Characterization of Two Mosquito STATs, AaSTAT and CtSTATDIFFERENTIAL REGULATION OF TYROSINE PHOSPHORYLATION AND DNA BINDING ACTIVITY BYLIPOPOLYSACCHARIDE TREATMENT AND BY JAPANESE ENCEPHALITIS VIRUS INFECTION*
Received for publication, September 3, 2003, and in revised form, November 5, 2003Published, JBC Papers in Press, November 7, 2003, DOI 10.1074/jbc.M309749200
From the ‡Graduate Institute of Life Science and §Institute of Preventive Medicine, National Defense Medical Center,Taipei 114, the ¶Department of Biochemistry, Taipei Medical University, Taipei 117, and the Institutes of �Zoology,**Biological Chemistry, and ‡‡BioAgricultural Sciences, Academia Sinica, Taipei 115, Taiwan
Two mosquito STATs, AaSTAT and CtSTAT, have beencloned from Aedes albopictus and Culex tritaeniorhyn-chus mosquitoes, respectively. These two STATs aremore similar to those of Drosophila, Anopheles, andmammalian STAT5 in the DNA binding and Src homol-ogy 2 domains. The mRNA transcripts are expressed atall developmental stages, and the proteins are presentpredominantly at the pupal and adult stages in bothmosquitoes. Stimulation with lipopolysaccharide re-sulted in an increase of tyrosine phosphorylation andDNA binding activity of AaSTAT and CtSTAT as well asan increase of luciferase activity of a reporter gene con-taining Drosophila STAT binding motif in mosquitoC6/36 cells. After being infected with Japanese enceph-alitis virus, nuclear extracts of C6/36 cells revealed adecrease of tyrosine phosphorylation and DNA bindingactivity of AaSTAT which could be restored by sodiumorthovanadate treatment. Taking all of the data to-gether, this is the first report to clone and characterizetwo mosquito STATs with 81% identity and to demon-strate a different response of tyrosine phosphorylationand DNA binding of these two STATs by lipopolysaccha-ride treatment and by Japanese encephalitis virusinfection.
Mosquitoes, like other insects, have efficient humoral andcellular defense system(s) that exhibit prominent similarity tothe innate immunity in vertebrates (1, 2). The innate immunityis the first line defense of insects and mammals to combatmicrobes. This ancestrally common defense system in Drosoph-ila and mammals depends on conserved intracellular signalingpathways that can respond rapidly to infection by transcrip-tional regulation (1, 3). Two distinct pathways are involved inthe regulation of the secretions of antimicrobial peptides in thefat body. First is the Toll/Dif pathway, which is activatedmainly by fungi and Gram-positive bacteria, and the second isthe Imd/Rel pathway, which is activated mainly by Gram-negative bacteria. These two conserved pathways mediate dif-
ferential expression of antimicrobial peptides via distinct nu-clear factor-�B-like transcription factors (4–6).
In addition to the Toll/Dif and Imd/Rel pathways, the Dro-sophila JAK1/STAT pathway, which is involved in several de-velopmental events, has also been shown to regulate the cellu-lar immune response (7). Recently, it has been demonstratedthat a complement-like humoral protein TEP1 was producedvia JAK/STAT pathway in Drosophila after bacterial challenge(8). Moreover, the Anopheles STAT could translocate into nu-clei of the fat body cells after bacterial challenge (9). Theactivation of c-Jun N-terminal kinase and JAK/STAT path-ways after LPS stimulation in addition to Toll/Dif and Imd/Relpathways was also detected by a genomewide approach inDrosophila (10). All data suggested that there is a tight con-nection among the pathways of Toll/Dif, Imd/Rel, and JAK/STAT in the innate immune responses in the insect. In thisscenario, the JAK/STAT pathway is most likely to be activatedto produce complement-like humoral factors after the activa-tion of the Toll/Dif or Imd/Rel pathway for secretion of antimi-crobial peptides to accelerate bacterial clearance. However, themolecular mechanism linking these two pathways remains tobe characterized.
In mammals, the interferon (IFN) signaling through JAK/STAT is an essential pathway to defense viral infection (11, 12).The well known IFN signaling pathway begins with the bind-ing of IFN-�/� or IFN-� to its cognate receptors on the surfaceof cells, followed by the activation of JAK/STAT and the trans-location of activated STAT dimer to the nucleus for binding tointerferon-stimulated response element or �-interferon-acti-vated sites on target genes and activates the transcription ofinterferon-stimulated genes that encode proteins with potentantiviral, antiproliferative, and anti-inflammatory effect (13–15). Although these IFN immune responses through the JAK/STAT pathway to combat viral infection are evolutionally con-served in mammals, there is no evidence to support whetherequivalent homologs of IFN signal transduction pathways arepresent in mosquitoes to combat virus.
Mosquitoes can transmit a variety of arboviruses that causedifferent diseases in mammals (16). Aedes albopictus (Skuse)and Culex tritaeniorhynchus Giles mosquitoes are known totransmit flaviviruses such as Dengue virus and Japanese en-cephalitis virus (JEV), which cause severe diseases in human
* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s)AY299686and AY299687.
§§ To whom correspondence should be addressed: Institute of Biolog-ical Chemistry, Academia Sinica, 128, Sec 2, Academia Rd., Taipei 115,Taiwan. Tel.: 886-2-2785-5696; Fax: 886-2-2788-9759; E-mail: [email protected].
1 The abbreviations used are: JAK, Janus kinase; Ab, antibody; CT,threshold cycle; EMSA, electrophoretic mobility shift assay; HA, he-magglutinin; IFN, interferon; JEV, Japanese encephalitis virus; LPS,lipopolysaccharide; mAb, monoclonal antibody; RACE, rapid amplifica-tion of cDNA ends; RT-PCR, reverse transcription-PCR; SH2 domain,Src homology 2 domain; STAT, signal transducers and activatorsof transcription.
(17). For their survival, arboviruses must have developed strat-egies to replicate in both vertebrate and invertebrate to avoidthe efficient immunity produced by these two different hosts(18), although mosquito does not have adaptive immunity. Weare interested in the regulation of the JAK/STAT pathway inthe immune reactions of these two mosquitoes in response toLPS stimulation and JEV infection. To our knowledge, thisstudy is the first time AaSTAT and CtSTAT have been clonedfrom these two mosquitoes. We investigated the different re-sponse in the tyrosine phosphorylation and DNA binding ac-tivity of these two mosquito STATs by LPS treatment in vivoand by JEV infection in C6/36 cells.
EXPERIMENTAL PROCEDURES
AaSTAT and CtSTAT Genes Were Isolated from A. albopictus and C.tritaeniorhynchus Mosquitoes, Respectively—Total RNAs were isolatedfrom the larvae of A. albopictus (Chungho strain) and C. tritaeniorhyn-chus (Peitou strain) using the RNAzol reagent (Tel-Test) following theinstructions of the manufacturer. First strand cDNA was synthesizedfrom 50–100 �g of total RNA using 10 pmol of oligo(T) primer andrandom hexamer in a 50-�l reaction volume containing 30 units ofRNasin (Promega, Madison, WI), 2 mM dNTP, 10 mM dithiothreitol, and300 units of reverse transcriptase superscript II (Invitrogen). Incuba-tion was performed at 42 °C for 1 h. Two degenerate primers, ST5F(5�-AA(A/G)CA(A/G)CCNCCNCA(A/G)GTNATNAA-3�) and ST5R (5�-GC(A/G)(A/T)ANAAGCA(T/C) TCCCA(A/G)AANGT-3�) were designedbased on the sequence of two well conserved regions, KQPPQV(M/I)Kand TFWEW(F/L)(F/T)A (these regions are indicated by underlines inFig. 1) in most mammalian and insect STAT proteins (19, 20). Withthese two primers and first strand cDNAs of the two mosquitoes, PCRwas performed to isolate STAT homologs cDNA fragment. The resultingPCR product of 700 bp was cloned into pGEM-T easy vector (Promega)and sequenced.
