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IRAV (FLJ11286), an Interferon-Stimulated Gene with Antiviral Activity against Dengue Virus ... PDF

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PATHOGENESIS AND IMMUNITY crossm IRAV (FLJ11286), an Interferon- Stimulated Gene with Antiviral Activity against Dengue Virus, Interacts with MOV10 D CoreyA.Balinsky,aHanaSchmeisser,aAlexandraI.Wells,aSundarGanesan,b o w TengchuanJin,cKavitaSingh,dKathrynC.Zoona n CytokineBiologySection,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth, lo Bethesda,Maryland,USAa;BiologicalImagingSection,ResearchTechnologiesBranch,NationalInstituteof a d AllergyandInfectiousDiseases,NationalInstitutesofHealth,Bethesda,Maryland,USAb;Structural e ImmunobiologyUnit,LaboratoryofImmunology,NationalInstituteofAllergyandInfectiousDiseases, d NationalInstitutesofHealth,Bethesda,Maryland,USAc;StructuralBiologySection,ResearchTechnologies fr o Branch,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,Bethesda,Maryland, m USAd h t t ABSTRACT Dengue virus (DENV) is a member of the genus Flavivirus and can cause p : / severe febrile illness. Here, we show that FLJ11286, which we refer to as IRAV, is in- Received17August2016 Accepted5 /jv duced by DENV in an interferon-dependent manner, displays antiviral activity against December2016 i. Acceptedmanuscriptpostedonline14 a DENV, and localizes to the DENV replication complex. IRAV is an RNA binding pro- s December2016 m tein and localizes to cytoplasmic processing bodies (P bodies) in uninfected cells, CitationBalinskyCA,SchmeisserH,WellsAI, . o where it interacts with the MOV10 RISC complex RNA helicase, suggesting a role for GanesanS,JinT,SinghK,ZoonKC.2017.IRAV r g IRAV in the processing of viral RNA. After DENV infection, IRAV, along with MOV10 (FLJ11286),aninterferon-stimulatedgenewith / antiviralactivityagainstdenguevirus,interacts o and Xrn1, localizes to the DENV replication complex and associates with DENV pro- withMOV10.JVirol91:e01606-16.https:// n teins. Depletion of IRAV or MOV10 results in an increase in viral RNA. These data doi.org/10.1128/JVI.01606-16. J a serve to characterize an interferon-stimulated gene with antiviral activity against EditorMichaelS.Diamond,Washington n DENV, as well as to propose a mechanism of activity involving the processing of vi- UniversitySchoolofMedicine ua Copyright©2017Balinskyetal.Thisisan r ralRNA. y open-accessarticledistributedundertheterms 6 IMPORTANCE Dengue virus, a member of the family Flaviviridae, can result in a life- oftheCreativeCommonsAttribution4.0 , Internationallicense. 2 threatening illness and has a significant impact on global health. Dengue virus has 0 been shown to be particularly sensitive to the effects of type I interferon; however, AddresscorrespondencetoKathrynC.Zoon, 1 [email protected]. 9 little is known about the mechanisms by which interferon-stimulated genes function b to inhibit viral replication. A better understanding of the interferon-mediated antivi- y g ral response to dengue virus may aid in the development of novel therapeutics. u e Here, we examine the influence of the interferon-stimulated gene IRAV (FLJ11286 ) on s t dengue virus replication. We show that IRAV associates with P bodies in uninfected cells and with the dengue virus replication complex after infection. IRAV also inter- acts with MOV10, depletion of which is associated with increased viral replication. Our results provide insight into a newly identified antiviral gene, as well as broaden- ingourunderstandingoftheinnateimmuneresponsetodenguevirusinfection. KEYWORDS IRAV,FLJ11286,C19orf66,UPF0515,dengue,flavivirus,interferon, MOV10,Pbody Denguevirus(DENV),amemberofthegenusFlavivirus,isapositive-senseRNAvirus comprised of a nucleoprotein core surrounded by a host-derived membrane. DENVistransmittedthroughthebiteofamosquitovectorandistheetiologicagent ofaspectrumofillnessesthatcanrangefrommildfevertothepotentiallyfataldengue hemorrhagicfever/dengueshocksyndrome.Duetogeographicexpansionofitsinsect vector, as well as increased travel and urbanization, dengue virus is of increasing March2017 Volume91 Issue5 e01606-16 JournalofVirology jvi.asm.org 1 