IDENTIFICATIONANDCHARA CIERlZATI ON OF A NOVEL CLASS OFRBCEP'IU R FOR ENCEPHAW MYOCARDITIS VIRUSON HUMANCELLS
by
c&lYi-MlngJin,M.B.
A thesissubmitted to the SchoolofGraduate Studies
in partialfulfilment ofthe requirementsfor thedegree of
Master ofScience
FacultyofMedicine Memorial Universityof Newfoundland
February 1993
51.John's Newfoundland
1+1
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Ac:quisiUlsand BbIiogf 3j:tlic SefvicesBfanch
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Abstract
Ence pbalo myocarditls(EMC) virus belongs 10 thegen uscnrd iovtrusof the picornavirusfamily.The virusisimportant in the etiologicalstudy ofsevera l virus- induced human diseases. However,littleisknownabout EMC virus attachment to nuclea ted cells. In this thesis, the results on the characte rizationof the biochemicalnatur e ofEMCvirusreceptors011two human nucleat ed cel l lines,K562 andHeL.,cells,arcpresent ed.The study showed that EMCvirusbound specificallytoboth cell typ es.The number of receptorsiteson l-IeL1. cellswas inthera ngeof 1.6x IUSper cel landthe dissociati on consta nt forvir usbindingwas 1.1 11M. Theseresult sarc consiste ntwithpreviouslydeterminedvaluesforthe bindingofEMCvirusto K562 cells.Aftertrypsin digestionof K562 andI-le Lacells, rcgcnc mtion of virus binding activitywasinhibitedby cycloheximide trcntmcut.suggesting that recovery ofthe EMC virus-specificreceptor isdepend entuponintracellular proteinsynthesis. Further,digestion with prot eases and ncuraminidase, as wellaslectintrea t ment ofintact cells,cell memb ran epreparationsand detergent-solubilizedcellmembran es, demonstra tedthat recept orsforthe virus on bot h K562 and He La cellsarc slnloglycop rotein s. Affinity chromatogra phyemployingEMCvir uscolumns isolat ed 70-kDrece ptor prote insfromK562 and HeLa cells.Virus overlayprotein assayrevealed that
EMCvirusspe cifically recogni zed onlythe 70-kD prote insinboththepurified rece ptor prep a rati on sandin detergent solubilizedcell memb ranes, sugges ting tha tvirus atta chmenttoK562 and He l.n cellscouldbe excl usively mediated hy theide nt ified recep to rmole cules.Using thechrom at ofoc usingte chnique, itwas found thnt the receptoron K562 cellsislikelymor e sia lylate d than tha t onHcL1. cells. Finally,West ernblotana lysis usin gantl-glycophorin A antibodyrevealed thatthe anti body doe snotrecogn iz etheEMCvirus70-kD receptor011K562 orHct,n cells(thelatterdoesnot expressglycophori nA).
This indicate sthnttheiden t ified recepto r prot ein smay not be glyco phori nA, bUIthey representnovel,not yet describ ed EMCvirusrecepto r mo lecu les.
iii
Acknowledgements
Iwould liketo express my sincere thanks tomy superv isor.Dr.111OI1Ias1.
Michala k, for his superv ision,helpnodguida nce.Also Iwouldlike to express deepappreciation tothemembers ofIllysupervisory committee,Drs.II.
Banfield You nghu sband andAlanPater, for theirconstructivesuggestionsand encouragement duringthis work.
Iwould like to express mywholeh eart edthankstomy ru nnersupervisor, Dr. AlfredT.H.Burn ess, who gavethis workexcelle ntguid ancenndsupport untilhisdeath. The thingsI learntfromhimwhet he r inscie nceorlifewill bevaluablefor therest of mylife.
Iamdeeplyindebtedto Ms.Ingrid. U.Pardoeforhe r valuable help, encourage ment, pat ien ceandsincere concernformy wellbeing duringmy program, especiallyforreadin g thedraftsof thismanuscript.Thanks nrcalso due toPahBaldeh,and Drs.Kan walK. Grewaland JawedHam idfortheir helpduring thisproject.
Igrate fullyacknowledge the financialsupport providedbytheFacultyof Medicine and School of Graduate St udies, Memorial University of Newfound land.
Finally,Iwould liketo thank my wifeand mypnrent sfortheir suppor t and encouragement.
iv
Contents
AllSTRACI' ACKNOWLEDGEMENTS TAIlI.EOF CONTENTS /.ISTOFTAIlI£~
/.1STOFFIG URr,s
/.1ST OF ABBREVIATIONSANDSYMBO LS USED
CIIAPTER 1:INTRODUCnON
Page ii
ix
xiii
1.1.General Background 1
1.2.Picoru a vir usesandEncephnlomyocarditls(EMC)Virus 3
1.3.Virus-Cell Interaction 6
1.3.1.Viral Attachm entProtei ns 7
1.3.2.PlasmaMembranes andCellular Receptors for Viruses10 1.3.2.1.Structur e andComponentsof the Plasma
Membrane 10
1.3.2.2.Cellular Receptor fortheVirus II 1.4.Obstacles inVirus ReceptorIsolati on 13 1.5. Meth ods of Isolat ionandCharacterizatio n of VirusRecept ors 14 IJl. Cellula rReceptorsfor Picoma viruses 18 [.6.1. General Propertie s ofReceptors for Plcornaviruses 18
1.6.2. ReceptorforPoliovirus 21
1.6.3. Receptors for Rhinoviruses 22
1.6.4. ReceptorsforOt her Picomaviruses 28
1.6.5. Receptor forEMCVirus 29
1.6.5.1.Glycophorin A - the Receptor for EMC
Virus onErythrocytes 29
1.6.5.2.SiteofAttac hmentofEMCViruson
GlycophorinA 31
