Sendai virus particle production: A more detailed role of F and HN through, namely, their association with M.

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Sendai virus particle production: A more detailed role of F and HN through, namely, their association with M.

SHEVTSOVA, Anastasia, ESSAIDI, Manel, ROUX, Laurent

Abstract

The Paramyxovirus membrane associated proteins are composed of two integral membrane glycoproteins, HN (H, G) and F, and of a matrix protein (M) carpeting the membrane inner layer. For Sendai virus (SeV), F and M have been proposed to form a complex at the endoplasmic reticulum that further migrates to the cell periphery where it represents a nucleation site for viral assembly completion (Essaidi-Laziosi et al., 2013). HN is recruited in the assembly complex once expressed at the cell surface. In contrast to F and M, HN appears dispensable for virus particle production. However, upon F suppression, concomitant HN suppression restricts viral particle production much more severely than F suppression alone, suggesting that HN plays a role as well. In this study, we demonstrate that the transmembrane and cytoplasmic regions of F are sufficient to promote virus particle production and incorporation of a foreign protein in viral particles. We further identify in the F cytoplasmic tail the site of interaction with M. We next confirm HN participation in viral particle production and we provide genetic evidence for a participation [...]

SHEVTSOVA, Anastasia, ESSAIDI, Manel, ROUX, Laurent. Sendai virus particle production: A more detailed role of F and HN through, namely, their association with M. Virus Research , 2015, vol. 199C, p. 31-41

DOI : 10.1016/j.virusres.2015.01.010 PMID : 25613008

Available at:

http://archive-ouverte.unige.ch/unige:46742

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Virus Research

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / v i r u s r e s

Sendai virus particle production: A more detailed role of F and HN through, namely, their association with M

Anastasia Shevtsova-Horoz

, Manel Essaidi-Laziosi

, Laurent Roux

DepartmentofMicrobiologyandMolecularMedicine,FacultyofMedicine,UniversityofGeneva,Switzerland

a r t i c l e i n f o

Articlehistory:

Received12November2014 Accepted9January2015 Availableonline19January2015

Keywords:

Sendaivirus

Virusparticleassembly Matrixproteininteractions

Mutantvirusandphenotypicreversion Deepsequencing

a b s t r a c t

TheParamyxovirusmembraneassociated proteinsarecomposedoftwointegralmembrane glyco- proteins,HN(H,G)andF,andofamatrixprotein(M)carpetingthemembraneinnerlayer.ForSendaivirus (SeV),FandMhavebeenproposedtoformacomplexattheendoplasmicreticulumthatfurthermigrates tothecellperipherywhereitrepresentsanucleationsiteforviralassemblycompletion(Essaidi-Laziosi etal.,2013).HNisrecruitedintheassemblycomplexonceexpressedatthecellsurface.IncontrasttoF andM,HNappearsdispensableforvirusparticleproduction.However,uponFsuppression,concomitant HNsuppressionrestrictsviralparticleproductionmuchmoreseverelythanFsuppressionalone,suggest- ingthatHNplaysaroleaswell.Inthisstudy,wedemonstratethatthetransmembraneandcytoplasmic regionsofFaresufficienttopromotevirusparticleproductionandincorporationofaforeignprotein inviralparticles.WefurtheridentifyintheFcytoplasmictailthesiteofinteractionwithM.Wenext confirmHNparticipationinviralparticleproductionandweprovidegeneticevidenceforaparticipation ofMintheprocess.WefinallyderiveobservationsthatmayprovideamechanismbywhichtheviralC proteinparticipatesinviralparticleproductionbymediatingHN–Minteraction.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction

Sendai virus (SeV)is an envelope virusthat belongs tothe Paramyxovirusfamily.Twoglycoproteins,HNandF,protrudefrom theenvelope forming spikes composedof HN tetramers and F trimers.Thematrixprotein(M)carpetstheinnersideoftheenve- lopeforminga twodimensionalorganizedarrayas seenatthe innersurface oftheinfectedcell(BuechiandBachi,1982).This arrayofMisthoughttoplayacentralroleinvirusparticleassem- bly,condensingthetwo glycoproteinsinpatches–which serve asattachmentsitesfortheviralnucleocapsids–andcompleting thereadytobudassemblycomplex.Moreover,Massociationwith Finthecytoplasmhasbeenrecentlyrecognized.Indeed,inthe casewhereFisrestrictedtotheendoplasmicreticulum,follow- ingmutationofaTYTLE/5Amotifinitscytoplasmictail,Mlevel isreducedatthecellperipheryleadingtoablockadeofviralpar- ticleproduction(Essaidi-Laziosietal.,2013).Thiscrucialroleof theFcytoplasmictailhasbeenotherwiserecentlydocumented (StoneandTakimoto,2013).Ontheotherhand,HNwasproposed

∗Correspondingauthor.Tel.:+41792796531.

E-mail addresses: shevtsova.anastasia@gmail.com (A. Shevtsova-Horoz), essaidimanel@gmail.com(M.Essaidi-Laziosi),laurent.roux@unige.ch(L.Roux).

1 Theseauthorshavecontributedequallytothedevelopmentofthisstudy.

tojointheassemblycomplexattheplasmamembranethrougha SYWSTmotifpresentinitscytoplasmictail(Takimotoetal.,1998).

Indeed,themutationofSYWST(substitutedwithAFYKD,thecor- respondingpeptideinmeaslesvirusHcytoplasmictail)induced accumulationofHNattheplasmamembrane,withoutincorpora- tioninviralparticles(Fouillot-CoriouandRoux,2000).Theroleof thethreemembraneassociatedproteinshasbeenrecentlyrevisited inexperimentswheretheyweresuppressedbysiRNAtechnology, individually, orin concertforHN and F(Gosselin-Grenetetal., 2010).Intheseexperiments,ForMindividualsuppressionledto similarmoderatebutsignificantdecreasesofvirusparticleproduc- tion.ThiscontrastedwithHNsuppressioneffect,confirmingHN dispensabilityforvirusparticleproduction(Portneretal.,1974, 1975;StrickerandRoux,1991;Fouillot-CoriouandRoux,2000).

Despitethiswellestablishedfact,FandHNconcomitantsuppres- sions ledtothelargestdropinvirusparticleproduction, larger thanthatobservedafterFsuppressionalone(Gosselin-Grenetetal., 2010).Theseresultsraisedquestionsabouttherespectiveroleof thetwoglycoproteinsandpointedtoyetlesswelldescribedfea- tures,namelytheirinteractionwithM.