Balinskyetal. JournalofVirology importancetopublichealth.Denguevirusinfectsanestimated390millionpeopleeach year,withmorethan2.5billionpeoplelivinginregionswheretheyareatriskofdengue virustransmission(1–4). Activation of interferon (IFN) signaling pathways results in the upregulation of hundreds of interferon-stimulated genes (ISGs) (5–7). Interferon-stimulated genes en- codingviperin,TRAIL,IFITM1,IFITM2,IFITM3,ISG20,andTRIM56haveallbeenshown to inhibit DENV replication through a variety of mechanisms (8–12). In addition, a number of ISGs, including IFIT2, IFIT1, ISG15, TRIM22, and TRIM79(cid:2)genes, have been foundtohaveactivityagainstotherflaviviruses(13–18).High-throughputscreenshave implicatedanumberofotherISGsininhibitionofflavivirusreplication(6,19). Previously,weidentifiedFLJ11286asoneofanumberofgenesthatwereupregu- lated in Daudi cells in response to treatment with IFN (20) and showed that they displayed antiviral activity against encephalomyocarditis virus (EMCV) (21). FLJ11286 D has also been shown by our laboratory and others to have antiviral activity against o w DENV (22, 23). FLJ11286, which we refer to here as IRAV (interferon-regulated antiviral n gene) (also annotated as C19orf66, UPF0515 , orRyDEN), encodes a protein 291 amino lo a acids(aa)inlengthwithacalculatedmolecularmassof33.1kDa.Analysisofpublished d microarraydatasuggeststhatIRAV(FLJ11286)isupregulatedinresponsetotypeIand e d typeIIIFNs(6,20,24–26).IRAV(FLJ11286)hasbeenshowninmicroarrayscreenstobe f r upregulatedinresponsetotheyellowfevervirusvaccine(27),aswellasafterinfection o m with a number of different pathogens, including adenovirus (28), influenza virus (29), h Lassa virus (30), and ebola and Marburg viruses (31), as well as after infection with t t p humanherpesvirus8andhumanherpesvirus1(32,33).Inaddition,proteomicanalysis : / / of the hepatitis C virus (HCV) interactome identified IRAV (FLJ11286) as one of 214 jv humanproteinsinteractingwiththeHCVNS3protein(34),suggestingaroleforIRAV i.a inthehostpathogenresponse. s m Here, we show that IRAV is upregulated in response to DENV infection in an . o IFN-dependent manner. Upregulation of IRAV in response to IFN-(cid:3)treatment can be r g blocked by disrupting the canonical ISGF3 pathway. CRISPR/Cas9-mediated knockout / o ofIRAVresultedinincreasedtitersofDENV,aswellasofEMCV.Wealsodemonstrate n thatIRAVassociateswithDENVproteinsandlocalizestotheviralreplicationcomplex. J a IRAVisanRNAbindingproteinandlocalizestoPbodiesinuninfectedcells.IRAValso n u associateswiththehostRNAbindingproteinsUPF1andHuR(ELAV1)andinteractswith a r MOV10 (a RISC complex RNA helicase), suggesting a role for IRAV in processing or y 6 stabilityofRNA.Furthermore,weproposeamechanismofactionforIRAVthatutilizes , 2 intrinsic RNA decay pathways. These pathways have been shown to be of increasing 0 importance to the life cycles of multiple viruses, as well as in an array of cellular 1 9 processes. b y g RESULTS u e IRAV is upregulated after dengue virus infection in an interferon-dependent s t manner.TodetermineifIRAVisupregulatedinresponsetoDENVinfection,A549cells were infected with DENV for 72 h. Quantitative real-time PCR (qRT-PCR) analysis showedupregulationofIRAVstartingat24hpostinfection,correspondingtoincreased expressionofDENVRNA,IFN-(cid:3),andISGs,includingIFIT3(Fig.1A).TodetermineifIRAV isregulatedthroughthecanonicalIFN(ISGF3)signalingpathway,IRF9knockouts(KO) were generated in A549 cells using CRISPR/Cas9. Knockout of IRF9 resulted in de- creased expression of IRAV after DENV infection (Fig. 1B), as well as after IFN-(cid:3) treatment (Fig. 1C), indicating that the canonical ISGF3 pathway plays a role in inductionofIRAV. To further characterize IRAV induction in response to IFN, HeLa cells were treated withvariousconcentrationsofIFN-(cid:3)for16horweretreatedwithIFN-(cid:3)ataconcen- trationof3ng/mlandcollectedattheindicatedtimepoints.Sampleswereanalyzed by Western blotting for expression of IRAV and IFIT3. Analysis showed IRAV to be detectableinunstimulatedHeLacellsandtobeupregulatedinresponsetotreatment withIFN-(cid:3)inbothdose-dependent(Fig.1D)andtime-dependent(Fig.1E)manners.To March2017 Volume91 Issue5 e01606-16 jvi.asm.org 2 