RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions

braz j infect dis.2014;18(4):441–444

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Brief

communication

RNA

interference

inhibits

herpes

simplex

virus

type

1

isolated

from

saliva

samples

and

mucocutaneous

lesions

Amanda

Perse

da

Silva,

Juliana

Freitas

Lopes,

Vanessa

Salete

de

Paula

LaboratóriodeDesenvolvimentoTecnológicoemVirologia,InstitutoOswaldoCruz,Fundac¸ãoOswaldoCruz,RiodeJaneiro,RJ,Brazil

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Articlehistory:

Received25November2013 Accepted6January2014 Availableonline15May2014

Keywords:

Herpessimplexvirustype1 Real-timePCR

siRNA

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TheaimofthisstudywastoevaluatetheuseofRNAinterferencetoinhibitherpessimplex virustype-1replicationinvitro.Forherpessimplexvirustype-1genesilencing,three dif-ferentsmallinterferingRNAs(siRNAs)targetingtheherpessimplexvirustype-1UL39gene (sequencesi-UL39-1,si-UL39-2,andsi-UL39-3)wereused,whichencodethelarge sub-unitofribonucleotidereductase,anessentialenzymeforDNAsynthesis.Herpessimplex virustype-1wasisolatedfromsalivasamplesandmucocutaneouslesionsfrominfected patients.Allmucocutaneouslesions’sampleswerepositiveforherpessimplexvirustype-1 byreal-timePCRandbyvirusisolation;allherpessimplexvirustype-1fromsalivasamples werepositivebyreal-timePCRand50%werepositivebyvirusisolation.Thelevelsof her-pessimplexvirustype-1DNAremainingaftersiRNAtreatmentwereassessedbyreal-time PCR,whoseresultsdemonstratedthattheeffectofsiRNAsongeneexpressiondependson siRNAconcentration.ThethreesiRNAsequencesusedwereabletoinhibitviralreplication, assessedbyreal-timePCRandplaqueassaysandamongthem,thesequencesi-UL39-1 wasthemosteffective.Thissequenceinhibited99%ofherpessimplexvirustype-1 replica-tion.Theresultsdemonstratethatsilencingherpessimplexvirustype-1UL39expression bysiRNAseffectivelyinhibitsherpessimplexvirustype-1replication,suggestingthatsiRNA basedantiviralstrategymaybeapotentialtherapeuticalternative.

©2014ElsevierEditoraLtda.Allrightsreserved.

Herpessimplexvirustype1(HSV-1)isamemberofthe Her-pesviridaefamilyandischaracterizedbyitsabilitytoestablish latencyafterprimaryinfectionandsubsequentlyreactivated.1

Herpessimplexvirus(HSV)isanenveloped,double-stranded (ds)DNAvirus.TheHSV-1genomeconsistsof152kbof lin-eardsDNAarrangedaslongandshortuniquesegments(UL andUS)flankedbyinvertedrepeatedsequences(TRL/IRLand

∗ _{Correspondingauthorat:LaboratóriodeDesenvolvimentoTecnológicoemVirologia,PavilhãoHelioePeggyPereira,InstitutoOswaldo} Cruz–Fiocruz,AvenidaBrasil,4365,Manguinhos,21040-360,RiodeJaneiro,RJ,Brazil.

E-mailaddress:vdepaula@ioc.fiocruz.br(V.S.dePaula).