ThepresentstudyshowsthattheFcytoplasmictailandtrans- membranedomainaresufficienttodriveparticleproductionandto allowincorporationofaforeignproteininvirusparticle.Secondly, itallowsidentificationofamotifinFcytoplasmictailpronetointer- actwithM.Thirdly,itconsolidatesthesurprisingobservationof http://dx.doi.org/10.1016/j.virusres.2015.01.010

0168-1702/©2015ElsevierB.V.Allrightsreserved.

HNparticipationinvirusparticleproductionunderFlevelrestric- tion,althoughHNappearstotallydispensableinnormalconditions.

Finally,itprovidesgeneticevidenceofMparticipationinHNincor- porationinvirusparticles.Inthisrespect,itpointstothepossibility thatHN–Minteractionbemediatedbyathirdparty,namelythe viralC protein,proposedpreviouslytoparticipate,amongother functions,in virusparticleformation (Kurotaniet al., 1998). In theend,theresultspresentedgiveamoredetailedpictureofSeV particleformationandopennewlinesofresearchthatmayhelp reconcilingpreviousdataconcerningtheroleoftheCproteins.

2. Materialsandmethods 2.1. Cellsandvirusinfection

MDCK and LLCMK2 cells were maintained in DMEM (Dul- becco’sModifiedEagleMedium,Gibco-Invitrogen)supplemented with5% fetal calf serum (FCS, Brunshwing) and penicillin and streptomycinantibiotics(Gibco).BSRT7cellsobtainedfromK.K.

Conzelmann (Max von Pettenkofer-Institute and Gene Center, Ludwig-Maximilians-University,Munich, Buchholz et al., 1999) were grown in GMEM (BHK-21 Glasgow minimum essential medium, Gibco-Invitrogen) with 5% FCS and a mix of peni- cillin/streptomycinantibiotics(Gibco)andtreatedwith0.5␮g/ml ofGeneticin(Gibco)onceeverytwopassages.MDCKandLLCMK2 celllinesexpressing␣-gfptsiRNAhave beendescribedindetail before(Mottet-Osmanetal.,2007).Allcelllinesweremaintained at37^◦Cunder5%CO2 atmosphere.Forinfection,DMEMdiluted Sendaivirus(SeV)stocks(multiplicityofinfection ∼=10)were addedtothecellsandadsorbedduring1hat33^◦C.Cellswerethen washedoncewithDMEMand2%FCS-DMEMmediumwasadded.

Cellswerethenincubatedforappropriatetimeperiodat33^◦C.After 24–48hofinfection(seelegendsoffigures)cellsandsupernatants werecollectedandusedforfurtherinvestigation.Virusparticles wereobtainedbypelletingtheclarifiedsupernatantsthrough25%

glycerol–Tris–NaCl–EDTAcushionat13Krpmat4^◦Cfor30min.

2.2. Constructionoffull-lengthcDNAplasmids

SeV full-length cDNA genomes were generated from FL5 throughinhousestandardmolecularcloningstrategiesasprevi- ouslydescribed(Mottet-Osmanetal.,2007).ThecDNAconstructs recombinant rSeV-Fg, rSeV-HNg,rSeV-Fg-HNg are as described previously(Mottet-Osmanetal.,2007;Gosselin-Grenetetal.,2010;

Essaidi-Laziosietal.,2014).Fordenominationsimplificationthe gfpt(standingforgreenfluorescentproteintarget)sequencewas indicated by “g”. rSeV-HN_AFYKD was generated by site directed mutationofthesequenceencodingtheSYWSTpeptideusingan insertflankedbytwouniquecloningsites,ClaI(intheFgene)and ApaI(intheLgene).ThesupplementalgenesencodingtheFor HNcytoplasmictailfusedtotheBiotppolypeptide(derivedfrom pcDNA6/BioEase-DEST,Invitrogenlifetechnologies)aredescribed inEssaidi-Laziosietal.(2013),withmodificationsimplyingaddi- tionsoftheC_1–23peptideandHAtag(seetextofSection3forfurther explanations).ThedTomatoprotein(TMT)encodinggenewasfrom Shaneretal.(2004).ThegeneencodingtheHAofH1N1influenza viruswasobtainedfromthelaboratoryofLaurentKaiser(Faculty ofMedicine,UniversityofGeneva,Switzerland).Thesesupplemen- talgeneswereinsertedintheuniqueMluIrestrictionsiteflanked bythegene-startandgene-endsequencesoftheadditionaltran- scriptionunitinsertedbetweentheMandFgenespresentinFl5.

SchematicrepresentationsoftherSeVgenomespreparedinthis paperwereshowninFigs.1,4A,5,7A,S1A,S2AandS3A.Allpre- paredrSeVcDNAconstructsweresequencedforattheleastthe modifiedregionsinvolved(seeTable1forfurtherinformation)or

Table1

PrimarydescriptionofHNAFYKDviruses.

No. Virusdescription^a Phenotype^b Motifsequence^c

wt wt/NF-Crescued wt SYWST

1 HNAFYKD/NF-Crescued Mutant AFYKD

2 HNAFYKD/NF-Crescued/ASG-G0passsaged Mutant AFYKD 3 HNAFYKD/NF-Crescued/ASG-G3passaged Revertant AFYKV 4 HNAFYKD/NF-Crescued/ASG-G4passaged Revertant AFYKV

aViruseswereobtainedthroughstandardrescueproceduresasdescribedinSec- tion2ofthispaperandofthepaperscitedhereafter.NF-C:asinFouillot-Coriouand Roux(2000).ME-L:asinManelEssaidi-Laziosietal.,2014.ASG-G:asinGosselin- Grenetetal.(2010).

bPhenotypeisestablisheduponinfectionofLLCMK2cellsandanalysisofthelevel ofincorporationofHNinvirusparticles:mutant=highlyrestrictedHNincorporation relativetowildtype(wt).

c SequenceanalysisoftheHNgeneonlyandoutlineofaminoacidsequenceinthe motif10aa14oftheHNcytoplasmictail.Numberofpassagesnotpreciselyrecorded.

intotalitywhenindicated.Theyallobeytheruleofsix(Calainand Roux,1993).

SupplementaryFigs.S1–S3canbefound,intheonlineversion, athttp://dx.doi.org/10.1016/j.virusres.2015.01.010.