1.6.5.3.Role ofSialicAcid in the Attachmentof EMC
Virus toEryt hrocytes 33
1.7.Purpose of Research 35
1.7.1.General Purpose of theVirusReceptor Studies 3S
1.7.2.Purpose of theProjec t .\6
1.7.3.Experime ntalStrategy 37
CHAPTER2:MATERIALSAND MET II ODS
2.1.GrowthandPurifica tion of EMC virus 41
2.2.Cells 41
2.2.1.K562Cells 41
2.2.2. BeL1 Cells 43
2.3.Prepar at ion and Solubilizat ionofKS()2 andHeL.1Cell
Membranes 44
2.3.1.Preparationof CellMembranes 44
2.3.2.Preparat ion of SolubilizedCell Membranes 45 2.4-Preparation ofErythrocyteMembra nesand Glyccphor tns 4<>
2.4.1.Human Erythrocy teMembranes 4<>
2.4.2.Preparationof Olycophorins 4H
25. Iodination of Proteinsbythe Iodogen Method 4"
2.6.Measure mentof RadioactivityIncor poratedinto Proteins 51 2.7.Attachmentof3H- L1beled EMC Virusto Cellsand
Membra nes 52
2.7.1.Measure mentof BindingofVirusto K562 Cells 53 2.7.2.Measure me ntofBindingof Virus to llcl,a Cells 54 2.7.3.Measure me ntof Bindingof VirustoCellMemb ranes 55 2.7.4.Measurementof Bindingof Virus toImmobilized
CellMembranes .15
2.8.Determ ina tionof Receptor Activity By Dot-BlotAssay 56 2.9.EnzymaticTreatmentofCells,Cell Membranesand
PurifiedVirusReceptorProteins .I"
2.9.1.EnzymaticTreatmentofIntactCells 59 2.9.2.Enzymatic Treatmentof CellMembranes (,1 2.9.3.Enzymatic Treatmentof1251_L'lbclcd,Affini tyPur ified
VirusReceptor Pro tein s 62
2.10.Effectof LectinsonBindingof EMC Virusto Cclls 63 2.11.Determinatio nofBiosynthesisof EMC VirusRecepto rAfter
TrypsinTreatment 63
2.12.PreparationofVirus -Se pharoscColu mns M 2.13.Vlrus-SepharoseAffinityChromatography (i,
2.14.Dye-BindingProtei nAssay (,7
vi
2.!5.SDS·l'o lyacrylamideGel Electrop ho resis 69
2.16.Coomasslc BlueGlelStaining 71
2.17.Autorndiography 71
2.!X. Western Blotting andVir usOverlay Prote in BioiAssay 72 2.1l).Chromntofocusi ngofEMCVirus Recepto rProteins 73
CHAPTER3,RESULTS
3.1. Kinet icsofAttachmentof EMCVirustoBela Cells 75 3.2. Biochemica lCha rac ter lzntlcnofEMCVirusReceptorson
K562 and11t:1..."1Cells 79
3.2.1.Effect of Protease andPhosph olipase C on
Attachmentof EMCVirus toIntactCells 82 3.2.2.Effectof Neura minidaseand Lecti ns on
At tachmentofEMC VirustoIntactCells 82
3.2.3. Effectof Trypsin andNeura minidaseonAttac hme nt ofEMCVirusto CellMembranePrep arat ion s 85 3.3.Effectof CycloheximideontheRegenera tio nofEMC Virus
Rece ptors onCells afte r TrypsinTreatment 86 3.4.Exa minationofAf fi nity ChromntogrnphyConditio ns 94
3.4.1. Det ergent Concent ration 94
.1 4.2.NnC IConcentration 98
35.lsclatl on of Putati ve EMCVirus Rece ptor Proteinsby EMC Virus-Scpharose Affini tyChroma tography 102 3.6.Molecular Weight s of Purified EMCVirus Receptor Prot ein s 105 3.7.Specific Bind ingofEMCVirusto AffinityPurifiedReceptors 108 J.x.AffinityChronmtogm pliyof Surface Labeled CellMembranes 111 3.1).Determina tionof Recepto rActivityin the Affin ity
Purified Materia l 117
.llll.BiochemicnlAnalysisof SolubilizedEMC Virus Rece pt orby
AffinityChromatogra phy 117
:'.1I.Ana lysisofPurifiedRecept orProteinsbySOS-PAGEAfter
Prote aseDigestion 123
3.12.lsocle ct ric PointsofPurif ied EMC VirusReceptors 123 3.13 .Western -Blot Analysisof EMCVirusReceptor Purified from
K562Cells withAnti-Glycoph orin AAntibody 127
vii
CHAPTER 4,DISCUSSION
4.1.Kineti cs ofEMCVirusAtt ach ment 1.l4
4.2.Bioche mical Nat ureof theRecept or forEMC Virus 1.17 4.3. Properties ofEMCVirus-Se pharoscColumns 140 4.4. Properties ofEMC Virus Rece ptor PurifiedbyAffinity
Chromat ograph y 14.1
4.5.Suggest ions forFurtherStudies ISO
4.6.Summary andConclusio n 152
REFERENCES 154
APPENDIX 174
viii
Usl of Tahles
Table I. Effectof enzymesandlecttnson EMC virusbinding to K562 and Hel ...1cells.
Table2. Bindingof EMC virus toK562 and HeLa cellmemb ran e preparationsinthe presenceof6 roMDOC.
TableJ. Elution of3H. lnbelcd EMCvirusbound to K562 and He l.a cell me mbranesbyvaryingconcentrations of Na'Cl.
ix
Listor Figures
Figure 1. Attachmentof EMC virustoHe1...,cells.
Figure 2 Saturation of EMCvirus receptors011BeL1cells.
Figure 3. Effectoftrypsin and neuramlnidnse digcstlon011bindingof EMC vir ustoKS62andHe1...,cell membranes.
Figure 4. TimecourseofEMC virusreceptorsynthesisinK5(.2and IlcL.1 cells.
Figure5. Inhibition ofEMC virus receptor synthesisbycycloheximideill K562and HeL.1cells.
Figure6. Evaluation of EMC virus receptor activity in sodium deoxycholat e-solubilized cell membran es of K562 and J-IeL, cells.
Figure7. EMCvirus-Sepharoseaffinitychromatographyofsolubilized cell membranes.
Figure8. Molecularweightdetermination ofEMCvirusreceptor purified fromK562and HeLacells.
Figure 9. Binding of EMC virustoreceptorproteinspuri1icd fromK.'i(12 and HeLa cellsinavirus overlayproteinbioiassay.