Rapid Amplification of cDNA Ends (RACE)—The 5�- and the 3�-endsof AaSTAT and CtSTAT mRNA were obtained by the RACE techniqueusing the Marathon cDNA amplification kit (Clontech, Palo Alto, CA)according to the supplier’s instructions. The 5�-RACE was performedwith a 27-mer sense primer (AP1) specific for the adaptor and anAaSTAT-specific antisense primer A5F1 (5�-ATTCATGATCTCTCCG-CACGACTGCTC-3�) or a CtSTAT-specific antisense primer C5F1 (5�-TTCATGATCTCGCCGCATGACTGCTCC-3�), then the second round ofPCR was carried out with a nested 23-mer sense primer (AP2) and anested gene-specific antisense primer A5F2 (5�-TGTTGAGCGTGTTGC-CGATCAGCAGTC-3�) or C5F2 (5�-TTGAGCGTGTTGCCGATGAG-CAGGCG-3�). Similarly, 3�-RACE was performed with two rounds ofPCR, first with the AP1 primer and an AaSTAT-specific primer A3F1(5�-GCCCTGAACACGAAGTTCCGTGCCTCG-3�) or a CtSTAT-specificprimer C3F1 (5�-TGCCGGACAAAGTCTCCTGGAACCAGC-3�) fol-lowed with the AP2 primer and a gene-specific primer A3F2 (5�-TC-GAACGACCCGACCATCACGTGGTCC-3�) or C3F2 (5�-GTCGCAGT-TCTGCAAGGAACCGCTGCC-3�). According to the sequences obtainedfrom 5�- and 3�-RACE, primers H3-A5-F (5�-GAAGCTTATGTCGCTGT-GGGCACGC-3�) containing a HindIII site and A3-R-Kpn (5�-TGGTAC-CGATCCCATTCGCCATCGGTG-3�) containing a KpnI site were usedto amplify the open reading frame of AaSTAT. Similarly, primers H3-C5-F (5�-TAAGCTTATGTCCCTGTGGGCCCGC-3�) containing a Hin-dIII site and C3-R-Kpn (5�-TGGTACCATGCCCGTTGACCACGGTTG-3�) containing a KpnI site were used to amplify the open reading frameof CtSTAT. The PCR products were cloned into pGEM-T easy vector,and their complete sequences were determined by DNA sequencing.
DNA Sequence Analysis and Phylogenetic Analysis—All clones in-cluding full-length clone were sequenced using PRISM Ready Reactiondye deoxy termination cycle sequencing kit (Applied Biosystems) on anApplied Biosystems 310 automated DNA sequencer. Sequence assem-bly and alignments were performed using the Genetics ComputerGroup software program. Phylogenetic analysis, based on the sequencesof the DNA binding domain, linker region, and SH2 domain from STATsof human, Anopheles, Drosophila, and Caenorhabditis were performedusing ClustalX program (21, 22).
RNA Isolation and RT-PCR—Total RNAs were isolated from C6/36cells (mosquito cell line of A. albopictus), embryos, larvae, pupae, adultmales, and adult females of A. albopictus and C. tritaeniorhynchususing the RNAzol reagent following the manufacturer’s instructions.First strand cDNA from tissue of each developmental stage was syn-thesized from 50–100 �g of total RNA using 10 pmol of oligo(T) primerand random hexamers in a 50-�l reaction mixture containing 30 units
of RNasin, 2 mM dNTP, 10 mM dithiothreitol, and 300 units of reversetranscriptase superscript II. Incubation was performed at 42 °C for 1 h,and 1–2 �l of the resulting reaction containing the single-strandedcDNA template was used for subsequent PCR amplification. The PCRswere performed in a 50-�l reaction mixture containing 25 ng of thespecific actin primers from A. albopictus (a-actin-F, 5�-TCGCCATCCA-GGCTGTCCTG-3�; and a-actin-R, 5�-GAGTTGTAGACGGTTTCGTGG-3�) or AaSTAT- specific primers (a-3F5, 5�-CCAGACCACCGGTGGAA-CGAC-3�; and a-3end, 5�-CCTCTCTACCGTCCCGATTAGATCC-3�,corresponding to sequences in the activation domain and 3�-untrans-lated region, respectively). Other PCRs were also performed in a 50-�lreaction mixture with the specific actin primers from C. tritaeniorhyn-chus (c-actin-F, 5�-TCGCTATCCAGGCTGTGCTG-3�; and c-actin-R, 5�-GAGTTGAACACGGTCTCGTGG-3�) or CtSTAT-specific primers (c-3F3, 5�-CAGATCCTGCACATCCAGCCGTTCACG-3�; and c-3end, 5�-ACAGTTCGCACCAAAACATGAGTACAC-3�, corresponding to se-quences in the SH2 domain and 3�-untranslated region respectively),1.5 mM MgCl2, 0.2 mM dNTP, and 0.5 unit of ExTaq (Takara Shuzo,Shiga, Japan). The conditions for amplification were denaturation at96 °C for 2 min, then 35 cycles of 96 °C for 1 min, 50 °C for 30 s, and72 °C for 30 s, and final extension at 72 °C for 5 min. A negative controlwas performed in the absence of RNA. The products were resolved on a1.2% agarose gel and stained with ethidium bromide.
Quantitative Real Time PCR Analysis—Gene expression of AaSTATwas also determined by quantitative real time PCR analysis on theDNA Engine Opticon® 2 System (MJ Research Inc., Reno, NV) withFastStart DNA master SYBR Green I (Roche Applied Science) as de-scribed previously (23). The reaction mixture contained 6 �l of templatecDNA, 2 �l of 10� SYBR Green Master Mix, 1.5 mM MgCl2, and 100 nM
primers (a-3end, a-3F5, and a-actin-F, a-actin-R for AaSTAT gene andactin gene, respectively) at a final volume of 20 �l. The reactions weredenatured at 95 °C for 10 min and cycled 50 times under the followingparameters: 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s. At the end ofthe PCR, the temperature was increased from 65 to 95 °C at a rate of1 °C/min, and the fluorescence was measured every 15 s to construct themelting curve. A nontemplate control was run with every assay, and alldeterminations were performed at least in duplicate to achieve repro-ducibility. The actin gene of A. albopictus was used as reference. Thethreshold cycles (CT) were recorded for all samples for both the AaSTATgene and the reference. The relative gene copy number was evaluatedfrom the CT of the AaSTAT gene and the reference gene. By subtractingthe CT of the reference gene from the CT of the AaSTAT gene, the �CT
value for the AaSTAT gene was determined.Plasmid Construction—The expression vector, pAaSTAT-HA and
pCtSTAT-HA were constructed by inserting the open reading frame ofAaSTAT and CtSTAT before a HA tag and between the Hind III andKpnI sites of pHA-YUN, which is derived from pcDNA3. The pHA-YUNplasmid was kindly provided by Dr. H. J. Kung (University of Californiaat Davis Cancer Center, Sacramento). The cJH1 domain of C. tritaenio-rhynchus JAK (235 amino acids, position 940–1175) (GenBank acces-sion number AY278117) was generated by PCR with primers containingEcoRV and NotI restriction sites on both ends and cloned into the Cterminus of pAaSTAT-HA and pCtSTAT-HA to generate pAaSTAT-HA-cJH1 and pCtSTAT-HA-cJH1, respectively. The resulting clones wereconfirmed by DNA sequencing.