2.3. Recombinantvirusrescueandvirusstockproduction

RescueofrecombinantSendaiviruses(rSeV’s)formfull-length cDNAgenomeswasperformedasdescribedinMottet-Osmanetal.

(2007).Shortly,FL5cDNAandpTM1basedplasmidsexpressingthe N,P/CstopandPviralproteinswereusedtotransfectBSRT7cells.

After48hours,transfectedcellswereincubatedwith1.5␮g/mlof acetylatedtrypsinin a mediumlacking FCS.Twenty-fourhours later,cellswerecollectedandinjectedinto9day-oldembryonated chickeneggs.After3daysofincubationat33^◦C,allantoicfluid(AF) wascollected.Thepelletof1mlofAFwasanalyzedonSDS-PAGE tocheckforthepresenceofvirusafterCoomassiebluestaining.

TitrationofvirusstockswasdoneasdescribedbeforeSugitaetal.

(1974).

2.4. Antibodies

ForWesternblotdetection,rabbitpolyclonalantibodies␣-HN,

␣-N,␣-MandantiFcytoplasmictail(␣-Fc)havebeendescribed (␣-HN_SDS, ␣-N_SDS, ␣-M_SDS (Tuffereau and Roux, 1988; Mottet- Osmanetal.,2007;Mottetetal.,1996).Monoclonalanti-HAtag isfromCovance(#16B12).Polyclonalrabbitanti-H1N1HAprotein (A/California/14/2009)isfromSigma–Aldrich(SAB3500059).Anti- dTMTisfromChemiconInternational(#AB3216).Thebiotinylated fusedproteinsweredetectedwithStreptavidin-HRP(Pierce).For immune-fluorescencestaining,␣-HN(cloneM68)and␣-F(clone M38)mousemonoclonalantibodieswereobtainedfromAllenPort- ner(StJude Children’s ResearchHospital,Memphis,Tennessee) andratmonoclonalanti-HA(Clone3F10,Rocheref11867423001).

TRITC-conjugateddonkeyanti-ratIgG(JacksonImmunoResearch) andAlexa488conjugatedgoatanti-mouseIgG(Invitrogen)were usedassecondaryantibodies.

2.5. Westernblotanalysis

Westernblotswereperformedaccordingtostandardprotocol.

Viralproteins,aftermigrationin12.5%acrylamideSDS-PAGEgel, weretransferredontopolyvinylidenedifluoridemembrane(PVDF, Millipore)usingTrans-BlotSDTransferCell(Biorad).Proteinswere visualizedusingthePNR2106ECLWesternBlottingDetectionSys- tem(Amersham).BlotimagingwasdoneusingaFujifilmLAS-40.

Fig.1. SchematicrepresentationofrSeV’sexpressingH1N1HAordTMT(TMT)inthecontextofconditionallysuppressedFandHN.1.OutlineoftherSeVgenomewithits sixgenesflankedbythegenomicpromoter(GP)andthecomplementoftheantigenomicpromoter(AGPc,nottoscale).TheFandHNgenesharborthegfptsequenceattheir 5end(blackbox),astargetsforsiRNAsuppression.2.OutlineofrSeV-HA-Fg/HNgvirus.Asin1,butwiththesupplementalgeneencodingH1N1HA.InFopenreadingframe (ORF),thesignalpeptideisshown(Fs,inmagenta).Theecto-(gray),trans-membrane(red)andcytoplasmicdomains(green)arehighlighted.NotethatHAandFaretypeI glycoproteins.Theschematicrepresentationsoftheirnon-cleaved(HA0orF0)andcleaved(HA1andHA2orF1andF2)formsareindicatedinthelowerrightframe.Arrows indicatecleavagesite.Hydrophobicpeptideisshownasasmallgreenrectangle.Yellowlinessymbolizedisulfidebonds.3.OutlineofrSeV-HAFt-Fc-Fg/HNgvirus.Asin2., exceptthatthetrans-membraneandcytoplasmicdomainsofFreplacetheHAcorrespondingdomains.4.OutlineofrSeV-TMT-Fg/HNgvirus.Asin2butageneencodingthe tomatoprotein(TMT,orange)isinsertedasasupplementalgene.5.rSeV-TMTFt-Fc-Fg/HNgvirus.Asin4,withtheFsignalpeptide(Fs),transmembrane(Ft)andcytoplasmic (Fc)domainsfusedtothedTMTORFasindicated.Onlyhighlightedparts(whiteonblack)ofthevirusdenominationsareusedforannotationsofthevirusesinfiguresand text.

2.6. Isotopicradio-labelingofcells

After 16h of infection, cells were overlaid with FCS free mediumcontaining1/10ththeamountofmethionineandcysteine and 30␮Ci/ml of ³⁵S-methionine and ³⁵S-cysteine (Pro-mix- [³⁵S]-Amersham,Biosciences).Twenty-fourhourslater,cellsand supernatantswerecollectedandusedforSDS-PAGEgelanalysis.

RadioactiveproteinswerethendetectedusingTyphoonFLA7000 Phospho-Imager(GEHealthcare).

2.7. Streptavidinagarosepull-downassays

Pull down with streptavidin agarose resins was performed accordingtothemanufacturer’sinstructions(PierceStreptavidin Agarose Resins, Thermo-Scientific) and as previously described (Essaidi-Laziosietal.,2013).Briefly,cellswereinfectedwithviruses expressingBiotpfusedtoForHNcytoplasmictails(represented inFigs.6-A,7-A,S1-AandS2-A)andincubatedinDMEMsupple- mentedwithbiotin1mg/ml(Sigma–Aldrich).Infectedcellswere washed three times withphosphate buffered saline(PBS), col- lected30hpost-infectionandlysedinNaCl10mM,Tris–HClpH 8.050mM,NP400.6%.Thecellularextractswerethenincubated with20␮lofstreptavidinagarosefor2hat4^◦C.Pulled-downpro- teinswerepelletedandwashedfivetimeswithNaCl150mM,EDTA 5mM,50mMTris–HClpH7.8,NP402%.Finally,theboundcom- plexeswereelutedwithSDSPAGEsamplebufferandanalyzedby Westernblotting.