Figure10. EMC virus-Sepharoseaffinity chro malogra phyo fce ll mem bra nes derived fromsurface-labeledK562andHel.acells orK562cell Dclone.
Figure11. SDS-PAGEorEMCvirusreceptorprote inspnriflcdfrom surfacc labe ledKS62nndHel,acells.
Figure12. Determinationofreceptoractivity inEMCvirusrece ptor prepa rat ionspurifiedfromK562andHel.acell membran es.
Figure 13. Effect of trypsinand neuram inidase digestionon the binding of K562and Hel,a cell membr an estoEMC vlrus-Sepha rosccolumn.
Figure14. SDS-PAGEofEMCvirusrecepto rprote inspurifiedfromK562 andIleLacellsafterpro teolyticdigestion.
FigureI.~ . Chromatofo cusingofEMevirusreceptor proteinspurified from K.'i62and IIcLacells.
FigllreHi. Anrt-glycopho rlnA antibodyreac tions in Wester nblotanalysis ofI~MCvirus receptor purifiedfrom K562 cells.
xi
BSA CRS CRU DMEM DOC
List or Abbreviation and Symbols Used
Bovine serumalbumin Cellular rece ptor site Cellularreceptorunit Dulbecco's modified eaglemedi um
Sodiumdeoxycholate EMCvirus
EDTA HBSS ICAM-l
Iodogen kD
Kd LFA-I LPA MIT
PBS pi PMSF RPMI medium
Encephalo myocarditisvirus Ethylenedla mtnetetraaceticacid Hanks'balancedsaltsolution Intercellular adhesionmolecule-I
1,3,4,6-tetrachloro-3a.6o:-<iiphcnylglycoluril Kilodalton
Equilibriumdissociationconstant
Lymphocytefunctio n- associa ted muigcu- I Limuluspolyphemus agglutinin
3-(4, 5 -dimethyl t hiazol-2 -y l)-2,5·
diphenyltetrazollumbromide Phosphatebuffered saline
Isoele ctr icpoint Phenylmethylsulfonyl fluoride Roswell ParkMemo r ialInst it ute mediu m
xii
SDS-PAGE
VAP
VO!'BA WOh
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Viral attachment protein
Virusoverlay proteinblotassay Wheat germagglutinin
xiii
Chapter 1 Introduction
1.1.Gener alBackg round
Perh ap s,Bcijcrtn ck(IH9H)W;"ISthefirstpersontolise1111:term"virus"III defln eakindofinfe ctiousage ntwhich canpassthro ughaboctcrin -proor fllrc r andretain infectivity.Howe ver ,several yearsbeforeBcijcrin ck'sdefinition ofn "vir us", asimllurpheno menonhadalre ady beenobservedhytheRussian biologist,lwanowskl (Dlnnnock&Primrose,!lJX7).
Viruses have been foundin every living creat ure, fromsingle-celled bacteria10mauuuals. Vimlnucleic acids arceitherDNAOfRNAbut1101 both. Viralnucleicncldscontain:'111 theIICCCS.~lIYgenetic infonuntionfur vim!re plica tion.Tbcviralcopsld is madeup ofruutiplcidenticalvimlprot cinscalledcnpsomcrcs.The viralcapsidencloseseitherauexte ndedorn condensed nucleic atid core toform a basic viralstructurecalled:I
nucleocapsid. Exce ptfor nfew virusInmlllcs,the com pleteinfectiousvirus part icle,orvirion, ha salipid-containing mcmbrnuccalled all"env elope"
enclosingitsnucleocapsid . '111e virusacquir esitsenvelopebybudding through the Iiml's cellula rrncrnbranc. The envelo peismodifiedbythe insertion of viral glycopro lci nsinto the cellmcmbnmcduring virion maturation.
The oldestclassifica tionsche me fo r viruses isbased on the clinic al syurptomxnudhistopa th ologicalcharacteristics ofthedisease s.Althoughthis oldschemeaidsthe cliniciansindealingwiththesympto msandepide miology ofviraldb-cases, itis no longersat isfactory becau se ma ny biologically unrelatedvirusescancause indistinguishable sympto msininfectedhosts.
Currently,viruses nrcclassified according tothesber neproposedbythe
"lntcnuulona lC0111Jl1iul'c011Taxono myofViruse s". Accordingtothis 1;1.~J10rny,nnimnl viruse saredividedinto 17families onthebasis ofthenatu re of thegenomicnucleicacid(R NAorDNA ).modeofviralreplicat ionand structureofthe virion(which re fers10 size,shape, symmetryofthe capsid, undpresen ceornoscuccofan envelope , etc. ). Clnsslflcat tons ofvir uses withinthefa milyintogener aandthenspecies,typesand stra insareusually hased011physico-chemi cal orserologica l differences.
Bydefinition.virusesca nno tberegardedasmicroor ga nisms,becau sethe y multiplyonlywithin living cells,whichsupplyall theene rgy,rep rod uctive machineryandmolecula r precursors. Ingene ral, thevirusre plicatio ncycl e Includesattachme nt.pencuntiou,unconttn g, transcript ion,re plicat ionof vira l n\lrlek adds.uunslation.assem bly nudre leaseofne w virions.
t\l;u I)'viruses. butnetall,canbe growninculturedcellsorfertileeggs.
The~rnwt hofvirusesincult uredcellsorferti le eggscan be easily detected
bynvarietyof methods. Observationofcyropar hiceffects in culturedcells nnddeathoftheembryo,additionof anindicato r virusto detectsomenon- cyto pathogenicviruses,and hcumggtnttnatlon:lSS;'YSwilldetectmost viruses.
Virusescanbe qunutunrcdbydirectcountingoftheviral pnrtictcsbyelec tron microscopy or marcoftenbybiological tltratfonssuchasplaquenudpock nssnys, andbyImmuuoassnys employing antibodiesspecific toviraluutigca s.
1.2. PlccmnviruscsandEnccplmlomyocarditis (E MC) Virus
Picomnviruscsnrcthesmallest RNA viruses. 2(1-30milin dlnmcrcr.