FLJ11286IsanISGwithActivityagainstDENV JournalofVirology A B i ii A n) 20 DENV2 * * * NA Rn) 45000000 IFN- * * Control IRF9 KO NR 15 Rd Relative RQuantity (d 1005 * Relative Quantity (1230000000000 * IIRRAFV9 0h 8h 16h24h 48h 72h 0h 8h 16h 24h 48h 72h IFIT3 iii iv Relative RNA Quantity (dRn) 1005 IRAV * * * Relative RNA Quantity (dRn) 1230000000 IFIT3* * * * GDAPEDNHHV - + - + D 0h 8h 16h 24h 48h 72h 0h 8h 16h 24h 48h 72h ow n lo C D a Control IRF9 KO IFN- N- IFN- Treatment (ng/ml) d o IF ed N IRF9 f IRAVV ro m IRAV IFIT33 h GAPDHH tt IFIT3 p : / / GAPDHH E IFN- 0 2 IFN4- T6r ea8tm e1n0t (h1) 6 24 jvi. -- +++ -- +++ a IFN- IRAV sm . IFIT3 o r g GAPDHH / o F n J Cell Type Monocytes U937 Daudi Detroit-551 A549 H1299 Hel-299 HepG2 Huh7 OVCAR3 HeLa MDA HEK293 anuary 6 , IRAV 30kDa 2 0 220kDa 1 Actinn 9 - ++ - ++ - ++ - ++ - ++ - ++ - ++ - ++ - ++ - ++ - ++ - ++ - ++ b IFN- y g FIG1 IRAVisaninterferon-stimulatedgene.(A)qRT-PCRanalysisofRNAexpressioninA549cellsafterDENVinfectionat0,8, u e 16,24,48,and72hpostinfection.CellswereinfectedwithDENVatanMOIof0.1for1h,andsampleswerecollectedattime s zeroandtheindicatedtimepoints.ThesampleswereanalyzedbyqRT-PCRforDENVRNA(i),aswellasforexpressionofIFN-(cid:3) t (ii),IRAV(iii),andtheISGIFIT3(iv).ThesampleswerenormalizedtotheHPRThousekeepinggene,andthechangeinexpression wascalculatedrelativetotimezero.Theerrorbarsrepresentstandarddeviations.*,P(cid:2)0.05.(B)WesternblotanalysisofIRAV expressionincontrolA549cellsorIRF9KOcellsafterDENVinfection(72h).Uninfected((cid:3))andDENV-infected((cid:4))A549orIRF9 KOcellswereanalyzedbyWesternblottingforIRF9,IRAV,orIFIT3.GAPDHwasusedasaloadingcontrol.(C)Westernblotanalysis ofIRAVexpressionincontrolA549orIRF9KOcellsaftertreatmentwithIFN-(cid:3).Thecellswereeitherleftuntreated((cid:3))ortreated withIFN-(cid:3)((cid:4))for16h,followedbyWesternblotanalysisforexpressionofIRF9,IRAV,orIFIT3.GAPDHwasusedasaloading control.(D)WesternblotanalysisofIRAVexpressioninHeLacellsaftertreatmentwith10-folddilutionsofIFN-(cid:3)(30to0.00003 ng/ml)for16h.SampleswereexaminedbyWesternblottingforexpressionofIRAVandIFIT3.GAPDHwasusedasaloading control.(E)WesternblotanalysisofIRAVexpressioninHeLacellsaftertreatmentwithIFN-(cid:3)(3ng/ml)attheindicatedtimepoints. SampleswereexaminedbyWesternblottingforexpressionofIRAVandIFIT3.GAPDHwasusedasaloadingcontrol.(F)Western blotanalysisofIRAVexpressioninvariouscelllines.Theindicatedcelllineswereeitherleftuntreated((cid:3))ortreatedwithIFN-(cid:3) ((cid:4))(10ng/ml)for16h,followedbyWesternblotanalysisforIRAV.Actinwasusedasaloadingcontrol. determine if IRAV was expressed in different cell lines, cells were left untreated or treatedwithIFN-(cid:3)(10ng/ml)for16h,followedbyWesternblotanalysisforIRAV.The resultsshowedthatIRAVwasupregulatedinallthetestedcelllines,withtheexception ofHEK293cells,whichproducedonlylowlevelsofIRAV.Inaddition,itwasobserved March2017 Volume91 Issue5 e01606-16 jvi.asm.org 3 Balinskyetal. JournalofVirology thatmonocytesandU937andDaudicellsproducedanadditionallow-molecular-mass band of approximately 26 kDa that matched the predicted molecular mass of an alternateIRAVisoform(Fig.1F). CRISPR-mediated knockout of IRAV results in increased titers of DENV and EMCV.TheCRISPR/Cas9systemwasusedtogenerateA549IRAVKOcells.Treatmentof A549cellswithIFN-(cid:3)resultedinupregulationofIRAV,aswellasofotherISGs.However, in the KO cells, IRAV was not detected at either the RNA (Fig. 2A) or protein (Fig. 2B) level,whileexpressionofotherISGsremainedunaffected.InfectionofA549orIRAVKO cells with DENV (Fig. 2C) resulted in increased titers of DENV (Fig. 2D), as well as significantincreasesinDENVRNArelativetocontrolcells(Fig.2E).Similareffectswere observedafterinfectionwithEMCV(Fig.2FandG).WhenDENV-infectedIRAVKOcells wereexaminedforexpressionofothergenes(Fig.2H),weobservedincreasedexpres- sionoftheIFIT3,Mx1,IRF9,andIFN-(cid:3)ISGsinIRAVKOcellsrelativetothecontrolA549 D cells. These results suggest that IRAV directly affects DENV replication rather than o w exertingitseffectsviaregulationofotherISGs. n IRAVassociateswiththedenguevirusreplicationcomplex.TodetermineifIRAV lo a hasadirectinfluenceonvirusreplication,HEK293cellswerestablytransfectedwithan d e