2.8. Immune-fluorescencestainingandconfocalmicroscopy

Immune-fluorescenceanalysiswasperformedaccordingtothe protocol previously described in details (Essaidi-Laziosi et al., 2013).Briefly, MDCK cells were grown on sterilized coverslips coatedwithpoly-lysine(Sigma).After24hofincubationinfected cells were washed with 20mM Hepes pH7.5-buffered DMEM and fixedwith4% PFA15min atroomtemperature. In case of surfaceimmunofluorescence(surfaceIF)stainingprimaryandsec- ondary antibodies were added on the PFA-fixed cells and for total immunofluorescence(total IF)staining, cells wereperme- abilizedusingmethanol(−20^◦C)beforestaining.Washingusing

PBSfollowedanyincubation.ThenucleuswasstainedwithDAPI (Boehringer Mannheim GMBH). Stained cells were mounted in Fluoromount-G(SouthernBiotech).Confocal microscopeLSM700 (CarlZeiss)via63×/1.4oilimmersionobjectivewasusedtotake imagesandZensoftwarefortheacquisitionandthentheanalysis andtreatmentofconfocalimages.

2.9. Analysisofrevertantviruses

Foranalysisofspontaneousreversion,viralRNAwasextracted fromallantoicfluid byTrizol(Life Technology)accordingtothe manufacturer’sinstructions.Reversetranscriptionwascarriedout withM-MLVreversetranscriptase(Promega).ViralgenomecDNA wasamplifiedbyPCR usingspecificprimers(primersequences available upon request).TheRT-PCR productwasthen purified (QiagenDNAgelextractionkit),fullysequencedandanalyzedusing theApplied Biosystemssoftware.Alternatively,1␮mofpurified viralRNAwasused fordeepsequencing.Librarieswerepooled andsequencedonanIlluminaGenomeAnalyzerGAIIx(Illumina).

Obtainedresults(reads)werevisualizedbyIntegrativeGenomics Viewer(IGV).PlasmidscontainingfulllengthcDNAsequencesof rSeV-wtandrSeV-HNAFYKDwereequallysequenced(seeTable2).

3. Results

3.1. TheFcytoplasmictail(+trans-membraneregion)isfully competentforvirusparticleproductionandglycoprotein incorporationintovirion

IftheFcytoplasmictail(Fc)representsaminimalrequirement forSeVparticleproduction,thenaproteinexpressingthisdomain intherightconformationshouldbesufficienttoreplaceF.Corol- lary,onewouldpredictthatthisproteinbeitselfincorporatedin particles.Atfirst,arSeV-Fg/HNgvirus,inwhichtheFandHNpro- teinsareopentosuppressionbysiRNAtechnology(seeSection2) wasconstructed,carryingthesupplementalH1N1influenzahea- magglutinin/neuraminidase(HA0)gene(Fig.1,line2).Inasecond versionofthisconstruct,theFtransmembrane(Ft)andcytoplas- mic(Fc)regionsreplacedtheoriginalHAcorrespondingregions, creatingHAFt-Fc (Fig. 1,line 3).Fig.2 shows that the two HA0

Table2

Summaryofthegenotypicandphenotypicanalysisofwildtype,mutatedvirusesandrevertantvirusesobtainedspontaneouslyorafterserialpassages.

No. Virusdescription^a Phenotype^b Motifsequence^c SequenceinM^d Sequencingfeatures^e

aa192 aa239

1 wt/ME-L wt SYWST Leu Arg Classic,HN/M

2 wt/NF-C wt SYWST Leu Arg Classic,HN/M

3 HNAFYKD/ME-L Mutant AFYKD Leu Arg Classic,HN/M

4 HNAFYKD/NF-C* Mutant AFYKD Leu Arg Classic,HN/M

5 HNAFYKD/NF-Crescued/ASG-G1 Revertant SYFKA Leu Arg Classic,HN/M

6 HNAFYKD/NF-Crescued/ASG-G2 Revertant SFYKN Arg Arg Classic,HN/M

7 HNAFYKD/NF-Crescued/ASG-G3 Revertant AFYKV Leu Gly Classic,HN/M

8 HNAFYKD/NF-Crescued/ASG-G4 Revertant AFYKV Leu Gly Classic,HN/M

9 HNAFYKD/NF-Crescued/ASP5* Revertant AFYKG His Arg Deep,total

10 HNAFYKD/NF-Crescued/ASclP6* Revertant AFYKG His Arg Deep,total

11 AScDNArescued* Mutant AFYKG Leu Arg Deep,total

aVirusdescriptionasinlegendtoTable1withtheadditionofviralcDNAandvirusespreparedbyAnastasiaShevtsova(AS).

b Phenotype:asinTable1.

c Sequenceanalysis:asinTable1.

d ThesequencehasbeenextendedtotheMgene,andtwopositions(aa192and239)havebeenoutlined.

eSequencingfeaturesrefertothemethodologyused.Classic:regularsequencingcoveringtheMandHNgenes(seeSection2).Deep:deepsequencingextendingoverthe entiregenome,beitofarescuedvirusorofafulllengthcDNApreparedforvirusrescue.“P5”and“clP6”forvirusesNo.9and10refertothenumberofserialpassagesin eggsstartingfromaviruswithcontrolledAFYKDgenotypeandphenotype(seetext).clP6:PlaquepurifiedvirusesfromtheP6virus.

Fig.2. RoleofFcytoplasmicandtrans-membranedomainsforformationofandproteinincorporationintoSeVparticles.MDCKcellsconstitutivelyexpressingornotanti gfptsiRNA(+/-␣-gfptsiRNA)weremock-infectedorinfectedwiththeindicatedrSeV’s(seefig.1).Cellularextracts(A–C)andvirusparticles(D–F)wereanalyzedbyWestern blotsusingindicatedantibodies.G.Quantification(ImageJsoftware)ofthelevelofvirusparticleproductionisfromthreeindependentexperimentsdoneinconditionsof expression(blackbars)orsuppression(graybars)ofHNandFglycoproteins.VirusparticlelevelsweretakenastheNlevelsinviralparticlesnormalizedtothelevelsof intracellularN.TheyareexpressedasapercentageofthosefoundinconditionsofHNandFexpression,takenas100%.pvaluesareindicated.

proteins(andHA1)areexpresseduponinfectionwiththesetwo viruses(Fig.2B,lanes5–8).WhenHAharborsthetransmembrane andcytoplasmicregionsofSeV-F(HA_Ft-Fc),itcanbedetectedwith

