2006. Host cell killingby the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficientto recruit caspase-8-based apoptotic pathway. Virology 345: 56–72.

24. Shafee, N., and S. AbuBakar. 2003. Dengue virus type 2 NS3 protease andNS2B-NS3 protease precursor induce apoptosis. J. Gen. Virol. 84: 2191–2195.

25. Vasquez Ochoa, M., J. Garcıa Cordero, B. Gutierrez Castaneda, L. SantosArgumedo, N. Villegas Sepulveda, and L. Cedillo Barron. 2009. A clinical

isolate of dengue virus and its proteins induce apoptosis in HMEC-1 cells:a possible implication in pathogenesis. Arch. Virol. 154: 919–928.

26. Varadi, K., S. Michelfelder, T. Korff, M. Hecker, M. Trepel, H. A. Katus,J. A. Kleinschmidt, and O. J. M€uller. 2012. Novel random peptide librariesdisplayed on AAV serotype 9 for selection of endothelial cell-directed genetransfer vectors. Gene Ther. 19: 800–809.

27. Chen, C. C., C. P. Sun, H. I. Ma, C. C. Fang, P. Y. Wu, X. Xiao, and M. H. Tao.2009. Comparative study of anti-hepatitis B virus RNA interference by double-stranded adeno-associated virus serotypes 7, 8, and 9. Mol. Ther. 17: 352–359.

28. Pierce, J. W., R. Schoenleber, G. Jesmok, J. Best, S. A. Moore, T. Collins, andM. E. Gerritsen. 1997. Novel inhibitors of cytokine-induced IkBa phosphory-lation and endothelial cell adhesion molecule expression show anti-inflammatoryeffects in vivo. J. Biol. Chem. 272: 21096–21103.

29. Doceul, V., B. Charleston, H. Crooke, E. Reid, P. P. Powell, and J. Seago. 2008.The Npro product of classical swine fever virus interacts with IkBa, the NF-kBinhibitor. J. Gen. Virol. 89: 1881–1889.

30. Le Breton, M., L. Meyniel-Schicklin, A. Deloire, B. Coutard, B. Canard, X. deLamballerie, P. Andre, C. Rabourdin-Combe, V. Lotteau, and N. Davoust. 2011.Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeasttwo-hybrid screen. BMC Microbiol. 11: 234.

31. Jacobs, M. D., and S. C. Harrison. 1998. Structure of an IkBa/NF-kB complex.Cell 95: 749–758.

32. Huxford, T., D. B. Huang, S. Malek, and G. Ghosh. 1998. The crystal structureof the IkBa/NF-kB complex reveals mechanisms of NF-kB inactivation. Cell95: 759–770.

33. Huxford, T., and G. Ghosh. 2009. A structural guide to proteins of the NF-kBsignaling module. Cold Spring Harb. Perspect. Biol. 1: a000075.

34. Malek, S., D. B. Huang, T. Huxford, S. Ghosh, and G. Ghosh. 2003. X-raycrystal structure of an IkBb 3 NF-kB p65 homodimer complex. J. Biol.Chem. 278: 23094–23100.

35. Khadka, S., A. D. Vangeloff, C. Zhang, P. Siddavatam, N. S. Heaton, L. Wang,R. Sengupta, S. Sahasrabudhe, G. Randall, M. Gribskov, et al. 2011. A physicalinteraction network of dengue virus and human proteins. Mol. Cell. Proteomics10: M111.012187.

36. Zaragoza, C., M. Saura, E. Y. Padalko, E. Lopez-Rivera, T. R. Lizarbe, S. Lamas,and C. J. Lowenstein. 2006. Viral protease cleavage of inhibitor of kBa triggershost cell apoptosis. Proc. Natl. Acad. Sci. USA 103: 19051–19056.

37. Yang, Z. Y., H. J. Duckers, N. J. Sullivan, A. Sanchez, E. G. Nabel, andG. J. Nabel. 2000. Identification of the Ebola virus glycoprotein as the main viraldeterminant of vascular cell cytotoxicity and injury. Nat. Med. 6: 886–889.

38. Wahl-Jensen, V. M., T. A. Afanasieva, J. Seebach, U. Stroher, H. Feldmann, andH. J. Schnittler. 2005. Effects of Ebola virus glycoproteins on endothelial cellactivation and barrier function. J. Virol. 79: 10442–10450.

39. Moraz, M. L., and S. Kunz. 2011. Pathogenesis of arenavirus hemorrhagicfevers. Expert Rev. Anti Infect. Ther. 9: 49–59.

40. Jessie, K., M. Y. Fong, S. Devi, S. K. Lam, and K. T. Wong. 2004. Localizationof dengue virus in naturally infected human tissues, by immunohistochemistryand in situ hybridization. J. Infect. Dis. 189: 1411–1418.

41. Ramos, C., G. Sanchez, R. H. Pando, J. Baquera, D. Hernandez, J. Mota,J. Ramos, A. Flores, and E. Llausas. 1998. Dengue virus in the brain of a fatalcase of hemorrhagic dengue fever. J. Neurovirol. 4: 465–468.

42. Hall, W. C., T. P. Crowell, D. M. Watts, V. L. Barros, H. Kruger, F. Pinheiro, andC. J. Peters. 1991. Demonstration of yellow fever and dengue antigens informalin-fixed paraffin-embedded human liver by immunohistochemical analy-sis. Am. J. Trop. Med. Hyg. 45: 408–417.

43. Balsitis, S. J., J. Coloma, G. Castro, A. Alava, D. Flores, J. H. McKerrow,P. R. Beatty, and E. Harris. 2009. Tropism of dengue virus in mice and humansdefined by viral nonstructural protein 3-specific immunostaining. Am. J. Trop.Med. Hyg. 80: 416–424.

44. Salgado, D. M., J. M. Eltit, K. Mansfield, C. Panqueba, D. Castro, M. R. Vega,K. Xhaja, D. Schmidt, K. J. Martin, P. D. Allen, et al. 2010. Heart and skeletalmuscle are targets of dengue virus infection. Pediatr. Infect. Dis. J. 29: 238–242.

45. Bray, M. 2005. Pathogenesis of viral hemorrhagic fever. Curr. Opin. Immunol.17: 399–403.

46. Wu-Hsieh, B. A., Y. T. Yen, and H. C. Chen. 2009. Dengue hemorrhage ina mouse model. Ann. N. Y. Acad. Sci. 1171(Suppl. 1): E42–E47.

47. Yu, C. Y., Y. W. Hsu, C. L. Liao, and Y. L. Lin. 2006. Flavivirus infectionactivates the XBP1 pathway of the unfolded protein response to cope with en-doplasmic reticulum stress. J. Virol. 80: 11868–11880.

48. Rodriguez-Madoz, J. R., A. Belicha-Villanueva, D. Bernal-Rubio, J. Ashour,J. Ayllon, and A. Fernandez-Sesma. 2010. Inhibition of the type I interferonresponse in human dendritic cells by dengue virus infection requires a catalyti-cally active NS2B3 complex. J. Virol. 84: 9760–9774.

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