IRS/TRS,respectively).2_{WorldwideprevalenceofHSVranges}

from 65%to90%.HSV-1givesrisetoaspectrumofclinical manifestationsand canstillbeamajorcauseofmorbidity and mortality.3 _{HSV-1 isthe causative agentof}

encephali-tis,cornealblindness,andseveralperipheralnervoussystem disorders.4 _{Beyond the neonatal period, most childhood}

herpes simplex virus infections are caused by HSV-1.

http://dx.doi.org/10.1016/j.bjid.2014.01.011

442

Theseroprevalence ofHSV-1antibodies increaseswithage, reaching20%bytheageoffiveyears.Noincreaseoccursuntil 20–40years ofage, when 40–60% ofindividuals are HSV-1 seropositive.Mortalityassociatedwithherpessimplexvirus isprimarilyrelatedtoperinatalinfection,encephalitis,and infectioninindividualswhoareimmunocompromised.4,5

Recently, RNA interference (RNAi) has emerged as a new therapeutic strategy against viral infection.6 _RNAi

is now widely used to knockdown gene expression, in a sequence-specific manner.6 _{It can inhibit the}

expres-sion ofcrucial viral proteins by targeting viral mRNAs for degradation instead of the proteins they encode.7 _{RNAi is}

mediatedby21–25nucleotide double-strandedsmall inter-feringRNA(siRNA)molecules.siRNAsareincorporatedinto theRNA-inducedsilencingcomplex(RISC),whichmediates mRNA sequence-specific binding and cleavage.8 _In

par-ticular, siRNAs, processed from double-stranded (ds) RNA precursors by the type III endoribonuclease Dicer, medi-atepost-transcriptionalgenesilencing(PTGS).9_Somestudies

confirm that siRNA-directed transcriptional gene silencing isconservedinmammaliancells.10 _{SmallRNAs mayguide}

mammaliantranscriptionalsilencinginmanydifferent bio-logical contexts.10 HSV-1 encodes its own ribonucleotide reductase(RR),whichreducesribonucleosidediphosphatesto thecorrespondingdeoxyribonucleotidesandisessentialfor replication.TheHSV-1RRisformedbyalargesubunit desig-natedICP6,encodedbytheUL39gene,andasmallsubunit whichisencodedbytheUL-40gene.TheHSV-1cannotutilize cellularRR andthereforeisdependentupon itsown reduc-taseforreplication.11 _{siRNA-basedantiviraltherapymaybe}

apotentialeffectivetherapeuticalternativeforpatientswith acyclovir-resistantHSVstrains.

Thisstudyevaluated theeffectsofsiRNAstargetingthe HSV-1UL39 geneonthe replicationofHSV-1 isolatedfrom mucocutaneouslesionsandsalivasamples.Infectedpatients with blisters and sores characteristic of herpes skin dis-ease who had HSV-1 DNA detected were included in this study.Forthispurpose,sampleswerecollectedbetween2009 and2010afterobtaininginformed consentstatementfrom eachindividual.ThisstudywasapprovedbyEthics Commit-teeofOswaldo Cruz Foundation(protocol number:544/09). ThesalivasampleswerecollectedusingChemBio(Medford, New York) and samplesfrom mucocutaneouslesions were collectedusingSalivette(Sarsdedt,Germany)devices, respec-tively.ThedetectionofHSV-1wasconfirmedbyvirusisolation incellcultureandreal-timePCR.Inbrief,samplesfrom muco-cutaneouslesionsorsalivasamplesweresuspendedin3mL ofmedium199(Sigma)containingantibioticsand antifungi-cal(2.5␮g/mLofeachonerespectively).Verocellcultureswere inoculatedwith300␮Lofsolutioncontainingmucocutaneous orsaliva samples.TheHSV-1 strain KOSwas usedas con-trolofinfection.12_{During15dayscellcultureswereobserved}

for viral cytopathic effect typical for HSV infection. HSV DNAwasextractedfromclinicalspecimensusingcommercial kits(Qiagen,Valencia,CA,USA)followingthemanufacturer’s instructions.HSV-1specificPCRanalysiswasconductedwitha SYBRGreenreal-timePCRassayaccordingtomanufacturer’s instructions.Thereal-timequantitativePCRwasperformed witholigonucleotideprimerpairsspecificforthecodingregion oftheglycoproteinD(gD)ofHSV-1,asreportedpreviously.13

TheprimersusedwereHSV-FP(5 -CGGCCGTGTGACACTATCG-3)andHSV-RP(5-CTCGTAAAATGGCCCCTCC-3).13_Astandard

curve was prepared by serial dilution (101_–107_{) from DNA}

extractedfromKOSstrain(108_copies/mL).