The reporter plasmid 2XDrafSTATwt was kindly given by Dr. M.Yamaguchi (Laboratory of Cell Biology, Aichi Cancer Center ResearchInstitute, Nagoya, Japan). This reporter plasmid contains the Drosoph-ila raf gene promoter with two recognition consensus sequences(TTCGCGGAA) for Drosophila STAT and a luciferase reporter gene(24).
In Vitro Transcription and Translation—The TNT-coupled tran-scription/translation system (Promega) was used to synthesizeAaSTAT-HA, CtSTAT-HA, AaSTAT-HA-cJH1, and CtSTAT-HA-cJH1proteins in vitro according to the manufacturer’s instructions. Briefly,0.2–1 �g of DNA plasmids and 1–20 �Ci of [35S]methionine were addedto the master mixture and incubated for 90 min at 30 °C. The synthe-sized proteins were analyzed by SDS-PAGE and visualized by autora-diography. For subsequent bandshift analysis and Western blotting,[35S]methionine was replaced by cold methionine (final concentration, 1mM).
Cell Cultures—Mosquito C6/36 cells (25, 26) were grown in RPMI1640 medium supplemented with 2% fetal bovine serum, 50 units/mlpenicillin G, 50 �g/ml streptomycin, 2 mM L-glutamine, 25 mM HEPESin a humidified atmosphere of 5% CO2 at 28 °C. All the culture regentswere purchased from Hyclone Laboratories (Logan, UT). To infect withJEV, an �80% confluent monolayer of C6/36 cells grown in 10-mm dishwas first absorbed with JEV at multiplicity of infection 0.2 for 1 h at
LPS Stimulation and JEV Repression of Mosquito STATs 3309
28 °C. After adsorption the unbound viruses were removed by gentlewashing with serum-free culture medium, and fresh medium contain-ing 2% fetal calf serum was added to the dish for further incubation at28 °C. Alternatively, after infection with JEV for 1 day, C6/36 cells weretreated with 50 �M sodium orthovanadate (Sigma) and harvested onday 3 postinfection.
Virus and Virus Infection—JEV strain NT113, isolated from mosqui-toes in Taiwan in 1985 (27), was inoculated onto a confluent monolayerof C6/36 cells in RPMI medium containing 2% fetal calf serum. Cellswere maintained for 5 days at 28 °C, and then supernatant fluids wereharvested and stored at �70 °C. This virus titer was 1 � 107 plaque-forming units/ml as determined by plaque assay on BHK21 cells. Thisvirus stock, from the Institute of Preventive Medicine, Taipei, Taiwan,was used to inoculate C6/36 cells for infection assay.
Mosquitoes—The colonized mosquitoes used in the study wereA. albopictus (Skuse) (Chungho strain) and C. tritaeniorhynchus Giles(Peitou strain), which were maintained on 10% sucrose solution in ahumidified (80%) insectary at 26–28 °C with 14/10-h light/dark cycles.Larvae were reared in water pans and fed on commercial Tetramin fishfood and yeast powder. These mosquitoes were kindly provided by S.Hung (Center for Disease Control, Department of Health, Taipei,Taiwan). Between 5 and 7 days after emergence mosquitoes were inoc-ulated intrathoracically (28, 29) with LPS (Escherichia coli serotype0127:B8; Sigma). The injection volume was 98 nl containing 9.8 ng ofLPS.
Preparation of Whole Cell Lysates and Nuclear Extracts—Whole celllysates were prepared as described previously (30). In brief, cells werewashed twice with phosphate-buffered saline and lysed in the lysisbuffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM
EDTA) containing 0.2 mM Na3VO4 and 2 mM phenylmethylsulfonylfluoride. The mosquito embryos, larvae, pupae, and adults were alsohomogenized with the same lysis buffer. Extracts were centrifuged at4 °C for 10 min at 13,000 � g, and the resulting supernatants were usedfor subsequent Western blotting. Nuclear extracts were prepared ac-cording to the procedures described previously (31). Briefly, cells werewashed twice with phosphate-buffered saline and solubilized withbuffer A (10 mM HEPES, 10 mM KCl, 1 mM EDTA, 1 mM dithiothreitol,0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM Na3VO4, 1 mM Na4P2O7,20 mM NaF, 10% glycerol, 1 �g/ml of aprotinin, pepstatin, and leupep-tin, pH 7.9) on ice for 30 min and then vortexed vigorously and centri-fuged at 12,000 � g at 4 °C for 1 min. The supernatant was thecytoplasmic part at this stage. The pellets were then extracted withbuffer B (20 mM HEPES, 350 mM NaCl, 10 mM KCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM Na3VO4,0.2% Nonidet P-40, 10% glycerol, 1 �g/ml aprotinin, pepstatin, andleupeptin, pH 7.9), rotated at 4 °C for 30 min. The extracts were cen-trifuged at 13,000 � g at 4 °C for 5 min, and these supernatants,nuclear extracts, were quickly frozen and stored at �70 °C for subse-quent use by electrophoretic mobility shift assay (EMSA) or Westernblotting. The extracts of adult female mosquitoes for EMSA were car-ried out by submerging the mosquitoes in liquid nitrogen for 5 min andthen homogenized in buffer A. All the protein concentrations weredetermined by the Bradford method (Bio-Rad).
Antibodies and Western Blotting—Monoclonal Abs against HA tagand phosphotyrosine (pY99) were purchased from Santa Cruz Biotech-nology (Santa Cruz, CA). Monoclonal Ab against �-tubulin was pur-chased from Sigma and OncogenTM (CN Biosciences, Inc., Darmstadt,Germany). Rabbit polyclonal antisera specifically recognizing AaSTATand CtSTAT were generated using a His-tagged AaSTAT or CtSTATfusion protein as antigen. The His-tagged AaSTAT and CtSTAT fusionproteins were generated by PCR amplification of the DNA fragmentencoding the C-terminal end of AaSTAT and CtSTAT (amino acidresidues 722–907 and 736–880, respectively), followed by cloning of thePCR products into the His tag expression vector pQE30 (Qiagen,Hilden, Germany). These His-tagged AaSTAT and CtSTAT fusion pro-teins were expressed in E. coli and purified using a nickel-nitrilotriace-tic acid agarose column (Qiagen, Hilden, Germany) under denaturingcondition (1.5% sarcosine in phosphate-buffered saline) as describedpreviously (32). The purified proteins were used to immunize NewZealand White rabbits by the intrasplenic immunization method (33).The polyclonal Abs were harvested according to a method describedpreviously (34). The in vitro transcription and translation products,extracts of cells or mosquitoes, were separated on 10% or 7% SDS-PAGE, then transferred to a nitrocellulose membrane and blocked with3% skim milk in phosphate-buffered saline. The membrane was incu-bated with indicated Abs, and visualized by the enhanced chemilumi-nescent detection system (PerkinElmer Life Sciences) according to themanufacturer’s instructions.