expressionvectorencodingIRAVwithanamino-terminal(N-terminal)greenfluorescent d protein (GFP) fusion or with a negative-control vector with an N-terminal GFP tag. fr o BecauseIRAVispoorlyexpressedinHEK293cells(Fig.1F),theyareanidealcelllinefor m overexpression experiments. Cells were infected with DENV (multiplicity of infection h t [MOI],0.1),andsupernatantswerecollectedattimezeroandat72hpostinfection,and tp : the titer was determined by plaque assay. While detectable in the negative-control / / group, no virus was detected in cells stably expressing IRAV (Fig. 3A). The IRAV- jv i. expressingHEK293cellswerealsotreatedwithasmallinterferingRNA(siRNA)target- a s ingIRAVorwithanegative-controlsiRNA.ThecellsweretheninfectedwithDENVat m . an MOI of 0.1 for 72 h. Virus titration showed a significant increase in virus titers in o r IRAV-expressing HEK293 cells after knockdown of IRAV (Fig. 3B), demonstrating that g / IRAVhasasignificantinfluenceonDENVreplicationevenintheabsenceofinterferon o n treatment. Because IRAV was previously identified by mass spectrometry (MS) as an J a interactionpartnerforHCVNS3(34),weexaminedwhetherIRAVwasinteractingwith n DENVproteins.Coimmunoprecipitation(co-IP)experimentswereperformedonDENV- u a infectedcellsusingGFP-IRAVasbaitandGFP-chloramphenicolacetyltransferase(CAT) ry as a negative control. Western blotting showed that the DENV protease/helicase NS3 6 , and the replication complex-associated protein NS4A interacted with IRAV, while the 2 0 DENVenvelope(E)proteindidnot(Fig.3C).InteractionsbetweenIRAVandNS3were 1 9 not affected by RNase A treatment (data not shown). To further substantiate our b findings, colocalization experiments were performed in A549 cells. Here, A549 cells y were infected with DENV serotype 2 (DENV-2) for 48 h, followed by fixation and g u immunostainingforIRAVandeitherDENVNS3(Fig.3D)orNS4A(Fig.3E)protein.While e s IRAVwaslocalizedtosmallpunctainmock-infectedcells,colocalizationbetweenIRAV t andNS3orNS4AwasobservedinperinuclearregionsofDENV-infectedcells(Fig.3F). ThissuggeststhatIRAVrelocalizestotheDENVreplicationcomplexafterinfection.In ordertodemonstrateassociationofIRAVwiththeDENVreplicationcomplexinprimary humancells,monocyte-derivedmacrophageswereinfectedwithDENV-2asdescribed above.AsshownforA549cells,colocalizationwasobservedbetweenIRAVandDENV NS3(Fig.3G)orNS4A(Fig.3H)inDENV-infectedmonocyte-derivedmacrophages. IRAVassociateswithhostRNAbindingproteins.TodetermineifIRAVinteracted with host proteins in DENV-infected HEK293 cells, co-IP experiments were performed using overexpressed IRAV as bait. The immunoprecipitated proteins were then ana- lyzedbyMS.Geneontology(GO)analysiswasperformedusingthePANTHERclassifi- cation system (http://pantherdb.org )3(5) on MS hits with a spectral count of (cid:5)50. Ontology derived based on the protein class revealed a strong preference (74%) for interactionswithnucleicacidbindingproteins(Fig.4A).Tocharacterizethenucleicacid bindingpropertiesofIRAV,fluorescencepolarization(FP)assayswereperformedusing March2017 Volume91 Issue5 e01606-16 jvi.asm.org 4 FLJ11286IsanISGwithActivityagainstDENV JournalofVirology A B C IRAV IRF9 No Treat IFN- Control IRAV KO n)25 n)10 R R d d ntity (20 ntity ( 8 IRAV IRAV ua15 ua 6 Q Q NA 10 NA 4 IFIT3 IFIT3 R R ve 5 ve 2 ati ati Rel 0 Rel 0 GAPDHH GAPDHH A5I4R9 ANVo KTrO eaNto TrAe5Ia4tR9 IAFV N-KO IFN- A5I4R9 ANVo KTrO eaNto TrAe5Ia4tR9 IAFV N-KO IFN- ontrol AV KO ontrol AV KO DENV - + - + Do C R C R w I I n lo D E F G a DENV Titer DENV RNA EMCV Titer EMCV RNA d * e 6 25 10 200000 * d Log10 PFU/ml45 ns * Relative RNA Quantity (dRn) 1120505 ns * Log10 TCID50/mL456789 ns Relative Quantity (dR)11055000000000000 ns http:/ from / 3A549 0hIRAV KO 0h A549 24hIRAV KO 24h 0A549 0hIRAV KO 0h A549 24hIRAV KO 24h 3A549 0hIRAV KO 0h A549 16hIRAV KO 16h 0A549 0hIRAV KO 0h A549 16hIRAV KO 16h jvi.asm H i IRAV ii DENV iii IFIT3 .or g / n)2.0 n)25 n)10 o R R R n uantity (d1.5 uantity (d1250 uantity (d 68 Janu Relative RNA Q001...050 Relative RNA Q 024 Relative RNA Q 024 ary 6, 20 A549 I0hRAV KO 0hA549 I16RhAV KO 16hA549 I24RhAV KO 24h A549 I0hRAV KO 0hA549 I16RhAV KO 16hA549 I24RhAV KO 24h A549 I0hRAV KO 0hA549 I16RhAV KO 16hA549 I24RhAV KO 24h 19 by g iv Mx1 v IRF9 vi IFN- u e s t n)140 n) 8 n) 140 R R R ntity (d110200 ntity (d 6 ntity (d 110200 ua 80 ua ua 80 Q Q 4 Q A 30 A A 15 N N N R 20 R R 10 e e 2 e ativ 10 ativ ativ 5 Rel 0 Rel 0 Rel 0 A549 I0hRAV KO 0hA549 I16RhAV KO 16hA549 I24RhAV KO 24h A549 I0hRAV KO 0hA549 I16RhAV KO 16hA549 I24RhAV KO 24h A549 I0hRAV KO 0hA549 I16RhAV KO 16hA549 I24RhAV KO 24h FIG2KnockoutofIRAVresultsinenhancedreplicationofDENVandEMCV.