␣-Fcantibodiesaswell(Fig.2A,lanes7and8).Thisexpressionis equivalentwhetherendogenousHNandFproteinsareexpressed orsuppressed(−/+␣-gfptsiRNA).AsfortheSeVproteins, their expressionisnotaffectedbytheexpressionofthesesupplemen- talproteins(forF,compareinFig.2A,lane3with5and7;forHN, NandMseeFig.2C).Moreover,FandHNlevelsrespondwellto suppression(comparelanes−/+inFig.2AandC,lanesFg/HNg), althoughthecorrespondingdropinviralparticleproductiondoes notexceed70%(Fig.2G,Fg/HNg),alimitationprobablylinkedto thecelltypeusedinthisseriesofexperiments(MDCKasopposed toLLCMK2).Ofnote,HAandHAFt-Fcaredetectedinvirusparticles (Fig.2DandE).Finally,expressionofHAFt-Fccontributesrescuing thehighestvirusproductionuponFandHNsuppression(Fig.2F, comparelanes7–8withlanes3–4,or5–6,andFig.2G).Fig.2Galso showsthatnormalHA,byitself,hasalreadyaboostingeffecton virusproductioninconditionofHNandFsuppression(graybars).

BecauseinfluenzavirusHAappearstoparticipatebyitselftoSeV particleproduction,likelybecauseitsharessomepropertieswithF, wenextusedaproteinthatshouldnotshareanyfeaturewithviral glycoproteins.ThegeneencodingdTomatoprotein(dTMT,seeSec- tion2)wastheninsertedasasupplementalgene,assuch(Fig.1, TMT),ormodifiedsothattheproteinharborstheFtransmembrane andcytoplasmicdomains(Fig.1,TMT_Ft-Fc).Also,theFN-tersignal peptide(Fs)wasaddedatthedTMTN-ter,toaddresstheproteinto ERandtotheexociticpathway.Fig.3Bshowsthatbothversionsof dTMTareexpressedininfectedcells(lanes5–8),withappropriate PAGEmigrationfeatures.TMT_Ft-Fcisalsodetectedby␣-Fcantibod- ies,whichdonotdetectdTMT(Fig.3A,lanes5–8).TMTFt-Fclevels, relativetothatofF,appearlowerthanthoseoftheHA_Ft-Fc(Fig.2A, lanes7and8).SeVproteins,however,areproperlyexpressedor suppressedwhenevertheyshould(Fig.3C).FandHNsuppression leadstosignificantdecreasesinvirusparticleproduction(Fig.3F, comparelanes3and4).Virusparticleproductionappearsnottobe affectedbyTMTexpression(Fig.3F,lanes5and6),butisslightly increasedbytheexpressionofTMTFt-Fc(Fig.3F,comparelanes6–8).

Thiseffect,althoughnotsubstantial,isconfirmedbystatisticalanal- ysis(Fig.3G).ThemodesteffectpromotedbyTMTFt-Fcmayresult fromitsreducedpresenceinthecell,likelyduetoafasterturnover.

Thislikelyexplainsalsothelackofitsdetectioninvirusparticles (Fig.3E).

Inconclusion,ectopicexpressionoftheFtransmembraneand cytoplasmicdomainsappearssufficienttopartlycompensatefor FandHNsuppressionintheviralparticleproduction,suggesting thattheinteractionsinvolvedinthisprocessonlyconcernsthese portionsoftheprotein.Inagreement,withthisconclusion,these twodomainsaresufficienttoleadtoquantitativeincorporationof aheterologousproteinlikeinfluenzaHA.

3.2. AregionoftheFcytoplasmictailproximaltothe transmembraneregionisinvolvedininteractionwithM

InapreviousstudybyFouillot-CoriouandRoux(2000),theF cytoplasmictail(of42 aa long)couldbetruncatedfromits 15 C-terproximalaminoacidswithoutaffectingvirusparticlepro- ductionandFincorporationintheseparticles.Thisresultsuggested thatcondensationofthetruncatedFintheassemblycomplex–a tasklikely devotedtoM–wasnotaffected.Asmutationofthe

24TYTLE₂₀ motifinthis cytoplasmictail, essentialforviruspar- ticleproduction,didnotdisruptF–Minteraction(Essaidi-Laziosi etal.,2013),itbecameplausiblethatthepointofM–Fcytoplas- mictailinteractionwouldlayintheregionflankedbyTYTLEand the transmembraneregion (see Fig. 4A, aa 25–42). The penta- peptide39SMLMG35wasthenmutatedto5A(Fig.4,C-Fcmut1)and

interactionwithMassessedaspreviouslyreported(Essaidi-Laziosi etal.,2013).Fcytoplasmictailinfusionwithapeptideproneto biotinylationinsitu(Biotp)wasexpressedfromasupplemental SeVgene(Fig.4A,C-Fcconstructs).Mpulldownassayswerethen testedusingstreptavidinbeads.Previously,thismethodallowedus toverifytheconservationofM-FcTYTLE/5Ainteraction(Essaidi- Laziosietal.,2013).Inthepresentstudy,wefurtheraddedaC_1–23 peptideandaHAtagattheN-terminalsideofthecytoplasmictail (Fig.4A,C-Fc_wt).C_1–23originatesfromtheSeV-CproteinN-terand representsanaddresstotheinnersideoftheplasmamembrane (Marqet al.,2007).Asexpected, C1–23 broughtthecytoplasmic tailtothecellperiphery,aconditionthatapparentlyimprovedits interactionwithM(Fig.S1,seealsoSection4).Ascontrols,regular infectionsnotexpressingthebiotinylatedFcytoplasmictail(Fig.4, Fg)orexpressingthecytoplasmictailofthemeaslesvirusHpro- teins(Fig.4,Hc)wereused.Analysisofthecytoplasmictailcarrying themutationTYTLE/5A(Fig.4A,C-Fc_5A)wasalsorepeated.Fig.4B andCshowsthat,aspreviouslypublished,TYTLE/5Amutationdid notcancelinteractionwithM(C-Fc_5A).Inthisseriesofexperiments, bindingofC-Fc_5AtoMwasratherboosted.Thiscontrastedwiththe reducedMpulldownbyC-Fcmut1,suggestingthatthisportionof thecytoplasmictailhaslostitsinteractionwithM.