Nouenvclopc d andIcosahedral,thevirioncomnlnsIIpositivesiuglc-sunudcd RNAgenome(Jackson,I98fi ;LIIO,]tJH7;ellen~'1 1l1.,l tJX9).Thevim!capsid is composedof60capsom crcs, eachcapsomerecontaining4polypeptid es VPI,VP1, VPJ andVP4,whichfife derivedfro m:1singleprecursor VPO (Pnlmcnbcrg&Rueckert, IlJI)2). ThelinearviralRNAwith a molecular weight
o r
about 2x11I(1dnltons,is polyadcnylntcdatthe 3'-tenllinus:1I1d contains a smallprotein (Vpgj nt the5'-terminus(AnnstrougcIIII.,1972;Yogo&Wimmer,]972:LeetHaI.,1977;Finnegan(!/al.,11)77).Theinfectious viralRNAcauactas:111IIInNAandhinddirectlyInribosomes furprotein rrunslaticnwithouta prioruunscriptlona!step.Therenrcfourgenerain the
plcurunvhusfa mily:enterovirus, ca rdlovirus. rhinovirus nod apht hovirus (Rueckert,IlJlJO),
Genus enterovirus includes polioviru ses,coxsacklevir uses,echovlr uses, humancnrcrovirus cs and hepa tit isAvir us, Theviruses inthis genusare Inrporunuluuunnpathogens(R uec kert,IWO).
Cardioviruscsinclude ColumbiaSKvirus,cncc plmlomyocarditis(EMC) virus. M:IlI.~ Elberfel d virus, MM vir us, mengo virus and The iler's l!1It"l!ph:Jlolll}'c1it is virus(Ruecker t,1990). However, sincethe virus es within till'~...'lII ISnrc serologically indi stinguisha ble,theyarcveryoften consid ered
[11bestrainsofErl'!Cvirus(R ueckert ,19lJO), Rhinovirusesinclud ehuman rhiuoviruscs witIIover 113 scro typcs,andbovinerhinoviruses. Huma n rhlnoviruscs arc one ofthemajo retiologicage nts of thecommoncold (Ruec kert . ]lNO).
Apluhoviruscsindudesevera lscrotypcsofthefoot-and-mou thdisease virus. These virusesnrc important in theagricultura leconomy sincethey causeahighlyinfectiousdisease incloven-footedanima ls(Rueckert,19( 0).
IIIthisproject,Et\tCvirusha sbeenusedwhichwasiniti a llyfou ndinmice dlll'illgthe investigationofpoliomyelitis ill IlJ43(Jungcblut&Dalldorf1(43).
:\h h\lll~hthevirusis generallyregar ded asnmurinevirus, it has been dl'lcl'lt.'d ill ma nyothe r speciesinclud ingswine, rhe su smonk eys, wild
mongoose,mosquitoes, andhumans (Adami~'\:.Littlcjuhns.11)7:-\;Denvtul., 199 1;Dick(,II/f"194H;Smit hburn,1948;Gnjdu sek,1955). IIImice. the virus causesseve reence phalit is and myocar ditis(D ick.19..j.l);war ren, 1965), whereasin human s.theinfe ctionnppcarsasa mildfebrileillnesswithcent ral nerv ous syste minvolvement(Warrell.1%5). The virusisan impor unu pnthcgc u,sinceite;ll ISCSvnrtousdiseasesinlabomroryanimals.thatmimic severalvirus-induc ed human diseases,inc\utlill&iusulln-dcpcndcn t~liahetes mellitus (Crutghc nd&Mel.nne, 1%8),vasculitis (Burch&Rayburn,11)77).
myocarditis (Gainer, 1974 ) andpolymyositis(Millert'lflf.•IlJK7).
In most cases.nsforothe rpicornnviruscs,replicat ion of EMCvirusin culture dcellswilleventuallylead tothe releaseof newvirion sfromlysed cells.There arcse veral EMCvaria n ts. amon g the m,the Dvarinut ofthevirus ca usesallInsufin -dcpcnd cu t dia bet esmellitus(ID DM )- Iike synd rome ill suscept ible mice.Thisvar iant wnsisolatedtogether witha uon-dinbctogcnic B varia nt (Yoon('II/ f.,11)77.Yoon &Not kius, IlJH3) from:I poolofa myocardiot ropicMvartnnt(Craighead,1% 6). Forthisproject,anoriginal virusstrain(K2)isolat edfro m infectedmicewasused(Hoskins&Sand e rs,
Il)S7 ).
(,
1.3.Viru s-CellInteract io n
'lhcCUllSC(IUClll'CSofviral infectionol ncell arc variousand the ydepend
OilIhe characteroftheInvadingvirus andthetypeof targetedcell.Alytic
infectionrcsullsilllysisund deathofthe infec tedcell.In anonlyticinfection , :1pcrsistcm inrcctloullIay occurinwhichtheinfectedcellsmaysurvivean d produce progeny virus;Itlow levelsora celll11:1ybecomemalignant.
However,10initiateinfectionof acell,thevirusmustattac h to its receptor
011Ihecell (Dales,1<)73;Lonbc rg-Hol m&Philipson , 1974;Lonberg-Holm, 11JH!;'Iurdlcu&weiner,IWl2;Marsh&Ilclcnfus,19H9; Lentz 19(0). There
:11"1,.'twoessent ial componentsinvolvedillthis initialinterac tion,thevir al al larlllw.::llI l'fIlh:ill (YA P )andthe hostcellular recepto r.TIle YAPis located uutheviralen velopefor en veloped viruses oron the capsidfor non- 1,."ll\'c1uped viruses,whichmedia tes theattac hm ent
or
thevirustothe cell...urracc.