(A)A549cellsorIRAVKOcellswereleftuntreatedortreatedwithIFN-(cid:3)for16h, followedbyqRT-PCRanalysisofRNAexpressionofIRAVorIRF9.SampleswerenormalizedtotheHPRThousekeepinggene,andthechangeinexpressionwas calculatedrelativetotimezero.(B)WesternblotanalysisofA549controlorIRAVKOcellsafterIFNtreatment.Thecellswereeitherleftuntreatedortreated withIFN-(cid:3)for16h,followedbyWesternblotanalysisforIRAVorIFIT3.GAPDHwasusedasaloadingcontrol.(C)WesternblotanalysisofA549controlorIRAV KOcellsafterDENVinfection.Thecellswereeitherleftuntreated((cid:3))orinfectedwithDENVfor72h((cid:4)),followedbyWesternblottingforIRAVorIFIT3.GAPDH (Continuedonnextpage) March2017 Volume91 Issue5 e01606-16 jvi.asm.org 5 Balinskyetal. JournalofVirology recombinant IRAV (with an amino-terminal maltose binding protein [MBP] tag and a carboxy-terminal truncation of 18 amino acids) and synthetic 20-mer nucleic acids. Here,dilutionsofIRAVwereincubatedwitheithersyntheticsingle-stranded(ssRNA)or double-stranded (dsRNA) RNA or single- or double-stranded DNA containing comple- mentarynucleicacidsequences.FPshowedIRAVtobeanucleicacidbindingprotein withahigheraffinityforssRNAandssDNAthanfordsDNA(Fig.4B). To further explore interactions between IRAV and proteins associated with RNA processing, MS hits were ranked based on the spectral count, and the top-scoring proteins were further characterized (see Table S1 in the supplemental material). We wereabletoverifyinteractionsbetweenIRAVandMOV10,UPF1,andHuRbyco-IPof overexpressedIRAV,followedbyWesternblotting(Fig.4C).BecauseMOV10,UPF1,and HuRareallknowntobeRNAbindingproteins,weexaminedtheinfluenceofRNaseA treatmentontheseinteractions.RNasetreatmentresultedineithercompleteorpartial D loss of interactions with IRAV (Fig. 4D). Partial loss of interactions due to RNase A o w treatmentwaspreviouslydescribedforinteractionsbetweenMOV10andUPF1(36). n IRAVcolocalizeswithPbodies.BecausemanyoftheRNAbindingproteinsshown lo a to interact with IRAV are associated with ribonucleoprotein (RNP) granules, we next d investigated whether IRAV colocalized with specific RNP granule markers. Here, we e d examinedA549cellsforthelocalizationofPbodymarkers(XRN1,DCP1a,andDDX6/ f r RCK),aswellasthestressgranulemarkerG3BP1(Fig.5A).Weobservedcolocalization o m betweenIRAVandallthreePbodymarkersinIFN-treatedcells;however,nostatistically h significantcolocalizationwasobservedbetweenIRAVandG3BP1,indicatingthatIRAV t t p associatedwithPbodiesinthecytoplasmofIFN-treatedcells(Fig.5B).Tocharacterize : / / theassociationofIRAVwithPbodycomponentsafterDENVinfection,A549cellswere jv infected with DENV, followed by staining for IRAV and XRN1 (Fig. 6A and B). As i.a s previouslydescribedforXRN1(37),IRAVrelocalizedtotheDENVreplicationcomplex, m colocalizingwithDENVNS3. . o IRAV interacts with MOV10. Due to its known role as a restriction factor for rg / retroviruses(38–41),hepatitisCvirus(6),andhepatitisBvirus(42),wefurthercharac- o terized interactions between MOV10 and IRAV. Co-IPs and reciprocal co-IPs were n J performed using HEK293 cells transfected with either IRAV (GFP-IRAV) or a negative a n control (GFP-CAT). Co-IPs were then performed using an antibody either to overex- u pressed IRAV or to endogenous MOV10, and Western blots were analyzed for the a r y presence of either MOV10 or IRAV (Fig. 7A). In addition, confocal microscopy and 6 fluorescenceresonanceenergytransfer(FRET)byfluorescencelifetimeimaging(FLIM) , 2 analyseswereusedtocharacterizeinteractionsbetweenendogenousIRAVandMOV10 0 1 inA549cells.ConfocalmicroscopyrevealedcolocalizationbetweenIRAVandMOV10, 9 as determined by Pearson’s linear correlation coefficient (Fig. 7B and C). Moreover, b y FRET-by-FLIM analysis demonstrated interactions between IRAV and MOV10, with g lifetime values of 1.8 to 2.1 compared to values of 2.5 to 2.8 for the donor control, u e representing25%to28%FRETefficiency.TheendosomalmarkerRab5wasusedasa s t negative control. Rab5 did not demonstrate significant colocalization with IRAV or a shiftinlifetimevalues(Fig.7DandE). ConfocalmicroscopywasalsousedtoexaminelocalizationofIRAVandMOV10in DENV-infectedandmock-infectedA549cells(Fig.8AandB).Here,itwasobservedthat, similar to XRN1, both IRAV and MOV10 relocalized to the viral replication complex, colocalizingwithDENVNS3.TofurthersubstantiatearoleforMOV10inrestrictionof FIG2Legend(Continued) wasusedasaloadingcontrol.