EMSA—EMSA was performed as described previously (35). Briefly,reactions were performed by the addition of nuclear extracts, adultmosquito extracts, or products of in vitro transcription and translationin the presence of 32P-labeled double-stranded oligonucleotide probes(10,000 cpm/�g) to the binding buffer: 20 mM HEPES, pH 7.9, 50 mM
KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 10 mM MgCl2, 0.5 mM ZnCl2,0.02% Nonidet P-40, 10 �g/ml bovine serum albumin, 10% glycerol, and2 �g of poly(dI-dC) in a 20-�l reaction volume. An excess of unlabeled ormutant oligonucleotides was added as competitor as indicated. 1 �l ofantiserum (1:2 dilution in phosphate-buffered saline) was also added asindicated. After incubation at room temperature for 15 min, the reac-tion mixture was loaded on 5% polyacrylamide gel in 0.5 � TBE and runat 200 V for 2 h at room temperature. The oligonucleotides probes usedin this study were commercially available, corresponding to the DNAbinding motifs for mammalian STATs (Santa Cruz). The sequences ofupper strand of the normal and the corresponding mutant oligonucleo-tides are listed in Table 3 of Ref. 30. The probes were prepared byannealing the upper and lower strands of oligonucleotides, one of whichwas end labeled with [�-32P]ATP by using T4 polynucleotide kinase(Promega).
Transactivation Assay—The plasmids used for transfections werepurified by Qiagen plasmid purification kit (Qiagen). For transactiva-tion assay, plasmid transfections were conducted by using the CELL-FECTIN kit (Invitrogen) in 6-well plates. For investigation of the en-dogenous AaSTAT activation by LPS, approximately 80% confluentC6/36 cells were transfected with 0.2 �g of 2XDrafSTATwt for 24 h;subsequently 10 �g/ml LPS was added into the culture medium. Afterstimulation with LPS for 30, 60, and 90 min, cells were harvested andextracted for luciferase activity assay. 2XDrafSTATwt contains the rafgene promoter of Drosophila with two recognition consensus sequencesfor Drosophila STAT and a luciferase gene (24). The C6/36 cells wereharvested at 24.5, 25, and 25.5 h post-transfection and assayed forluciferase activity using luciferase assay kit Firelite according to themanufacturer’s instructions (Packard). Final luciferase activity wasobtained after normalizing protein concentration of each sample.
RESULTS
Isolation and Characterization of AaSTAT and CtSTATGenes—Two STAT homologs were isolated from mosquitoesA. albopictus and C. tritaeniorhynchus by RT-PCR amplifica-tion using degenerate primers containing sequences corre-sponding to two stretches of amino acid residues, KQP-PQV(M/I)K and TFWEW(F/L)(F/T)A, which are highlyconserved between mammalian and insect STATs (underlinesin Fig. 1). The first strand cDNA from larvae of these twomosquitoes was used as template. Only one PCR product of700 bp was obtained, the sequences from which were used todesign the primers A5F1, A3F1, C5F1, and C3F1 for further5�- and 3�-RACE to obtain full-length cDNA of the AaSTATand CtSTAT. The full-length AaSTAT cDNA encompasses3579 bp including a 2724-bp coding region, which encodes aprotein of 907 amino acid residues, as well as a 524-bp5�-untranslated region and 331-bp 3�-untranslated region(Fig. 1). The full-length CtSTAT cDNA consists of 4014 bpincluding a 2643-bp coding region, which encodes a protein of880 amino acid residues, plus 843 bp upstream and 528 bpdownstream of the open reading frame. These two sequenceswere deposited in GenBank with accession numbersAY299686 and AY299687, respectively.
The overall organization and the deduced amino acid resi-dues of these two mosquito STATs compared with knownSTATs of Anopheles, Drosophila, and human are shown in Fig.1. Sequence alignment shows that AaSTAT and CtSTAT havethe same functional domain organization as the known STATsand highly conserved sequences between the DNA bindingdomain and SH2 domain and lower homology in the N-terminalprotein interaction domain, coiled-coil domain, and C-terminaltransactivation domain. The conserved amino acid residues arealso annotated. For example, a tyrosine at residue 685 to bephosphorylated by JAKs during activation (20) in the C-termi-nal transactivation domain of both AaSTAT and CtSTAT isconserved. An arginine at residue 31 in the N terminus whose
LPS Stimulation and JEV Repression of Mosquito STATs3310
FIG. 1. Comparison of deducedamino acid sequences of AaSTAT andCtSTAT with those from AgSTAT,DmSTAT, and human STAT5a. The en-coded amino acid sequences were alignedusing the PILEUP program (GeneticComputer Group). Gaps are introduced tooptimize alignment and are shown asdashes. Arrowed brackets indicate theboundaries of functional domains of theSTAT polypeptide. The putative phospho-rylated tyrosine residue is indicated by ablack star. Black underlines indicate thetwo conserved regions from which the de-generate oligonucleotides were designedand used to clone STAT homologs fromcDNA of A. albopictus and C. tritaenio-rhynchus. Black shadows indicate iden-tity in amino acid residues. Accessionnumbers for the five sequences are thesame as in Fig. 2.
LPS Stimulation and JEV Repression of Mosquito STATs 3311
methylation was reported affecting STAT dephosphorylation isconserved through all the STATs (36), suggesting that regula-tion of these STATs from different species may be similar.Interestingly, the length of C-terminal activation domain ofAaSTAT and CtSTAT is much longer than that of any otherSTATs.
AaSTAT and CtSTAT Are in the Ancient Class of the STATFamily—Based on the amino acid sequence comparison in allprotein sequences, AaSTAT and CtSTAT are 81% identical toeach other and show 35 and 28% identity to those of Anophelesand Drosophila. Moreover, AaSTAT and CtSTAT displayed 30and 27% identity to human STAT5 and STAT6, respectively,but with only 18–20% identity to another class of human STATfamily (Table I). To investigate further the evolutionary rela-tionship between AaSTAT, CtSTAT, and other STAT familymembers, phylogenetic analysis was performed in the centralconserved region from the DNA binding domain to the SH2domain. As illustrated in Fig. 2, AaSTAT, CtSTAT, AnophelesAgSTAT, Drosophila DmSTAT, and human STATs5 andSTAT6 are more closely related to each other with about 50%identity (data not shown) and constitute an ancient class of theSTAT family. In contrast, human STAT1, STAT2, STAT3, andSTAT4 form another diverged group. These analyses raised thepossibility that an ancestral STAT gene had been duplicated inthe split between insects and vertebrates to create these twoclasses of STAT family.