Thercllularreceptoris ncomplcmcmnrystructureto YAP,towhich till'virusIlilllh..unlisculled thecellula rreceptorsite (CRS)(Lonbcrg-Holm, IlISI ),lIowcw r. bccnnsctheCRS isoftencomposedofmultipleunits,the rc nu cctlularreceptorunit(CR U) isused10 refertotheseunits (Lonberg- llolm.IIJSI), Virusescallbind ron cellularrecept ordirect lyor indirectly thnlll~h:1t1intermediatemolec ulewhic h mayhenssoclmc d with the cell surtacc. ;\lllihn dk':;1I.I\'c beenindcnriflcdasinte rmedia te moleculesfornuacluuc utofseveral virusestocells (Webster&Askonus. 1~)HO;l'c irist'l III.•
19tH;Mcrfulre1.'11/1.,19H6). Poiyalbumiuandthereceptorforpolymerized human albuminappare ntlyfacilitatesthebindingof hepatitisB virus to hcpatocytcs (Irnni1:1ul.,1979;Thuug.'\;, Gerber,19H4). Virus cellular receptor scallbeallyhostcellsurface moleculeoreven:Iviralprotein produc edhya previousvirusinfection. IIW:1Sreported thaiinfe ct ion of hepatitisdeltavirusreq uire s thepresenceoran ongoing tnrccuonwit h he patitis U virus (Rizzcuo d "I., 11)911), and cellsu rfncc iufhrcnzn hemagglu tininmediatesinfectionof other viruses10cells{F uller('Iul.,IIJHS).
Following theinteractionbetween VAl' anditsreceptor , virus entry into thecytoplasm is usuallybydirectfusion wit htheplasmame mbran eor by receptor-mediat edendocytosis(Marsh&llclcnlus ,IIJ!ltJ). Inthecase of piccnm vl ruscs, the pen etration into hostcellsisbyreceptor-mediated endocytosis (Crowell&Landau. 1!JIB).
1.3.1.ViralAttachmen tProteins
Asmentioned before,viralattachment proteins arc locatedeitheron the viral envelopeor onthecapsid. Recently, seve ralsuch viral attachment
prut cfushave be endesc r ibed. For example. in reovirus.theV APhasbeen i,lelltific unsthc01prote in,the viralhc mnggtutlnln(Bnssel- Dubyetal.,1986).
The spcclfkityufthis protein forrec ognition ofcellular receptor s was demonstr atedby changingitsreceptor specificity bythereplaceme nt of gt ur.unlc ncid wit hlysine a1amin o acid419 all th eVAl'polypeptide(Bessel- Bllhy l'lat.•IlJI{(I). Simila rly.a singleamino acidchangeinthe in fl uenzavirus lrcmaggluttuintedtoacha ngeillrecepto rspeci ficity (Pnufsonctul.•1986),
'I"'lesefindings1101onlyrevcnlthchighspecificityofVAPbut also suggest the Involvement(Ifsecondaryandter tiarystructuresin thedetermina tionof YAP slh:rilkily .InIlWllycases.VAP possesse sotherfuncti on s besid es bind ingto hostrclls. Furexam ple,influcu zn virushcmagglu rlnlnpa rticipatesinfusion all dviruspenetrationintothe ce ll(Whitectat.,19M);theYAPof vesicu lar srounuhls virus,asoleglycosylatc dspikeprotein on the virussurface. can Function ;ISa hemolysin,hemngglutlni n ,and cytotoxin (Sch legel, 1986)., Al thuu,ghsomeviruses suchasiu fluenznvirus andIII V·Ihaveveryhigh mnt.niou r.ucs.thehimli ng domains of VAP nppcnrtobeconse rvedaslong astill'virusesllla intainthesame cclltro plsm (Laskyaat.,1987 ; Weisetal.
tl}~S ).Theconser vednatureofthe bindlngdomalnsofYAPprovides a good la Q:.etfi ll"intervcutioninviralinfe ction.
Studies orplcorunvlrusVAPsresultedin unimpo rtanthypothesis. The researchers inthisField were perplexedbytheIncttll:lt severa l VAl'shad beeniden tifiedinother viruses.butnot forplcom aviruscs.However. hyusing Xvrnycrys tallogro phynudnuctc!c acid seq uence :lll;llysis.:1deep "canyon"or
"pit"W:ISfoundinthe viralcapsid(Rossmann("at.,I()M;;lloglc("et.,19M; ; Luo('/at.,l1JM7:Jenkins ("I/f.•19M?)."11 1Cdeepnessand narrownessofthe
"c,II1YOI\"UJ'"pit"nuikcsncccssof 'lIl lihu dicstothe floor ufthe'cnuyou"
Impossible.Thenmlucaddslillingthe surface(If the"canyon"showgrc atcl cons ervat ionthan those atthe surfncc.ondallowviruses10rcm!n their re ce ptor specificity,whileat the sa me timepermit viruses 10 nvokt thehost's Immune syslcmbymutation orre sid ues about the"C:lllYOII"rim (Rossmnuu~~
Palmcnbc rg.ItlXX).The"canyon"hypot bcslscxptalns scvcrul(aile d «ncmpu toisolate picornaviru sreceptorsbyusingnnti-idictyplc antibodies. Howe ver, unlike othe rpicomnviruscs.the foot-an d-mouth diseusevirus receptor nunclnncntsiteuppcnrs10beaproject ing"loop"011its capsidinsteadof a
"cnuyon"or"pit"(Achary:l('I//1.,19S1J).Therefore ,the Failure to isolatethe receptorfo r thefoot-and-mouthdisease virusnppcursfloltobe explainedby thismecha nism(sec svctlon1.5).
lO
1-1.2. l'lnsruaMembranes andCell u larRecept ors forViruses 13.2.1.Str uctureandComponentsofthePLumn Membr ane
III lIlus t("'ISCS,virusreceptorsnrc cellularmoleculeslocated on the sur face orIhe ,,'as ma membrane. The ptasmn membrane consists ofthree ouupoucms,lipids,protein sandcarbobyd rntesthat form3Fluid st ructu r e.
The re:11"1,'three major classes oflipids in theplasmamembrane:
1,llllSlll llIJi 'l il ls,l'!ltl!cstcl'ulsnudglyc o lipids. Thenmphlpa thlc pro per tiesof theselipidscunblc them10formn contin uousbltnycrcdstructure ,the basic srtuvturcofthe rncm bmnc.