(D)A549orIRAVKOcellswereinfectedwithDENV,andsampleswerecollectedattimezeroand24h,followedbytitrationusing theplaqueassaymethod.(E)A549orIRAVKOcellswereinfectedwithDENV,andsampleswerecollectedattimezeroand24h,followedbyqRT-PCRanalysis ofDENVRNA.(F)A549orIRAVKOcellswereinfectedwithEMCV,andsampleswerecollectedattimezeroand16h,followedbytitrationusingthe50%tissue cultureinfectivedose(TCID )method.(G)A549orIRAVKOcellswereinfectedwithEMCV,andsampleswerecollectedattimezeroand16h,followedby 50 qRT-PCRanalysisofEMCVRNA.(H)qRT-PCRanalysisofA549orIRAVKOcellsinfectedwithDENV.Sampleswerecollectedattimezeroand16hand24h postinfectionandanalyzedforIRAV(i)andDENVRNA(ii),aswellasforexpressionofIFIT3(iii),Mx1(iv),IRF9(v),andIFN-(cid:3)(vi).Sampleswerenormalizedto theHPRThousekeepinggene,andthechangeinexpressionwascalculatedrelativetotimezero.Theerrorbarsrepresentstandarddeviations.*,P(cid:2)0.05;ns, notsignificant. March2017 Volume91 Issue5 e01606-16 jvi.asm.org 6 FLJ11286IsanISGwithActivityagainstDENV JournalofVirology D o w n lo a d e d f r o m h t t p : / / jv i. a s m . o r g / o n J a n u a r y 6 , 2 0 1 9 b y g u e s t FIG3IRAVassociateswiththeDENVreplicationcomplex.(A)HEK293cellsstablyexpressingIRAVoranegativecontrolwereinfectedwith DENVatanMOIof0.1,andsampleswerecollectedattimezeroand72hpostinfection,followedbytitrationusingtheplaqueassay method.TheinsetrepresentsexpressionofIRAVintransfectedHEK293cellscomparedtothenegativecontrol,asdeterminedbyWestern blotanalysis.GAPDHwasusedasaloadingcontrol.(B)HEK293cellsstablyexpressingIRAVornegative-control(NC)cellsweretreated witheithernegative-controlsiRNAoransiRNAspecifictoIRAV(IRAV_I).ThecellsweretheninfectedwithDENVfor72h,followedby titrationonVerocells.(C)IRAVcoimmunoprecipitateswithDENVproteins.HEK293cellswereeitherleftuninfected((cid:3))orinfectedwith DENV((cid:4))for48h,followedbytransfectionwithaplasmidexpressingGFP-IRAV(IRAV)orGFP-CAT(Control)foranadditional48h.The celllysateswerethencollected,andco-IPexperimentswereperformedusingantibodiestoGFP.InputandIPsampleswerethenanalyzed byWesternblottingforthepresenceofDENVenvelope(DENV-E),DENVNS3,orDENVNS4Aprotein.(D)ConfocalmicroscopyofIRAV colocalizedwithreplicationcomplexesinDENV-infectedormock-infectedA549cells.Green,IRAV;red,DENV-NS3.Thenucleuswasstained withDAPI(blue).ColocalizationbetweenIRAVandDENV-NS3isshowninwhite.LowMag,alower-magnificationfieldshowingacluster ofinfectedcells.(E)ConfocalmicroscopyofIRAVcolocalizedwithreplicationcomplexesinDENV-infectedormock-infectedA549cells. Green,IRAV;red,DENVNS4A.ThenucleuswasstainedwithDAPI(blue).ColocalizationbetweenIRAVandDENVNS4Aisshowninwhite. (Continuedonnextpage) March2017 Volume91 Issue5 e01606-16 jvi.asm.org 7 Balinskyetal. JournalofVirology B 150 A nucleic acid binding P) rdeecfeenpstoer/immunity protein on (m surfactant ati100 ceyxttorasckeellleutlaalr pmroatteriixn protein olariz ctrhaanpspeorortneer ce P ssRNA hydrolase en 50 dsRNA c enzyme modulator es ssDNA transferase or dsDNA transcription factor Flu 0 0 1 2 3 4 5 IRAV Protein (µM) C Input IP D Inpput IP D IIRRAAAAVV CCoonnttrrooll IIRRAAAAVV Control IIRRAAAAVV Control IIRRAAAAVV Control o w n IRAV GFP lo a d e d MOV10 MOV10 f r o m UPF1 UPF1 h t t p : HuR / HuR /jv i. DENV -- ++ -- ++ -- ++ -- ++ RNase AA -- ++ -- ++ -- ++ -- ++ as m . FIG4IRAVinteractswithRNAbindingproteins.(A)GeneontologyanalysisofIRAVinteractionpartnersasidentifiedbyco-IP o r ofGFP-IRAVfromDENV-infectedHEK293cells,followedbyMSanalysis.Geneontologyanalysiswasperformedusingthe g PANTHERclassificationsystem(http://www.pantherdb.org),basedontheproteinclass.