3.3. HNdoesparticipatetoSeVparticleformationinconditionsof Frestriction

The largerdropof virusparticleproduction upon F and HN suppression(exceedingthat followingFsuppressionalone)was interpretedastheHNparticipationinconditionsofreducedFlevel (Gosselin-Grenetet al.,2010).Alternatively, HN co-suppression couldproduceahigherviralglycoproteinturnover,resultingin amoreefficientFdepletion.ToresolvethisuncertaintyHNwas replacedbyHN_AFYKD,excludedfromviralparticle(Fouillot-Coriou and Roux, 2000), hence likely not to participate in viral parti- cle formation.A recombinantSeV wasthenproduced encoding HN_AFYKDinthecontextofanFgeneopentosuppression(Fig.5, rSeV-Fg/HN_AFYKDanddescriptionofalltherSeV’susedintheexper- iment). Eachinfectionwasstudiedinnormal conditions(Fig.6,

−␣-gfptsiRNA)orinconditionsofeffectiveproteinsuppression (Fig.6,+␣-gfptsiRNA).Viralparticleproductionwasrecordedby direct ³⁵S-labeled viral proteins analysispurified from thecell supernatants(Fig.6A).Intracellularviralproteinsweremonitored byWesternblotsusingappropriateantibodies(Fig.6B).Totalradi- olabelledcellextractswerealsodirectlyanalyzedbySDS-PAGE servingasloadingcontrols(Fig.6C).Fig.6A,lanes3and4,show restrictionofVPproductionuponFsuppression.Assownprevi- ously,this restrictionincreases upondual suppressionofF and HNsothatVPproteinsarebarelydetected(lane8).Interestingly, similarrestrictionsareseenuponFsuppressionaloneinthepres- enceofHN_AFYKD (lane10). So,for VPproduction, expressionof HNAFYKDisequivalenttosuppressionofHN.Notethat,asexpected frompreviousresults,HN_AFYKDexpressioninconditionsofnormal Fexpression,doesnotperturb VPproduction(lanes11and12).

Intheend,inconditionsofFsuppressionalone,butwithexpres- sionofanon-functionalHN_AFYKD,thenegativesynergisticeffect thatsuppressionofbothglycoproteinsexertsonvirusproductionis reproduced.ThisratherconfirmstheHNparticipationinviruspro- ductionwhenFisreduced;whenFisfullyexpressed,HNremains fullydispensable.

3.4. EvidenceforMinvolvementinHNincorporationintovirion

InthemodelofSeVassembly(Essaidi-Laziosietal.,2013),HN isrecruitedattheplasmamembranebyinteractionwithafraction ofMthatisfoundbydefaultatthatlocation.Thisrecruitmentdoes nottakeplacewhentheHNcytoplasmictail10SYWST14motifis

Fig.3.RoleofFcytoplasmicandtrans-membranedomainsinincorporationofavirallyirrelevantprotein(dTMT)intoSeVparticles.AsinFig.2exceptthattheanalyzed virusesarethosedescribedinFig.1,lines1,4and5withappropriateantibodies.␣-TMT:anti-tomatoproteinantibody.InpanelG,pvaluesareindicated.

changedforAFYKD(Fouillot-CoriouandRoux,2000).Onthisbasis, itwashypothesizedthatSYWSTisdirectlyinvolvedinMinterac- tion.SimilarexperimentsasthosedescribedinFig.4weretherefore setupforHNcytoplasmictail.Inafirstseriesofexperimentsapep- tidecontainingonlytheHNcytoplasmictail(HNc)fusedtoBiotp failedtoshowanypulldownofM(Fig.S2B,HNcwt).Additionof theC_1–23peptidetotheHNcconstructbroughtthecytoplasmic tailtotheplasmamembrane(Fig.S2C,HNcwt-C)andimprovedthe efficiencyofMpulldown(Fig.S2B,HNcwt-Clanes).Becauseitwas previouslyshownthatadeletionofthefirstN-ter10aminoacidsof HNchadnodeleteriouseffectonHNincorporationintoviruspar- ticles(Fouillot-CoriouandRoux,2000),twomotifsdownwardsof SYWSTwerenextmutated(₁₇SGSTT₂₁and₂₄SGWE₂₈)intoalanine tocreateHNc_mut1-CandHNc_mut2-Crespectively(Fig.7A).Noneof thethreemutantsexhibitedadecreasedabilitytopulldownM.It isparticularlysurprisingthatevenmutationHNc_AFYKD-C,known tobeassociatedwithlackofHNincorporationinvirusparticles, appearstoperformbetterthanHNcwt-C.Intheend,notonlythe datadonotsupportthenotionthattheSYWSTmotifparticipatesin HN–Minteraction,butalsonomotifofinteractionappearstoexist intheHNcytoplasmictail.Consequently,lackofHNincorporation intovirusparticlecanapparentlynotbeexplainedbyadefectof HNinteractionwithM(inthelimitofthemotifstestedhere).

Confronted with this conundrum, we developed another approachbasedonnaturalselectionofHNAFYKDphenotypicrever- tants. We rationalized that poor HN incorporation into viral particleswouldcertainlylowerviralfitness, afitnessthatcould onlyimprovewithviruspassages.Besides,asthemutationinvolves

5aminoacids(ofwhichtherelevantoneswerenotknown),rever- sionwouldnowtargettherelevantsite(s).Andlastbutnotleast, theprobabilitythat reversionwouldinvolvea siteontheputa- tiveproteininteractingwithHNwaspossible.In fact,sequence data limited to themutated region had shown increased vari- abilitycorrelatingwithviruspassages(Fig.S3B).Itwastherefore not surprising that two out of three repeatedly passaged SeV- HN_AFYKD virusesexhibitedrevertant phenotypes(Fig.S3C,lanes 3and 4),in contrasttoSeV-HNAFYKD analyzed just afterrecov- eryfromcDNA(lane1).Interestingly,sequenceanalysisshowed thatthetworevertantvirusescarriedthesubstitutionAFYKDto AFYKV(Table 1).Thisresultwasfully consistent,butcontribu- tionoftheputativeinteractingMproteinwasnotaddressed.More SeV-HN_AFYKDviruses,includingrandomlypassagedviruses,were consideredwithsequence analysis extendingtotheMgene as well(Table2).Moreover,arSeV-HN_AFYKDvirus,justobtainedfrom rescue,wasrepeatedlypassedinacontrolledwayuntilthepheno- typicreversionwasobserved(Fig.8AandB).ThefulllengthcDNA plasmidusedforrescue(notshown),therescuedvirusgenome (Fig.8A,wtandTable2,No.3)andthephenotypicrevertantvirus genomes(Fig.8AandB,Table2,No.9,P5andNo.10,P6)were submittedtodeepsequencing.Theresultsofthedeepsequencing areshowninTables3and4.Onlythesequencedatathatdiffer fromthoseoftherSeV-HN_AFYKDareoutlined,i.e.theremainingof theentiregenomesequencewasfoundunchanged.Interestingly, theAFYKDmotif,whichlikelyprovokesalossoffitness,showed signsofvariabilitywithminorchangescomparedtotheoriginal ateverysinglenucleotideposition.Onlyinposition44,thechange