I'IaSl1l:1mcurluuucproteins nrcembeddedin thelipid bilayerorboundto carhother. Mciubrnuc proteinspinymajo rrolesinactive transpor t,cell ular
Si~I I;llli l1f.mcmbrunc-ussockucd enzymat iccatalysis,etc. Many membrane
pnuctusarctermedrrnusmc mbmncproteinsbcC.1USCtheirpolypeptidech a ins passthrough thelipid bilayer.MembraneproteinsCo1"alsobind tothelipid hilap..'I"hycovalcmbo ndswithone sideorthelipidbilayer orbynon-covalent tll,1I1ds with theuunsmcmbrnneproteins. TIle transmembra n e prote ins IIl~c ll l l'r\\"ilhollie!"covalentlyboundmcmbrnucproteinscanbereleasedfrom membraneshydisrupting thelipid bilayer withdete rgents. Thesemembrane prcrclnsarco(ICIlreferred 10 as in tegral membra neproteins. TIleno n- owalcnuyhoumlplnsmamembmneproteinscallbe easilyreleasedundermild
II
conditions, suchas changesin ionicstreng thor piI, without disrupting the lipid bilayer . These plasma memb rane protein s arc termed pcrlphcrul IIlcl~lbrnnepro teins(Karp,1984)
Carbohydrmesnrc presenton eukaryoriccellsurfacesand arccovalently boundto membraneproteins orlip ids.Increasing evidence shows lhalthe carbohydrate groupsall glyccprotcius playlmportnnt roles ill V:lriOUS biological activitiesof theplasmamernbrauc,suchastheinte ra ction of ligand- cellula r recep tors,proteintargeting, cell-ce llIutcmctlons(Paulson ,IlJK9).In addit io n, most cukaryctlc cellshave a cnrbohydnuc-richzone atthe cell surfac e,calle dtheglycocalyx (Kar p,1984).'111eblologlcnlfunction ofthe carbo hydra t esillthe glycocalyxhas notyetbeenelucidated ,However,thei r complexitynudloca tion onthe cellsurface suggesttluuthey may playSUllie roleincell-cell nndcell-ma trixintcmcrio ns (KMp,ItJX4 ).
1.3.2.2 Cell ularRece ptorsfortheVirus
Obvi ously,cellula rrece p to rsarc not synthesized forthe purp usc of providing receptors(orvir usbinding. Allthree majorclass e s of plnsmn memb ranecomponents, proteins,lipidsandcarbohydnncshavebeen found to netasviru s recepto rs. Many of these cellularreceptorshave:1150other biologicalfunctions. Viruse s mayusc hormoneorncurotr nnsmiuc r recepto rs
12
as their cellu larreceptors. Forexample, reovirusutilizesthe~'lldrcllcrgic rcccp t,... \ Co1:1til"1985u),nndvacciniavirus usestheepide rmal growthfuctor rece pto rforbinding 10hostcells (Epp stclnct al.,]tJH5).Ma rcrecently,CD4, ICAM· ! and class I MHC molecules, all of which belong to the Immunoglobulin super family,weredefined ascellularreceptorsforhuma n immunodeficiency virus(1-!lV)(Dalgleish ctul.,1984;Klatznmnn('Iul.,1984), major scrotypcsof human rhinoviru s (G reve('Ial.,!9XlJ;Stnunron ('(al. ,Il)Hl);
Tomassinictet.,!lJSl)a )and simianvirus 40 (Breau ctat..•191)2), respectively.
Themembers of theimmunogfobullufumilyactively engageill immune responses. For example, endogenouslysynthesi zedforeignantigens such as viral pcptldcs canberecognizedby COM+cytotoxicTlymphocytes,onlywhe n theyarenssochucd withclassIMI-IC molecules.whereas CD4+helper T cells recognizethe exogenous antigens onlywhenthenurigcnsarc nssochuc dwith clnssIIMHCmolecules. Although, proteinsarcmostofte nfound tobevirus receptors,lipidsandcnrbolrydnucsalsoserveasvirus receptors, Sitch as phosph a tidylinosit olfor vesicularstomatitis virus(Mastrcmnrinoeral.,ItJH7);
sialyloligosncclm rldcs for scndaivirus(PaulsenC!/al.,11)71), Newcast le disease virus(Paulsonctal.,1979), potyonmvlrus (Friedetal.,ItJHI) nndinfluenza virus(Rogers, 191'6).
13 J.4.Obs taclesin VirusReceptor Isolation
Isolationofvir usreceptors has alwaysposed conslde rcbl edifficulties.The majorobstaclesinvir usrece ptorisolation are:
(I) Virusesmayattach 10 cellsnon-speclrlcnlly,sometimessuch non-spe cific uttnchmcutmayevenlend toinfecti on of cells,as obse rvedinthe bindingof vesicularstomat itisvirus 10 baby hamsterkidney cells (Baileyelal.,1984), ma kingitextremelydifficultto distinguishspecificbindingfrom non-specific billding.
(2) VirusesIllOlyutilize differentcellularrece ptors in differen tcell lines.This isthe('<IS\:intheattachmentofcoxsacklcvirus03 (RDvariant) toHel.acells amirbabdomyosnrcornacells {Rea ga n, 19K4). A virion may have morethan one recepto r binding dctc nnin nnt, end each determina ntcouldbindto differentcellularrece ptors, although thesedete rmina nts may belocated on lite samemoleculeofthc vir ion(Reaga net «I.,19X4;Tignoraal.,1984).
(.l)Weak;lmnilybetween viruses andtheir cellularreceptors.With afew cxccpttons,virus-recep torbends arcunsta ble in theprese nceofrela tivelylow ronrcntmtiousuf det e rgent required bymostbiochemicalpurification methodsto solubilizecellmembranes(Dales,1973;Knipe,1990).Thisfeatu re lllgc lhcrwith thesmallquantityofvirus receptorsmakesitdifficult to apply
14
most biochemicalmethods forthe pur poseofvir us re ceptorpurification , (4) ViruslllOl ybind\0int erm edi a temole cules, whichillturnbind tothe cell ns mentioned(section1.3.2.2). Thismakesiteven more diffi cult10idcuttfy nnd purify virus re cept o rs .