The mRNA and Protein Expression Profiles of AaSTAT andCtSTAT at Different Developmental Stages—The mRNA ex-pression level of AaSTAT and CtSTAT at the embryonic, larval,pupal, adult stages, and C6/36 cells was analyzed by RT-PCR.As showed in Fig. 3A, a 305-bp DNA fragment could be ampli-fied from all stages examined in A. albopictus. The quantitativereal time PCR analysis (Fig. 3B) also revealed that equivalent
mRNA expression of AaSTAT was found at all stages exceptpupal and male mosquito, in which their AaSTAT mRNA levelswere higher. In Fig. 3C, an 819-bp DNA fragment was alsoamplified at all stages examined but lower at embryonic stagein C. tritaeniorhynchus. A similar conclusion was obtained byquantitative real time PCR (data not shown). These resultsshowed that AaSTAT and CtSTAT were ubiquitously tran-scribed at all developmental stages, comparable with the ex-pression patterns of Anopheles STAT gene (9). When the pro-tein expression of AaSTAT was analyzed by Western blottingusing anti-AaSTAT polyclonal Abs, a major band of about 102kDa was shown in C6/36 cells, pupae as well as adults of A.albopictus, but not found in embryos and larvae (Fig. 4A, up-per). Similarly, anti-CtSTAT Abs detected a protein band ofabout 100 kDa in pupae and adults of C. tritaeniorhynchus,lower amount in larvae, but not in embryos (Fig. 4C). Thedetectable bands were not observed when the membranes werereprobed with the preimmune sera at the same dilution (datanot shown). The above membranes were reprobed with mAbagainst �-tubulin, a band about 55 kDa and 58 kDa was ob-served in equal amounts in C6/36 cells and adult stages ofA. albopictus (Fig. 4A, lower) and adult stages of C. tritaenio-rhynchus (data not shown), respectively. The antibody couldnot detect the corresponding proteins in the protein samplesfrom embryo, larva, and pupa possibly because of the presenceof other isoforms of �-tubulin. The integrity of all protein sam-ples was showed by Coomassie Blue staining (Fig. 4B). Thesedata demonstrated that AaSTAT and CtSTAT proteins wereexpressed with detectable amount at pupal and adult stages,suggesting that they may play some roles during developmen-tal stages guarding against environmental stress.
In Vitro Translated AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 Fusion Proteins Are Tyrosine-phosphorylated and Bound
TABLE IPairwise amino acid sequence comparisons of AaSTAT, CtSTAT, and other known STATs
All protein sequences were aligned pairwise using the Geneworks nucleic acid and protein sequence analysis software 2.5 from Intelligenetics,Inc. The numbers represent percent amino acid identity. Accession numbers for all sequences are the same as in Fig. 2.
to Mammalian STAT5-responsive Element—To investigate thebiochemical properties of the AaSTAT and CtSTAT proteins,we adopted the strategy developed by Berchtold et al. (37) togenerate constitutively active AaSTAT and CtSTAT variantsand to characterize their DNA binding properties in a cell-freesystem. AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 fusion pro-teins were generated by fusing the JH1 domain of the JAK fromC. tritaeniorhynchus mosquito into the C-terminal end ofAaSTAT and CtSTAT proteins, respectively, as described pre-viously (30). In vitro transcription and translation products ofAaSTAT-HA, AaSTAT-HA-cJH1, CtSTAT-HA, and CtSTAT-HA-cJH1 were all recognized by mAb against the HA tag, butonly AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 were recognizedby anti-pY99 mAb (Fig. 5A), demonstrating that the JH1 do-main of JAK from C. tritaeniorhynchus can autophosphorylateor transphosphorylate AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 in vitro, respectively.
We next investigated whether AaSTAT, CtSTAT, AaSTAT-
HA-cJH1, and CtSTAT-HA-cJH1 bind DNA in vitro. As shownin Fig. 5B, AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 formedcomplexes with the STAT5 binding motif (Fig. 5B, lanes 2 and6). In contrast, AaSTAT and CtSTAT, without cJH1 domain,did not bind to the STAT5 binding motif (Fig. 5B, lanes 1 and5). The shifted bands were not seen in the presence of a 50-foldexcess of cold probe (Fig. 5B, lanes 3 and 7) but sustained in thepresence of mutant competition (Fig. 5B, lanes 4 and 8). Theseresults indicate that tyrosine phosphorylation of AaSTAT andCtSTAT by JAK is required for the DNA binding of theAaSTAT and CtSTAT. Several commercial available mamma-lian DNA binding motifs for mammal STATs (sis-inducibleelements, STAT 1, 3, 4, 5, 6) were applied in similar EMSAexperiments. In agreement with the phylogenic analysis in Fig.2, among the STAT response elements tested, AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 displayed specific and strong bind-ing to the STAT5 binding motif, weak binding to STAT 3, 4, 6binding motifs, and not at all to either sis-inducible elements orSTAT1 binding motif (data not shown). Therefore, the STAT5binding motif was used further to test the DNA binding activityof the endogenous STAT proteins in adult mosquito or itsderived C6/36 cells in EMSA.
AaSTAT and CtSTAT Were Activated in Response to LPSStimulation—C6/36 cells transfected with 2XDrafSTATwt re-porter plasmid for 24 h and then were treated with LPS for 30,60, and 90 min before being harvested. There was a 2–3-fold
FIG. 2. The phylogenetic tree analysis of AaSTAT, CtSTAT, andother STAT family members. The amino acid sequences betweenDNA binding, linker domain, and SH2 domains of AaSTAT and CtSTATwere aligned with those of 10 known STAT proteins. The phylogenetictree was constructed by using the NEIGHBOR-JOINING program to-gether with bootstrap analysis using 1,000 replicates provided by Clust-alX. Branch lengths are proportional to sequence divergence. Branchlabels record the stability of the branches more than 1,000 bootstrapreplicates. GenBank accession numbers of the sequences used are asfollows: Caenorhabditis elegans (CeSTAT), Z70754; Drosophila melano-gaster (DmSTAT), Q24151; human h-STAT1 (HsSTAT1), P42224; h-STAT2 (HsSTAT2), P526301; h-STAT3 (HsSTAT3), P40763; h-STAT4(HsSTAT4), Q14765; h-STAT5a (HsSTAT5a), P42229; h-STAT5b(HsSTAT5b), NP036580; h-STAT6 (HsSTAT6), P42226; Anophelesgambiae (AgSTAT), AJ010299; A. albopictus (AaSTAT), AY299686;C. tritaeniorhynchus (CtSTAT), AY299687.
FIG. 3. RT-PCR and real time PCR analyses of AaSTAT andRT-PCR analysis of CtSTAT transcripts at different develop-mental stages. Total RNAs (2–5 �g/reaction) from tissues of A. albop-ictus and C. tritaeniorhynchus were subjected to RT-PCR analysis. Theresulting PCR products were electrophoresed on 1.2% agarouse gelcontaining ethidium bromide. A negative control was run simulta-neously. A, a DNA fragment of 305 bp was amplified from tissues ofdifferent developmental stages using AaSTAT-specific primers (upper).�-Actin mRNA was used as an internal control and amplified fromdifferent developmental tissues of A. albopictus using �-actin-specificprimers (lower). B, in quantitative real time PCR analysis, the relativegene copy number was evaluated as �CT values by subtracting the CTof the reference �-actin gene from the CT of the target AaSTAT gene. C,a DNA fragment of 819 bp was amplified from tissues of differentdevelopmental stage by using CtSTAT-specific primers (upper), and�-actin was used as and internal control.
LPS Stimulation and JEV Repression of Mosquito STATs 3313
increase in luciferase activity after 30-min LPS stimulationand declined gradually (Fig. 6A). In Fig. 6B we show thatanti-AaSTAT and anti-pY99 detected equivalent signals inC6/36 cells under LPS treatment, suggesting a high basal levelof tyrosine phosphorylation in the culture cells. However, LPStreatment can still enhance the transcriptional activity of theAaSTAT. These results were in agreement with the previousreport that the Gram-negative bacterial cell wall (LPS) inducedthe activation of endogenous STAT in Drosophila cells (10).