Fig.4.InteractionsoftheFcytoplasmictailwithMprotein.(A)SchematicrepresentationsofrecombinantSeV’sharboringendogenousFproteinopentoconditional suppressionby␣-gfptsiRNAandadditionaltranscriptionunits(line1)expressingtheFcytoplasmictail(Fc,green)initswt(Fcwt,line2)ormutated(Fc5AandFcmut1,lines 4and5)configurations.Fcisfurtherfusedtoapeptide(Biotp,yellow)opentobiotinylation,toaHAtag(HA,gray)andtoa23aminoacidpeptideoriginatingfromthe SeVCproteinN-terminus(C1–23,purple).IntheFcsequence(line3,highlightedingreen),motifs24TYTLE20and39SMLMG35aresubstitutedby5alaninestocreateC-Fc5A

andC-Fcmut1respectively(lines4and5).Lightgreen:partofFcthatcanberemovedwithoutaffectingglycoproteinincorporationinviralparticleandvirionproduction (Fouillot-CoriouandRoux,2000).Hc(red/green,line6),cytoplasmictailofmeaslesvirusHproteinfusedtoBiotp,usedascontrol.(B)Anti-gfptsiRNAexpressingMDCKcells wereinfectedwiththeindicatedvirusesinmediumsupplementedwithbiotin.Sixtyhourspostinfectioncellularextractswerepreparedandincubatedwithstreptavidin agaroseresinstopulldownFcfusedtoBiotp(seeSection2).Leftpanelsandrightpanels:Westernblotanalysisofpulldownsamplesandcellularextracts,respectively,probed with␣-Mantibodiesandstreptavidin-HRP.Fg:Fgvirus,seeFig.5,line2.(C)QuantificationoftheratioofMproteintoBiotp’susingImageJsoftwarefrom2independent experiments.pvaluesareindicated.

A44GbecamepredominantandledtoanaminoacidchangeD₁₄G (Table3).TheotherchangeobservedconcernstheMORF,where, atnucleotideposition574,aTtoAchangewasevident(93%ofthe reads),leadingtoaL₁₉₂Haminoacidchange(Table4).Notethatthe

positionssurroundingthemutationshownovariabilityatall,100%

ofthereadscorrespondtothoseoftherSeV-HN_AFYKDparentstrain.

Table2presentsasummaryofallthesequencingdataandmany featuresareremarkable.Allrevertantvirusescarryamutation(s)of

Table3

DeepsequencingresultsoftheHNAFYKDviruses:AFYKDmotifhighlighted.HNAFYKDandclP6asinTable2virusesNo.3and10,respectively.ForHNAFYKD,the%oftotalnumber ofreadsforthepositionshighlightedis>99%(notshown).

HNAFYKD

NtpositioninHNORF 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

ntsequence G C G T T C T A C A A G G A T

aasequence A10 F11 Y12 K13 D14

%totalread inclP6

A 1 1 1 1 2 1 2 75 7 82 87 17 12 4 3

C 2 96 3 2 2 92 4 5 81 8 7 3 3 5 6

G 96 2 95 6 3 2 2 5 6 1 3 73 80 83 11

T 1 1 1 90 93 4 91 13 1 7 2 5 3 6 80

clP6 ntsequence G C G T T C T A C A A G G G T

aasequence A10 F11 Y12 K13 G14

Boldvaluesarestressingthehighpercentageofthemajorgenomespecies.

Fig.5. SchematicrepresentationofrSeV’susedtoconfirmtheroleofHNinvirusparticleformation.1.OutlineoftheparentalSeVgenomewithits6genesflankedbythe genomicpromoter(GP)andthecomplementoftheantigenomicpromoter(AGPc).2.OutlineofrSeV-Fgvirus.TheFtranscriptionunitisflankedbythestart/stoptranscription signals/).InFopenreadingframe(ORF),theecto-(gray),trans-membrane(red)andcytoplasmic(green)domainsareshown,withthe24TYTLE20motifoutlined.Inthe5 noncodingregionofthegene,thesequencederivedfromthegreenfluorescentproteingeneservingastargetforsiRNAbasedsuppressionisshown(gfpt,black).3.Outline ofrSeV-HNgvirus.AsfortheFgenein2.DomainsofHNORFaswellasgfptareoutlinedusingthesamecolorcodeasin2.TheHNcytoplasmicdomaincarriesthewildtype motif10SYWST14.4.OutlineofrSeV-Fg/HNgvirus.BothFandHNgenesaretargetedwithgfptsequence.5.OutlineofrSeV-Fg/HNAFYKDvirus.Asin2,buttheSYWSTmotif isreplacedwithAFYKD.6.OutlineofrSeV-HNAFYKDvirus.Asin3,withtheSYWSTmotifisreplacedwithAFYKDandwithoutgfptintheFgene.Virusdenominations,asin Fig.1.

Fig.6. HNAFYKDbehavesasHNsuppressioninthenegativesynergypromotedbyF suppression.LLCMK2cellsconstitutivelyexpressingornotantigfptsiRNA(+/−␣- gfptsiRNA)wereinfectedwithindicatedviruses(seeFig.5).Sixteenhourspost- infection,theinfectedcellswereradiolabelledwith³⁵S-methionineandcysteine (seeSection2).(A)AutoradiogramofvirusparticlesamplesanalyzedbySDS-PAGE.

(B)CellularextractsanalyzedbyWesternblotusing␣-HN,-F,-Nand-Mantibodies.