1.5.Methods of Isolationfind Chnmctcri zntionof VirusReceptors Despite thedifficultiesmentioned above, var ious meth odshavebeen successfullyusedto isolate andchnrnctcrlzcvirus rece ptors. Somesuccessful cases or isolatingvirus receptorstookndvamngc of a particularprop e rty of cellularreceptors or thevirus-recep to r interac tion s. For example, the receptorfor coxsnckicvirus011IIc L1cells hasbe en successfully isolatedby laking ndvamngc ofthe stability ofrecepto r-viruscomplexesillthepresenc e of non-ionic detergent. Usingdifferentia lsucrosegradien tcentrif ugatio n,11
conventionalvirus purification method, Mnpolcs andhilico-workers(19X() achievedgreaterthanlO4.fol<lpurificationofthe rece ptor .
Exrunlnlng thehistory of virus recep tor studies,oneseestha t numerous methods have beenuse d for isolatio nandcharac terizationoftherecept or s.
The meth ods describe dbelowhavebeenmost commonlyusedboth ill thepast andpresently.
15
(r)Physlco-chcmicnlmethodsinvir us receptoridentificatio n
Snturution andcompe titionbindingassaysnrctwo of the most commonly usedme th odsillthestudyof thephysicalparametersof virus attachment.
Thesemethodswereoriginallyusedinneuropharmaco logynnd endocrino logy.
Theynrc not ncccssnrlfysuitable for the studyofvirus-rece ptor interactions, because vir usesarclargeparticlescontainingmultiplecopies of YAP,and in ma lty cases thebinding ofviruses to cells isvirt uallyirreversible(Lonberg - llohu.llJSI;Turdicue/(/1.,19H2).Nevert he less, theseme thods have beenand stillarcbeing used inalmosteveryvirusreceptor study,andmerega rded as aIrumcworkfor the defin itionof avirus receptor(Ta rd ieuel al.,1982).
(2) Chemicalandenzymat ic modificationofvirusrecep tors.
Manychemicalandbiochemica lagentshavebeen usedto alter thevirus n:n'ptor1111plasma membranesillattempt stocharacte rize the biochemical natureorvir usreceptors. Inthisca tegory,enzymeshaveperhapscontributed themost10ourearly knowledge aboutthenatureofmoleculesinvolved in vir usbinding.Ascnrtyas1949,VcrtindcanddeBnanfoundthattreat ment or erythrocytes with neuraminidase,purifiedfromVibrio choterae,preven ted subse quent 1::t-.-t C vil'lls ind ucedhe magglut ina tion (Verflnde&de Baan, 1949).
16
for thisreason,neuraminidasewas called arece ptor-destroying enzyme (R O E).TIlesensitivity of therece p tor 10nc urnmiuidns c treatment suggested tha tsialic acidresidues, whichwerecleavedfrom thesinlylmcdreceptor molecules ,wereinvolved in virusnuachrncn t.Similarly,the eliminationof receptorsforgroup Bccxsnckicvhuscsfindrhinoviruscsbychy motrypsin trcntmcntrevealedthatthoserecepto rsnrc proteins.nudthaithe receptor polype ptides conta in one or mo re resid uesof tyrosine, phcuylnlantnc, uyptopbau. leucine , methi onine, nsparngin cand glutamine (Crowell &
1.audau,19H3).However,besidescleavingpolype p tidechains.treatmentof cellswithpretenses causes other ind irecteffectsOilcells,suchas changing thecellmembrnne conformationandincreasingprot ea se secretionfromcells (Spcl ketal.,1972;Werb&Aggcler,1978),therefore,extra cautionisneeded when interpretingthedata (Colonna,19K?).
(3)Chara cterizatio n nnd isolationof virusreceptorsusingspecificbiological liga nds.
Inthis ca tegory,lcctinshave been widelyusedfor virusrecepto rstud ies.
Lccuuswere origina lly isolatedfrom plantseeds.'111eyrecognizelindbindto specificsugar residuesonpolype p tidechains. For example,whea tgerm
17
agglutinin(WGA) recognizes andbindstoNca cctylgluccsamineorsialicacid residues,whereasConcnnnvnlinA bindstoe-mnnnosyl.Takingadvantageof the specificityof lectinbindingto sugarresid ues, aconside rableamountof inform ationabou t the role of carbohydra teson rece ptorsfor viruseshasbeen otnulnc d(section 1.6). Attempt shavenlsc beenmadeto isolate virus recept ors,includingEMC virusreceptors,byaffinity chromato graph y on lcctfu-columns(Pardoe""'" Burness,ItJHI;Mischak1:/at.,1988).
(.l)Isolationnndclmracrcrlzatlonofvirusreceptorsusingspecificanti- n.:n:llh1rnmibodics.
Till:ret'CIIIUTfurthemajo r groupofrhinoviruseswassuccessfullyisolated hyaffinitychro uuuograph y emp loyingmonoclon al anti-recep toranti body cotumu s (Tomasslui&Colonna, 19R6).
(5)lsulaliull
or
virusrece pto rsusingnrnl-Idicryptc nnttbod les.Thismethodis basedonIe rne'sinternalimagetheory(Jeme,1974)_
An :l11"lIillJ;tothistheory ,auti-idioty picantibod iesraisedagainstan antibody IIIthe viral unacluncntproteinmaymimic the biologicalpropertiesof theviral utuwtnuc utproteinandbind tothecellula rreceptor, An anti-id lotyplc
IN
antibody raised again stantibod iesto thereovirusnuocluu c m proteinW:15 successfully used10isolate the receptorfor reovirus(Co('/(II.• lIJSSa). Unfortunately, Ihis method is not consideredsuitable for isolationofthe receptorformostpicomaviruscs,including EMCvirus,:15their receptor bindingsites arclikelyto be embeddedinthe"ca nyon" audarephysic:l!ly lnacccsslble10antibodie s(sec section1.3.1).
(6)Transfcc uou of virusreceptorgenefromrece ptor-positive\0receptor - negativecells.
Usingthisrcclmiquc. the gene for poliovirusreceptorhas beensuccessfully iden tified (Mendelsohnct al.•19M; MendelsohnetIlJ.,IlJX9 ).However, since EMCvirus infectsthe cell lines (romnmny differentspecies.the techn ique pro bably cnl11101beapplied for cloning the EMCvirusrece ptor gene.