(B)FPassayswereperformedusing / o various concentrations of recombinant IRAV and FAM-labeled 20-mer single-stranded or double-stranded DNA or RNA n oligomers.Sampleswererunintriplicate,andFPwasmeasuredonaHidexsensemicroplatereader(Turku,Finland).(C)Co-IP J experiments performed on HEK293 cells left untreated ((cid:3)) or infected with DENV ((cid:4)). The cells were transfected with a expressionvectorsforGFP-IRAV(IRAV)orGFP-CAT(Control),followedbyIPwithanantibodyforGFP.Westernblottingwas n u performedforIRAVputativeinteractionpartnersMOV10,UPF1,andHuR.(D)Co-IPexperimentswereperformedonHEK293 a cellsafteroverexpressionofGFP-IRAV(IRAV)orGFP-CAT(Control).Celllysateswereeitherleftuntreated((cid:3))ortreatedwith r RNase A ((cid:4)), followed by IP with an antibody for GFP. Western blots were then performed for IRAV putative interaction y 6 partnersMOV10,UPF1,andHuR. , 2 0 1 DENV replication, siRNA-mediated knockdown experiments were performed in A549 9 b and IRAV KO cells (Fig. 8C). Depletion of MOV10 resulted in an increase in virus y replication for DENV, as well as for EMCV, which was significantly greater in IRAV KO g u cellsthaninIRAV-expressingA549cells(Fig.8DandE),indicatingthatbothIRAVand e s MOV10playrolesintherestrictionofvirusreplication. t DISCUSSION Interferon-stimulated genes are key mediators of antiviral immunity and play a pivotal role in the immune response to DENV infection. While the IFN response is an innate and broad-spectrum response to many pathogens, ISGs have been demon- stratedtohaveremarkablespecificitybothforthevirusestheytargetandthepathways throughwhichtheyinhibitviralreplication(6).HundredsofISGshavebeenshownto be upregulated in response to induction of IFN signaling pathways, illustrating the FIG3Legend(Continued) (F)ColocalizationcoefficientsofIRAVandDENVNS3(NS3)orDENVNS4A(NS4A)inDENV-infectedA549cellsasdeterminedbyPearson’s linearcorrelationcoefficient.(G)ConfocalmicroscopyofIRAVcolocalizedwithreplicationcomplexesinDENV-infectedormock-infected monocyte-derivedmacrophages.Green,IRAV;red,DENV-NS3.ThenucleuswasstainedwithDAPI(blue).(H)ConfocalmicroscopyofIRAV colocalizedwithreplicationcomplexesinDENV-infectedormock-infectedmonocyte-derivedmacrophages.Green,IRAV;red,DENVNS4A. ThenucleuswasstainedwithDAPI(blue).Theerrorbarsrepresentstandarddeviations. March2017 Volume91 Issue5 e01606-16 jvi.asm.org 8 FLJ11286IsanISGwithActivityagainstDENV JournalofVirology A B RNP Marker IRAV Merge ROI 0.8 DCP1a efficient 0.6 o s C 0.4 n` o s DX6 Pear 0.2 D 0.0 DCP1a DDX6 XRN1 G3BP1 D 1 o N w R X n lo a d e d f P1 ro B m 3 G h t t p FIG5IRAVassociateswithPbodiesinIFN-(cid:3)-treatedcells.(A)ConfocalmicroscopyofIRAV;PbodymarkersDCP1a, :/ / DDX6,andXRN1;andthestressgranulemarkerG3BP1ainA549cellsaftertreatmentwithIFN-(cid:3)(10ng/ml)for16h. jv Green,IRAV;red,RNPmarkers.ThenucleuswasstainedwithDAPI(blue).Regionsofinterest(ROI)areboxedinwhite. i. a (B) Colocalization coefficients of IRAV with DCP1a, DDX6, XRN1, and G3BP1 as determined by Pearson’s linear s correlationcoefficient.Theerrorbarsrepresentstandarddeviations. m . o r g scopeandcomplexityoftheIFNresponse.Todate,onlyafractionofthesegeneshave / o beencharacterized. n Here, we show that IRAV is an IFN-stimulated gene with antiviral activity against J a DENV.KnockoutoftheIFNsignalingcomponentIRF9resultsinasignificantreduction n u of IRAV expression after IFN treatment, suggesting regulation through the canonical a r ISGF3 complex; however, it should be noted that there appears to be some baseline y 6 constitutive expression of IRAV. In addition, IRAV homologues were identified in , 2 hemichordates and echinoderms, organisms that lack a “classical” IFN response, sug- 0 gestingthepossibilityofIFN-independentfunctionsforIRAV-likeproteins. 