(C)AutoradiogramofcellularextractsanalyzedbySDS-PAGE.

theAFYKDmotif.Withoneexception(No.5,whichexhibited4out of5mutationspresentinthemutatedAFYKDmotif,regainingthe SYwtmotif),alltherevertantvirusescarryasecondsitemutationin theMprotein,targetingeitherLeu192orArg239.Basedonthedeep sequencingresultsofP5andP6(forwhichnoothermutationscould bedetectedintheentiregenome),aviralcDNAwasconstructed thatonlycarriedthemutationAFYKGmotif(No.11).cDNANo.11 yieldedaHN AFYKGviruswithaconserved mutantphenotype, stronglysuggestingthatthemutationLeu192HisinMwasrespon- sibleforphenotypicreversionoftheHNAFYKGP5andP6viruses.

Intheend,therevertantvirusanalysisbroughtinterestinginfor- mationsupportingalinkbetweenHNandMforHNincorporation intoviralparticle,aswellasitprovidedamoredetaileddescription oftheSYWSTmotif(importanceofSY,seemoreinSection4).

4. Discussion

Insummary,thisstudyprovidesafewdifferentsetsoforiginal informationconcerningtheprocessofSeVparticleassemblyand production.Ingeneral,itmakesampleuseofsupplementalpro- teinexpressionandillustratesthepossibilitiesoftheintegrated suppressioncomplementationsystem(ISCS)toperformviralpro- teinstructure/functionanalysisinthecontextoftheviralinfection.

At this point, it is fair to point to the major drawback of this approach,whichresidesintheimpossibilitytogenerallysuppress morethan95%ofthesiRNAtargetedprotein.Ontheotherhand,this methodologyallowsexpressionofsupplemental(complementing) proteinstolevelscorrespondingtothoseoftheotherviralpro- teinsand inthesame“context”.By this,one refersnotonlyto thegeneralcontextofaninfection(incomparisontoviralprotein expressionresultingfromplasmidtransfection),butmoreprecisely tothesamemolecularmechanismsand tothesamesubcellular topography,featuresthatarenotfullyappreciatedyetforParamyx- oviruses,butarebetterdescribedforpositivestrandRNAviruses (VanKuppeveldetal.,2010).

Thecriticalinvolvement ofFwastargeteddowntoitscyto- plasmicandtransmembranedomains.Ectopicexpressionofthese twodomainswasenoughtorestoreasignificantpartofthedrop inparticleproductionthatfollowsFandHNsuppression.Inview ofthefactthatthecytoplasmictailaloneappearstocontainthe siteofFinteractionwithM,onewouldtendtoconcludethatthe transmembraneregionmaynotbecritical.Interestingly,influenza virusHA appeared topartiallyrescue thedrop ofviral particle

Fig.7. InteractionofHNcytoplasmictailwithMprotein.(A)SchematicrepresentationofrecombinantSeV’sharboringendogenousHNproteinopenedtoconditionalsup- pressionandadditionaltranscriptionunitsexpressingtheHNcytoplasmictail(HNc,blue)initswt(HNcwt-C)ormutated(HNcAFYKD-C,HNcmut1-C,HNcmut2-C)configurations.

Biotp,HAandC1–23,asinFig.4.Inline3,theHNcsequence(highlightedinblue)motifs10SYWST14and17SGSTT21and24ASGWE28whichweresubstitutedwithAFYKDand5 alaninestocreateHNcAFYKD-C,andHNcmut1-CandHNcmut2-Crespectively.Lightblue:partofHNcthatcanberemovedwithoutaffectingglycoproteinincorporationinviral particleandvirionproduction(Fouillot-CoriouandRoux,2000).(B)AsinFig.4,butwiththeindicatedviruses.

productioncausedbyFandHNsuppression.Whetherthisreflects partialfunctional similarity of SeV F and influenza HA, and/or partiallackofspecificityintheinteractionsrequiredforthepro- cessremainsopen;noprimarysequencesimilaritybetweenSeV-F (42aa)andH1N1HA(16aa)cytoplasmictailscouldpossiblybe identified.Regardless,inthesamewaytheHNcytoplasmicand transmembraneregionsallowedmeaslesvirusH (typeIIglyco- protein)uptakeintoSeVparticle(Essaidi-Laziosietal.,2014),the correspondingdomainsofF(typeIglycoprotein)favoredincorpo- rationofaglycoproteinofthesametype.Uptakeofheterotypic glycoproteinsinSeVparticlesappearsthentorequirethehomo- typiccytoplasmic and trans-membranedomains, in accordance withthehumanparainfluenza3(HPIV3)requirementtobesub- typedbythehumanparainfluenza2(HPIV2)glycoproteins(Tao etal.,2000).Thisrequirement,however,maydependonthesource

oftheheterotypicproteins,sinceHPIV3couldbereadilysubtyped withthehumanparainfluenza1(HPIV1)glycoproteins without substitution(Taoetal.,1998).

TheresolutionoftheapparentparadoxicalroleofHNinvirus particleformationreceivedfurtherclarificationwiththeHN_AFYKD expressionaccompanyingFsuppression.Inthissituation,anega- tivesynergyforvirionproductionwascreatedbetweenpresence ofthenon-functionalHN_AFYKDandFsuppression,similartothat observed upon simultaneous suppression of both proteins. The coherenceofthetwosetsofdatafirmlyvalidatesthenotionof partialandunilateralredundancyofthetwoproteinsinviruspar- ticleformation,afindingthatwasalreadymadeusingthevirallike particle(VLP)system(Sandersonetal.,1993,1994).WhenFcan fullyreplaceHN,thislattercanonlypartiallysubstituteforF.The higherdeficitinviralparticleproductioncreatedbyFsuppression

Table4

DeepsequencingresultsoftheHNAFYKDviruses:criticalpositionsintheMproteinarehighlighted.AsinTable3butthepositionsaroundleucine192inMareshown.These highlightedpositionsaretheonlypositionsshowingvariationintheentireviralgenome.

HNAFYKD

NtpositioninMORF 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581

ntsequence G C A G A C C T T G C A T T G

aasequence A190 D191 L192 A193 L194

%totalread inclP6

A 0 0 100 0 100 0 0 97 0 0 0 100 0 0 0

C 0 100 0 0 0 100 100 0 0 0 100 0 0 0 0

G 100 0 0 100 0 2 0 0 0 100 0 0 0 0 100

T 0 0 0 0 0 0 0 3 100 0 0 0 100 100 0

clP6 ntsequence G C A G A C C A T G C A T T G

aasequence A190 D191 H192 K193 G194

Boldvaluesarestressingthehighpercentageofthemajorgenomespecies.