To examine further the activation of AaSTAT and CtSTAT byLPS in vivo, the adult females of these two mosquitoes wereinjected intrathoracically with LPS for 3 h, and the whole cellextracts were prepared for EMSA analysis. As showed in Fig.7A, the DNA-protein complexes were formed in the LPS-treated mosquitoes, but not in the control (lanes 1 and 6 versuslanes 5 and 10). These complexes were competed completely inthe presence of a 50-fold excess of unlabeled probes (lanes 2 and7) but not with an excess of mutant probes (lanes 3 and 8).Supershift of the DNA-protein complexes was observed in thepresence of anti-AaSTAT or anti-CtSTAT Ab, respectively(lanes 4 and 9). The same extracts from LPS-treated or controlmosquitoes were inspected by Western blotting using anti-AaSTAT or anti-CtSTAT polyclonal Abs and anti-pY99 mAb.As shown in Fig. 7B, the AaSTAT and CtSTAT proteins weredetected with equal amounts, and they were tyrosine phospho-rylated mainly in the group of LPS treatment. Taken together,the intrathoracic injection of LPS induces AaSTAT and Ct-
STAT proteins in vivo along with an increase of their specificDNA binding activity and tyrosine phosphorylation.
JEV Infection Inhibits the Activation of AaSTAT, and So-dium Orthovanadate Treatment Reverses This Effect in C6/36Cells—To investigate whether virus infection would influencethe activity of AaSTAT, the nuclear extracts of JEV-infectedC6/36 cells were examined by EMSA. In normal C6/36 cellsculture, there was basal activity of endogenous AaSTAT as ablank control demonstrated in Fig. 6A. As shown in Fig. 8A, theDNA binding activity decreased upon 1 day JEV infection com-pared with that of the control (lane 2 versus lane 1). Theinhibitory effect induced by JEV infection was even more ap-parent on days 2 and 3 postinfection (lanes 3 and 4). Decreasedtyrosine phosphorylation of AaSTAT was also noted in nuclearextracts from JEV-infected C6/36 cells when probed with anti-pY99 mAb (Fig. 8B, lower). These data suggested that JEV
FIG. 4. Western blots of homogenates of A. albopictus andC. tritaeniorhynchus from different developmental tissues. Anti-AaSTAT (A, upper) or anti-CtSTAT (C) Abs were used to probe homo-genates of different developmental tissues from A. albopictus and C.tritaeniorhynchus. Approximately, 15 �g/lane was run in 10% SDS-PAGE, transferred to nylon membrane, and probed with the indicatedantiserum, followed by ECL detection. The same membrane was rep-robed with anti-�-tubulin mAb (A, lower). An equal amount (15 �g) oftissue homogenate of A. albopictus at different developmental stage wassubjected to SDS-PAGE and stained with Coomassie Blue (B).
FIG. 5. AaSTAT-HA-cJH1 and CtSTAT-HA-cJH1 fusion pro-teins were tyrosine phosphorylated and bound to STAT5 probein vitro. A, in vitro transcription and translation products ofAaSTAT-HA (first and third lanes), AaSTAT-HA-cJH1 (second andfourth lanes), CtSTAT-HA (fifth and seventh lanes), and CtSTAT-HA-cJH1 (sixth and eighth lanes) were detected by Western blot analysisusing anti-HA mAb (first, second, fifth, and sixth lanes), or anti-pY99mAb (third, fourth, seventh, and eighth lanes). In B, the same fusionproteins were incubated with labeled STAT5 binding motif and followedby EMSA. In vitro transcription and translation products ofAaSTAT-HA and CtSTAT-HA were used as control (lanes 1 and 5). Thebinding of fusion protein to STAT5 binding motif was completely abol-ished by the addition of a 50-fold molar excess of cold competitor (lanes3 and 7) but remained unchanged when mutant oligonucleotide wasadded at a 50-fold molar excess (lanes 4 and 8).
LPS Stimulation and JEV Repression of Mosquito STATs3314
infection in C6/36 cells compromises tyrosine phosphorylationof AaSTAT protein and thus suppressed its activation as de-creased DNA binding ability. To study further whether thereduced tyrosine phosphorylation of AaSTAT involves phos-phatase activities, a tyrosine phosphatase inhibitor, sodiumorthovanadate, was added into C6/36 cells infected with JEV(Fig. 9, lane 3), AaSTAT tyrosine phosphorylation was restoredby such a treatment.
DISCUSSION
In this study, we cloned two STAT homologs from A. albop-ictus and C. tritaeniorhynchus mosquitoes and investigatedtheir functional responses to LPS stimulation and JEV infec-tion from the prospect of DNA binding activity and proteintyrosine phosphorylation status. Approximately 50% of se-quence identity of these two STATs compared with those ofAnopheles, Drosophila, and human STAT5 were found in theconserved regions from DNA binding domain to SH2 domain.They are much more similar to each other with 81% identity inthe overall protein sequences (Table. I). Their C-terminaltransactivation domains, however, are unusually long and sig-nificantly more divergent (Fig. 1). Although two STAT geneswere annotated in the complete genome sequence of A. gambiae(38), only one STAT gene has been isolated in each mosquito inthis study. Similarly, there is only one STAT gene in the anal-ogous insect Drosophila (19, 39). The possibility of having an-other STAT gene requires further investigation.
Recently, a number of constitutively activated mammalianSTATs have been identified and constructed (40, 41). A fusionprotein consisting of STAT5 and the kinase domain of JAK2 orJAK1 was also reported to be constitutively active (30, 37). Inthis study, the JH1 domain of JAK from mosquito C. tritaenio-rhynchus was fused to the C termini of AaSTAT and CtSTATproteins to generate their constitutive active forms, namelyAaSTAT-HA-cJH1 and CtSTAT-HA-cJH1. Similarly, each fu-sion protein showed tyrosine kinase activity and was phospho-rylated on tyrosine (Fig. 5A). They also exhibited specific DNA
binding activity to mammalian STAT5 binding motif (Fig. 5B).The core sequence of STAT5 binding motif is TTCTAGGAA,which resembles the binding motif of human STAT5 (TTCTNA/GGAA) (42) and Drosophila DmSTAT (TTCNNNGAA) (19).These results indicate that the biochemical properties of thesemosquito STAT proteins are more akin to mammalian STST5and Drosophila STAT, consistent with the higher homologybetween the DNA binding domains of these proteins (Fig. 2).
So far, only two insect STAT genes have been isolated andcharacterized from Drosophila and malaria mosquito, Anoph-eles gambiae. In Drosophila, a single JAK homolog, hopscotch(hop) (43) and a STAT protein have been identified (19, 39).Thus, the existence of an invertebrate JAK/STAT system withone JAK and one STAT has been established. The receptor ofJAK/STAT signal transduction pathway in Drosophila hasbeen identified recently (44, 45). The JAK/STAT pathway inDrosophila is not only involved in larval hematopoiesis but alsoinvolved in a number of developmental events (7, 46, 47).Moreover, the Drosophila STAT protein plays important rolesin regulating the mitogen-activated protein kinase cascadethrough D-raf gene activation in the immune response. A STAT
FIG. 6. Endogenous AaSTAT was activated in response to LPSstimulation in C6/36 Cells. C6/36 cells were treated with 10 �g/mlLPS for 30, 60, and 90 min before harvesting. The reporter plasmid (0.2�g of 2XDrafSTATwt) was transfected into the C6/36 cells 1 day beforeLPS treatment (A). The nuclear extracts of C6/36 cells treated with LPSor blank control were probed with anti-AaSTAT Ab (B).