AfterconfirmingHSV1infection,thereplicationofHSV-1 was inhibitedusingthreesiRNAmoleculesagainstthe UL-39genefromHSV-1(si-UL39-1,si-UL39-2,si-UL39-3).14_One

sequencenottargetinganyknowngenewasusedasa neg-ative controlofsiRNA(AppliedBiosystems,FosterCity,CA, USA).Verocellsweregrownin6-wellplatesto80%–90% con-fluenceand thentransfectedwithspecificorcontrolsiRNA usingthecommercialkitsiPORTTM _{Amine(Ambion/Applied}

Biosystems,Fostercity,CA,USA).Afterfourhours,thecells were infected with HSV-1 from the infected patients (25 PFU/mL). At 48–72h post-infection, plates were fixed with 10% paraformaldehyde for two minutes and then stained with 1% crystal violet for 30minutes to count the num-ber of plaques per well. The effects of siRNA in infected cells were also measured by quantification of HSV DNA. After 48h, the DNA from the infected cells was extracted usingacommercialkit(Qiagen)followingthemanufacturer’s instructions. Real-time PCR relative quantitative reactions wereperformedusingSYBRGreenreal-timePCRMasterMix (Roche,NewJersey,USA)and18SRNAwasusedasthe endoge-nous control. Thestatistical analysiswas performed using the programs “Graph Pad Prism” 5.0 and Excel. The data werereportedasmean±standarddeviation(SD)andthe lev-els ofsignificancewere evaluatedusingtheStudent’st-test

and ANOVA.Differences were considered significant when

p<0.05.

All samplesofmucocutaneouslesions were positivefor HSV-1byrealtime-PCRandbyvirusisolation.Theviralload inmucocutaneouslesionssamplesrangedfrom3.85×103_to

9.78×104_{copies/mL.Thesalivasampleswereallpositivefor}

HSV-1byreal-timePCR,andonly50%ofsampleswere posi-tivebyviralisolation.Thevirusloadinsalivasamplesranged from2.44×103_to1.54×104_{copies/mL.Previousstudieshave}

shownthattheisolationofHSVDNAfromsaliva,usingthe methodofviralisolation,ishamperedbythepresenceof sub-stanceswithanti-HSVactivityinsaliva.15,16_{Usingthehighly}

sensitivetechniqueofreal-timePCR,wedetectedtheHSV-1 eveninthosesampleswithlowviralload.Theconcentration ofsiRNAisimportanttosuppressvirus replication.Aiming toevaluatethebestconcentrationofsiRNArequiredto sup-pressHSVreplication,cellsweretransfectedwithsiRNAsin differentconcentrations(finalconcentration3nM,6nM,9nM, 12nM,15nM,18nM,21nM).Inthisexperimenttwocontrols wereused:(1)cellsinfectedwithHSV-1thatweretransfected withnon-specificsiRNA(NC)andcells infectedwithHSV-1 andnottransfected(CIN).

TheresultsdemonstratedthatthesesiRNAscouldpotently inhibitHSV-1 replicationinvitroandwasobservedthatthe concentrationofUL39specificsiRNAstoachievethehighest inhibitionofHSVtranscriptionwas6nM(Fig.1).This concen-trationisconsiderablylessthanthoseusedinotherstudies of genesilencing inmammalian cells,which has typically rangedfrom20to200nM.17–19 _{However,lowconcentrations}

around 10nMhavealsobeenshown tobesufficientforan effectivesilencingofgenes.20,21_{Followingtheestablishment}

brazj infect dis.2014;18(4):441–444

443

Fig.1–EffectofdifferentsiRNAsontheexpressionlevelsof

HSV-1DNA.TheexpressionlevelofHSV-1DNAincells

treatedwithdifferentconcentrations(3–21nM)ofsiRNAs

(si-UL-391,si-UL-392,si-UL393)wasdeterminedby

real-timequantitativePCR.Theresultwasnormalizedto

thehousekeepinggene18SRNA.NC–cellstransfected

withnon-specificsiRNA(negativecontrol).CINcells

infectedwithHSV-1butnottransfected.