1 9 IRAVassociateswithPbodies,asevidencedbyinteractionswithPbody-associated b proteins(MOV10andUPF1)andcolocalizationwithPbodymarkers(DCP1a,XRN1,and y g DDX6).PbodiesarediscretecytoplasmicstructurescomposedofRNAandproteinsthat u e are associated with various aspects of RNA turnover, including nonsense-mediated s t decay,adenylate-uridylate-richelement(ARE)-mediateddecay,andgenesilencing(43). Notably, both DENV and West Nile virus (WNV) have been shown to associate with components of P bodies. Subgenomic flavivirus RNA (sfRNA) inhibits the activity of XRN1andTRIM25(44–46),andDDX6interactswithDENVuntranslatedregions(UTRs) andlocalizestoDENVreplicationcomplexes,possiblyplayingaroleinvirusreplication (47).Furthermore,WNVinfectionofHeLacellsresultsinadecreaseofboththesizeand number of P bodies, along with a relocalization of P body components (including GW182, DDX3, and XRN1) to WNV replication complexes, colocalizing with NS3 (37). ThisissimilartowhatweobservedwithIRAVduringDENVinfection,withIRAVpuncta becomingmorediffuseandcolocalizingwithNS3indistinctperinuclearcomplexes.A numberofotherviruses,includinghepatitisCvirus(48),poliovirus(49),influenzavirus (50), and bunyaviruses (51, 52), have been shown to interact with P bodies or with P bodycomponents(53). MOV10andUPF1arebothmembersoftheSF1familyofhelicasesandhavebeen previouslyshowntointeractwithoneanotherandtoformcomplexeswithAPOBEC3G March2017 Volume91 Issue5 e01606-16 jvi.asm.org 9 Balinskyetal. JournalofVirology IRAV XRN1 DENV-NS3 Merge A IRAV V N DE XRN1 DENV-NS3 IRAV k c XRN1 o M D DENV-NS3 o w n B 0.8 lo a nt d n's Coefficie00..46 ed fro o m s Pear0.2 h t t 0.0 p IRAV-NS3 IRAV-XRN1 ://jvi. a FIG6IRAVrelocalizestothereplicationcomplexafterDENVinfection.(A)ConfocalmicroscopyofXRN1colocalizedwithIRAV s andDENVNS3atthereplicationcomplexinDENV-infectedormock-infectedA549cells.Green,IRAV;magenta,XRN1;red, m DENVNS3.ThenucleuswasstainedwithDAPI(blue).ColocalizationbetweenIRAV,XRN1,andDENVNS3isshowninwhite. . o Regions of interest (ROI) are boxed in white. (B) Colocalization coefficients of IRAV and DENV NS3 or IRAV and XRN1 in r g DENV-infectedA549cellsasdeterminedbyPearson’slinearcorrelationcoefficient,demonstratingcolocalizationbetweenIRAV / andbothXRN1andDENVNS3inDENV-infectedcells.Theerrorbarsrepresentstandarddeviations. o n J a and Argonaute 2 (40, 54), as well as with the antiviral protein ZAP (55). Interactions n u betweenMOV10andUPF1arepartiallysensitivetoRNasetreatment(36),aswehave a r also demonstrated for IRAV interactions with MOV10 or UPF1. Knockdown of MOV10 y 6 resultsinanincreaseinthehalf-livesofmRNAtargets(36).MOV10hasbeenshownto , 2 be involved in retrotransposition of LINE elements (56–58), as well as in microRNA 0 pathways(54,59).MOV10mayalsoplayaroleinmediatingantiviralresponse(60)and 1 9 restricts replication of retroviruses (38–41), hepatitis C virus (6), hepatitis B virus (42), b and influenza virus (61). UPF1 is a key component of nonsense-mediated RNA decay y g pathways(62)andhasbeendemonstratedtoplayaroleinviralreplicationcycles(63, u e 64).IRAValsoassociateswiththeRNAbindingproteinHuR.HuRhasbeenshowntobe s t involved in RNA decay pathways (65) and has been linked to viral replication and modulationofthehostresponse(66–69).Inaddition,IRAVhasbeenpreviouslyshown tointeractwithRNAbindingproteinsLARP1andPABPC1,bothofwhichhavealsobeen showntobeinvolvedinRNAdecay(23).Takentogether,IRAV’sroleasanRNAbinding protein, its association with proteins linked to RNA decay, and its localization to P bodies (sites of RNA decay) and the marked increase in viral RNA observed after knockout of IRAV all suggest a role for IRAV in degradation of viral RNA. The role of MOV10 in IRAV-mediated antiviral activity remains unclear; however, given MOV10’s roleasanRNAhelicaseinvolvedinRNAdecaypathways,aswellasitslocalizationat the viral replication complex and the increase in viral RNA observed after its knock- down, MOV10 may function in conjunction with IRAV and other proteins to aid in destabilizationofviralRNA. Inconclusion,weshowthatIRAVisanISGthatisregulatedthroughthecanonical type I interferon signaling pathway. IRAV displays antiviral activity against DENV and EMCVandinteractswithDENVproteins.IRAVisanRNAbindingproteinthatlocalizes March2017 Volume91 Issue5 e01606-16 jvi.asm.org 10

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retroviruses (38–41), hepatitis C virus (6), and hepatitis B virus (42), we further charac- terized interactions between MOV10 and IRAV. UniProtKB Swiss-Prot, and the common contaminant proteins found in the GPM.org's cRAP database, all downloaded in March 2014. The data were searched with 2
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