FIG. 7. AaSTAT and CtSTAT were activated in response to LPSstimulation in vivo. Adult females extracts of A. albopictus andC. tritaeniorhynchus mosquitoes inoculated with LPS for 3 h were usedfor EMSA (A). Approximately 10 �g of extracts was incubated withSTAT5 probe. Binding complexes were completely abolished by unla-beled probes in a 50-fold molar excess, but not by an excess of mutantprobes. Anti-AaSTAT and anti-CtSTAT polyclonal Abs partially de-creased the intensity of the binding and created a supershifted band.Almost no complexes were formed in those phosphate-buffered salinecontrols. Identical protein extracts were probed with anti-AaSTAT oranti-CtSTAT Abs to show equivalent amounts of protein (B, upper) andreprobed with anti-pY99 mAb (B, lower) to monitor the phosphorylationof STAT.
LPS Stimulation and JEV Repression of Mosquito STATs 3315
binding motif was identified in the promoter region of Drosoph-ila Raf gene (24). The second member of insect STAT family(AgSTAT) with a possible role in immune response has beencloned from the human malaria vector A. gambiae. The Ag-STAT was shown to translocate into the nuclei of the fat bodycells after bacterial challenge (9). Also, microarray analysis oftranscription pattern of Drosophila SL2 cells after LPS treat-ment revealed sequential activation of c-Jun N-terminal kinaseand JAK/STAT pathways in addition to Toll/Dif and Imd/Relpathways, which might be transmitted through the DrosophilaTak1 (10). Moreover, the gene encoding the complement-likeTEP1, which might bind to the surface of bacteria and promotephagocytosis by hemocytes, had been identified as a target ofthe JAK/STAT pathway through the Toll cascade (8). All aboveobservations provide direct evidences that the STAT pathwaymay participate in the immune response and contribute toantimicrobial infection. Our results are that 1) the AaSTATand CtSTAT are activated at the level of protein tyrosine phos-phorylation and DNA binding after LPS treatment (Fig. 7A);and 2) the D-raf reporter activity was elevated in C6/36 cells
after LPS treatment (Fig. 6). These results are consistent withthe previous observations mentioned above. Taken together,LPS stimulation in vivo and in vitro could activate the tran-scriptional activity of AaSTAT and CtSTAT, which are involvedin immunity.
The mosquito cell line C6/36 was established from A. albop-ictus (25, 26) and used to culture many flaviviruses (48–50).JEV, a mosquito-borne flavivirus, inflicts more than 35,000fatal cases annually, causes permanent neurological sequelaein survival patients (17), and infects a broad range of verte-brate species (51). JEV must develop unique strategies to rep-licate in both mosquitos and many vertebrate hosts that haveefficient immune system against the virus. In previous studies(52, 53), JEV appeared to be lifelong and was presented in mostorgans of mosquito while infected, indicating that JEV hadescaped barriers and the innate immune responses in thismosquito. Although A. albopictus is not the major vector incontrast to C. tritaeniorhynchus for JEV, the JEV (54, 55) andWest Nile virus, an encephalitic flavivirus (56), had indeedbeen isolated from A. albopictus. We then investigated theimmune response of mosquito to the virus in JEV-infectedC6/36 cells to study the regulation of AaSTAT pathway. Adiminished DNA binding activity as well as decreased tyrosinephosphorylation of AaSTAT were observed in nuclear extractsof JEV-infected cells (Fig. 8), suggesting that JEV infectionmay interfere with the tyrosine phosphorylation of AaSTAT,probably through the induction of cellular phosphatase(s) orthe inactivation of JAK or other tyrosine kinase(s) by viralproducts. Our data also showed that the addition of sodiumorthovanadate, a tyrosine phosphatase inhibitor, to the JEV-infected cells could restore the tyrosine phosphorylation ofAaSTAT protein (Fig. 9A), suggesting that at least the induc-tion of cellular phosphatase(s) may be in part the cause ofdecreasing AaSTAT tyrosine phosphorylation. These resultswere comparable with a previous report that human cells in-fected with Sendai virus showed blocking of interferon signal-ing by inhibiting the tyrosine phosphorylation of Tyk2, whichresulted in the subsequent failure in tyrosine phosphorylationof the STATs (57). However, it is unclear whether an analogousIFN is present in mosquitoes or whether a parallel pathway ispresent which could be activated by JEV through mosquitoAaSTAT/CtSTAT signaling to induce antiviral effects. In sum-mary, two mosquito STATs were cloned and their biochemicalproperties, such as tyrosine phosphorylation, DNA binding,and transactivation activity, were characterized by LPS treat-ment and by JEV infection.
Acknowledgments—We thank S. Hung for providing the mosquitocolony strains. We also thank Drs. M. Yamaguchi and H. J. Kung for
FIG. 9. Effects of orthovanadate on the phosphorylation ofAaSTAT in JEV-infected C6/36 cells. After JEV infection for 24 h, 50�M sodium orthovanadate was added into the culture medium, and thecells were harvested on day 3 for Western blotting. 5 �g of nuclearextracts with orthovanadate treatment (lane 3) or not (lanes 1 and 2)was probed with anti-AaSTAT Ab (upper) and reprobed with anti-pY99mAb (lower). Lane 1 was normal control, and lanes 2 and 3 wereJEV-infected.
FIG. 8. Complexes were formed in nuclear extracts of C6/36cells with STAT5 probe but inhibited by JEV infection. In A, thenuclear extracts of JEV-infected C6/36 cells (lanes 2–4, or control lanes1, 5–8) were incubated with STAT5 probe. The multiplicity of infectionwas 0.2 in lanes 2 and 3. Lanes 1 and 5–8 were control on days 1 and 3,respectively. The complexes were abolished by cold probes in a 50-foldmolar excess (lane 6) but not by an excess of mutant probes (lane 7). Asupershifted band was observed at lane 8 when anti-AaSTAT Ab wasadded. In B, the identical nuclear extracts, 4 �g/lane, of JEV-infectedC6/36 cells from A were probed with anti-AaSTAT Ab (upper) andreprobed with anti-pY99 mAb (lower).
LPS Stimulation and JEV Repression of Mosquito STATs3316
providing the reporter plasmid 2XDrafSTATwt and the pHA-YUN plas-mid, respectively. We are grateful to Drs. H. Chen and T. C. Meng forcritical reading the manuscript and helpful discussions.
REFERENCES
1. Hoffmann, J. A., Kafatos, F. C., Janeway, C. A., Jr., and Ezekowitz, R. A. B.(1999) Science 284, 1313–1318
2. Elrod-Erickson, M., Mishra, S., and Schneider, D. (2000) Curr. Biol. 10,781–784
3. Borregaard, N., Elsbach, P., Ganz, T., Garred, P., and Svejgaard, A. (2000)Immunol. Today 21, 68–70
4. Khush, R. S., Leulier, F., and Lemaitre, B. (2001) Trends Immunol. 22,260–264
5. Lemaitre, B., Reichhart, J. M., and Hoffmann, J. A. (1997) Proc. Natl. Acad.Sci. U. S. A. 94, 14614–14619
6. Hultmark, D. (2003) Curr. Opin. Immunol. 15, 12–197. Zeidler, M. P., Bach, E. A., and Perrimon, N. (2000) Oncogene 19, 2598–26068. Lagueux, M., Perrodou, E., Levashina, E. A., Capovilla, M., and Hoffmann,
J. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11427–114329. Barillas-Mury, C., Han, Y.-S., Seeley, D., and Kafatos, F. C. (1999) EMBO J.
18, 959–96710. Boutros, M., Agaisse, H., and Perrimon, N. (2002) Dev. Cell 3, 711–72211. Samuel, C. E. (2001) Clin. Microbiol. Rev. 14, 778–80912. Shtrichman, R., and Samuel, C. E. (2001) Curr. Opin. Microbiol. 4, 251–25913. Lau, J. F., and Horvath, C. M. (2002) Mt. Sinai J. Med. 69, 156–16814. Sen, G. C. (2001) Annu. Rev. Microbiol. 55, 255–28115. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and Schreiber, R. D.