effectonUL-39transcription,asamplefrommucocutaneous lesionswiththehighestviralload(9.78×104_{copies/mL)was}

usedtoevaluatetheefficiencyofgenesilencing.The concen-trationof6nMofsiRNAswasusedfortransfectionandgene silencingwasevaluated48hourspost-infection.Analysisby real-timePCRshowedthat99,90and88.5%ofgene silenc-ingwere achievedupon transfectionwith siRNAstargeting si-UL39-1,si-UL39-2,andsi-UL39-3sequences,respectively (Fig.2).Inourstudy,thesiRNAspecifictotheUL-39-1sequence showedthehighestlevelofsilencing.Byplaqueassaysfrom 63to69%inhibitionofviralplaque formationupon silenc-ing the UL-39 gene. Previous study demonstrated that the inhibitionratesofsiRNA1andsiRNA2onHSV-1plaque for-mationwere35.51and51.62%.14_{Thedifferencesinthelevel}

ofsilencingdetermined bythese siRNAsequences may be duetotheconcentrationofsiRNA,conditionsoftransfection, viralstrains,typeofcellusedanddifferencesinthe thermo-dynamicpropertiesofthesiRNAs.Currently,therehasbeen increasingnumberofstudiesexploringthepotentialforRNAi

100 80 60 40 20 si UL39-1 si UL39-2 Percentage of HSV -1 DNA (%) si UL39-3 NC CIN 0

Fig.2–InhibitionofHSV-1replicationinaninfected

patient.TheinhibitionofUL-39genewasperformedusing

siRNAs(si-UL-391,si-UL-392,si-UL393).NC,negative

control;CIN,cellsinfectedwithHSV-1butnottransfected.

approachestoHSV-1.ThesiRNAiusedinthisstudysilenced specificallytheHSV-1UL39gene,whichencodesthelarge sub-unitofribonucleotidereductase,ICP6.14,22_{RNAihasalsobeen}

reportedtoinhibitHSV-1replicationbyusingsiRNAs target-ingglycoproteinEthatplayskeyroleincell-to-cellspreadand virus-inducedcellfusion20_{;DNApolymerasegeneandVP16}

playvitalrolesininitiationofviralgeneexpressionandviral proliferation22_{andICP4isamajorregulatorygenerequired}

forefficienttranscriptionofearlyandlateviralgenes mak-ingitessentialforlyticinfection.19_{Thesestudiesthatapplied}

RNAitointerfereHSV-1infectionsuggestedthatthesesmall sequencesmighthavethepotentialforeffectivetherapeutic alternative inpatientswithHSV-1infection.Oneimportant aspectofusingsiRNAactivityagainstDNAvirusesistheneed to show the inhibitionof viral DNA replication, and when the amounts of viral DNA were quantified almost no viral DNA couldbedetected,whichdemonstrated theinhibition ofgenomereplication. Herein,the siRNAiwere effectiveto inhibit replicationofHSV-1 ina strainadaptedin cell cul-ture(KOS)andalsoinwild-typevirusisolateddirectlyfrom aninfectedpatientwithhighviralload.Theinhibitoryeffects wererelatedtotheconcentrationofsiRNAitransfected,thus determiningtherightconcentrationsiRNAicanimprovethe inhibitionofvirusreplication.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

NationalCouncilofTechnologicalandScientificDevelopment (CNPq),478979/2009-6 andFundac¸ãodeAmparoàPesquisa doEstadodoRiodeJaneiro(FAPERJ),E-26/111.561/2010were acknowledgedforfinancialsupport.

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