(1998) Annu. Rev. Biochem. 67, 227–26416. Bres, P. (1988) in The Arboviruses: Epidemiology and Ecology (Monath, T. P.,
ed) Vol. 1, pp. 1–17, CRC Press, Boca Raton, FL17. Burke, D. S., and Monath, T. P. (2001) in Fields Virology (Knipe, D. M.,
Howley, P. M., Griffin, D. E., Lamb, R. A., Martin, M. A., Roizman, B., andStraus, S. E., eds) 4th Ed., Vol. 1, pp. 1043–1126, Lippincott Williams &Wilkins, Philadelphia, PA
18. Diamond, M. S. (2003) Immunol. Cell Biol. 81, 196–20619. Yan, R., Small, S., Desplan, C., Dearolf, C. R., and Darnell, J. E., Jr. (1996) Cell
84, 421–43020. Darnell, J. E., Jr. (1997) Science 277, 1630–163521. Higgins, D. G., Thompson, J. D., and Gibson, T. J. (1996) Methods Enzymol.
266, 383–40222. Higgins, D. G., and Sharp, P. M. (1988) Gene (Amst.) 73, 237–24423. Konnai, S., Usui, T., Ohashi, K., and Onuma, M. (2003) Vet. Microbiol. 94,
283–29424. Kwon, E. J., Park, H. S., Kim, Y. S., Oh, E. J., Nishida, Y., Matsukage, A., Yoo,
M. A., and Yamaguchi, M. (2000) J. Biol. Chem. 275, 19824–1983025. Singh, K. R., and Paul, S. D. (1968) Indian J. Med. Res. 56, 815–82026. Igarashi, A. (1978) J. Gen. Virol. 40, 531–54427. Jan, L. R., Yueh, Y. Y., Wu, Y. C., Horng, C. B., and Wang, G. R. (2000) Am. J.
Trop. Med. Hyg. 62, 446–45228. Imler, J. L., Tauszig, S., Jouanguy, E., Forestier, C., and Hoffmann, J. A.
(2000) J. Endotoxin Res. 6, 459–46229. Gubler, D. J., and Rosen, L. (1976) Am. J. Trop. Med. Hyg. 25, 146–15030. Sung, S. C., Fan, T. J., Chou, C. M., Leu, J. H., Hsu, Y. L., Chen, S. T., Hsieh,
Y. C., and Huang, C. J. (2003) Eur. J. Biochem. 270, 239–252
31. Ariyoshi, K., Nosaka, T., Yamada, K., Onishi, M., Oka, Y., Miyajima, A., andKitamura, T. (2000) J. Biol. Chem. 275, 24407–24413
32. Chang, M. S., Chang, G. D., Leu, J. H., Huang, F. L., Chou, C. K., Huang, C. J.,and Lo, T. B. (1996) DNA Cell Biol. 15, 827–844
33. Hong, T. H., Chen, S. T., Tang, T. K., Wang, S. C., and Chang, T. H. (1989)J. Immunol. Methods 120, 151–157
34. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, pp. 53–137,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
35. Schmitt-Ney, M., Doppler, W., Ball, R. K., and Groner, B. (1991) Mol. Cell.Biol. 11, 3745–3755
36. Zhu, W., Mustelin, T., and David, M. (2002) J. Biol. Chem. 277, 35787–3579037. Berchtold, S., Moriggl, R., Gouilleux, F., Silvennoinen, O., Beisenherz, C.,
Pfitzner, E., Wissler, M., Stocklin, E., and Groner, B. (1997) J. Biol. Chem.272, 30237–30243
38. Christophides, G. K., Zdobnov, E., Barillas-Mury, C., Birney, E., Blandin, S.,Blass, C., Brey, P. T., Collins, F. H., Danielli, A., Dimopoulos, G., Hetru, C.,Hoa, N. T., Hoffmann, J. A., Kanzok, S. M., Letunic, I., Levashina, E. A.,Loukeris, T. G., Lycett, G., Meister, S., Michel, K., Moita, L. F., Muller,H. M., Osta, M. A., Paskewitz, S. M., Reichhart, J. M., Rzhetsky, A., Troxler,L., Vernick, K. D., Vlachou, D., Volz, J., von Mering, C., Xu, J., Zheng, L.,Bork, P., and Kafatos, F. C. (2002) Science 298, 159–165
39. Hou, X. S., Melnick, M. B., and Perrimon, N. (1996) Cell 84, 411–41940. Onishi, M., Nosaka, T., Misawa, K., Mui, A. L., Gorman, D., McMahon, M.,
Miyajima, A., and Kitamura, T. (1998) Mol. Cell. Biol. 18, 3871–387941. Daniel, C., Salvekar, A., and Schindler, U. (2000) J. Biol. Chem. 275,
14255–1425942. Ehret, G. B., Reichenbach, P., Schindler, U., Horvath, C. M., Fritz, S., Nabholz,
M., and Bucher, P. (2001) J. Biol. Chem. 276, 6675–668843. Binari, R., and Perrimon, N. (1994) Genes Dev. 8, 300–31244. Brown, S., Hu, N., and Hombria, J. C. (2001) Curr. Biol. 11, 1700–170545. Chen, H. W., Chen, X., Oh, S. W., Marinissen, M. J., Gutkind, J. S., and Hou,
S. X. (2002) Genes Dev. 16, 388–39846. Luo, H., and Dearolf, C. R. (2001) Bioessays 23, 1138–114747. Baksa, K., Parke, T., Dobens, L. L., and Dearolf, C. R. (2002) Dev. Biol. 243,
166–17548. Leake, C. J., Ussery, M. A., Nisalak, A., Hoke, C. H., Andre, R. G., and Burke,
D. S. (1986) Trans. R. Soc. Trop. Med. Hyg. 80, 831–83749. Ledger, T. N., Sil, B. K., Dunster, L. M., Stephenson, J. R., Minor, P. D., and
Barrett, A. D. (1992) Vaccine 10, 652–65450. Randolph, V. B., and Hardy, J. L. (1988) J. Gen. Virol. 69, 2199–220751. Endy, T. P., and Nisalak, A. (2002) in Japanese Encephalitis and West Nile
Viruses (Mackenzie, J. S., Barret, A. D. T., and Deubel, V., eds) pp. 12–48,Springer-Verlag, New York
52. Lamotte, L. C. J. (1960) Am. J. Hyg. 72, 73–8753. Leake, C. J., and Johnson, R. T. (1987) Trans. R. Soc. Trop. Med. Hyg. 81,
681–68554. Huang, C. H. (1982) Adv. Virus Res. 27, 71–10155. Vythilingam, I., Oda, K., Chew, T. K., Mahadevan, S., Vijayamalar, B., Morita,
K., Tsuchie, H., and Igarashi, A. (1995) J. Am. Mosq. Control Assoc. 11,94–98
56. Holick, J., Kyle, A., Ferraro, W., Delaney, R. R., and Iwaseczk, M. (2002)J. Am. Mosq. Control Assoc. 18, 131
57. Komatsu, T., Takeuchi, K., Yokoo, J., Tanaka, Y., and Gotoh, B. (2000) J. Virol.74, 2477–2480
LPS Stimulation and JEV Repression of Mosquito STATs 3317