1.6. Cctlolnr Recepto rsforPicornaviruscs
UJ.!.GcncmlPropcnics of Recept orsforPicomnvituscs
Early physico-chcm icnlst udiesalltheuttuchmcntofplco rnavlruscsto cells reveale d thateach host cellbindsabo u t 104toll~virio ns(Lon bcrg-Hohnel al.,I976a).and thatse veralphys icaland chemicalfactors suchastcmp cmtu rc,
19
I'll, ionic strengt h IIIld cell concentrationaffect virusnunchment (Lonberg- llulm, llJXI). Furexample,the ntmchmcut of EMC virus toHe La-S3 cells W;ISrepo ned10betempe ratureindependentover therange
au
to400C,whereas theaunchmcntof thesamevirustoL-929 murine cells was progressivelyreducedwith increasingtemp eratu re over thesametem pe rature wlt£e(McClintockt'ltil.•19S0). The at tachmentof plcoma vir uses to cells dues nuloccurillthe absence of catlous(Hollandl.'XMcl.aren,1959), and divalcurcations,(;;12+orMg2+,nrcrequiredforattachme ntbyseveral picorunviruscs.includingrhinoviruscs [Finln&Kenn y,1967;Lonbe rg-Holm
&Whitele y.JIJ7fl),two scrotypcs ofcoxsackicvir us A (Mcl.nren ctat.,1960;
("ortis('/al..lin .' )andIoor -nnd-mourhdiseasevirus(Brownf!lal, 1962).
B;lscl! on virus competition stud ies, itwas discoveredthatalthoughthere lire morethan1211 scrorypcs ofhuma nrhinovirus,90%ofthem(the majorgro up) sha rea singlecellular recept or (Abraham& Colonna,1984),whilethe remainingsc rotypcs (theminorgroup) withoneexcept ion,compete for a scccuulrece ptor (Colonuo("nl.,1986; Uncapherel(1/.,19(1).Similar studies onFoot-and-mouthdisease virusshowedIhatsixscrotypesof thevirussha re
.. l"l,111111l0 11receptor , which isdistinct from the recept orforpolioviru s and the
recept or fur Et\ICvirus (Sckiguc hlf:/(/1., 19X2). The discove ry that
zu
picorna virus receptorsnrcdividedintogroups suggeststhat thebinding domainsin the VAl'amongmanydifferentscrotypcsof virusesarchighly conserved (Colon na,J~S7).Thesefindingsraiseanew therapeuticposslbtltty
(0 block virusinfectionbyblockingvirusnrtuchmcm toits cell surface receptors.
Ea rly informatio n abo ut the blocbcmlcu! nnurrc of a receptor I'Of plcomavlruscswasobta ine d mainlybytrcrumcntof cells or cellularplasma membraneswithvariousenzymesandchemicalreagents,undthcuexamining theeffectsof such treatmentallvirusattachment. Inmost cases,itnppcnrcd that the recep tors forpicornnviruscsarcglycoprotcin slindtheyarclocated 011rue plusma membranes(Lonbcrg-Hclm&Crowell,llJ1'i();COIOIl110, )91'17).
Furt herbioche micalchnractcr iznrlcnof thevirusreceptorsre vealedthatsome carbohydrategroupsaunchcdto theproteinpolypeptide chainsnrciutcgtul componentsofthevirus receptor. ConcnuvnlinA,nlectin,which hindsto componentsonncellsurfacecontaininge-Dcnumnopyra uosyl-Hkcresid ues, inhibits attachmentand infe ctionofboth rhinovirustype-2nudpoliovir ustypc 2tollc l.n cells (Lonberg-Holm,1( 75). Snnilnrly, whentgermagglutinin, bindingtocompo ne ntscoutniniu g Nnctylglucosautincor sialicacidresidues , preventsthebinding ofrhinovirustype15 tohostcells(Colonuo,19H7).
These resultssuggeststhatthe sugunrresidues, whichthelcctlns recognize
21
and bind to, arc involvedilltheattachmentofviruses tothe receptors.
Knowledge aboutreceptorsforpicornavir uscshasrapidly expandedsince thedevelopment of monoclonal antibody and recombinan tDNAtechnologies.
Thesetechn iques led to theisolation and identifica tion of two receptors for picornavirusfamily.therecept orfor poliovirus andthe recepto r forthe rhinovir us.
1.6.2. Recept orforPoliovirus
Poliovirusis nne of the mostextensivelystudiedplcomavlruses.Asearly asIIJ.'iIJ,itwasnlreudyfoundthnt poliovirus rcp llcntesDillyin pri ma te cell lilies(Mcl.nrcnCII/I.,1951).Late rstud ies de monstratedthat onlycelldebri s derivedFrompoliovirus-susce pt ibleprimatecel lscaninhibit infectivity of poliovirus(Holland&Mcl.nren ,1959 ),and incculatlcn of purified poliovirus RNAtounturcresistantcell linesfrom speciesothertha npri matescan produce onlyone cycle of re plicat ionin those ce lls(DeSomerelal.,1959;
llollnnd ("III"11J:'i9).Thesefindings reveale d thatresista ncetopolioviru s lntccrlonillnon-prinuuc cells lmppeus.1\thelevel ofvirusentry ra t he r tha n .utheinunccttularlevel.
Thefirstuucmp r\0searchfortherece ptorforpoliovirusbyagenet ic
22
appro a ch was accomplishedbyMillerand hisco-workers(197-1). An:llyting the chromosome complements oflunuan/ mcusc hybrid cells linesby chromosome ba nding methods,theyconcludedthatthe poliov irus receptor gene is carriedbyhumanchromos o me19. lnrcrcstln gly,altho ughscvcrnt mon oclonalami-re cep tornuubodles wereprodu cedbydiffere ntgroups(e.g.•
Minoraat.,I~R";Nobisef(/1.,19H5 ;She pleyctat.,!9HH),itwnsthe genetic npprcnch,thaifinally led totheisolationof therece ptorfor poliovirus.By usingDNAuunslccticn ,the genefor thepoliovirusrecept or wassuccessfully transf erredfrontthe virus-susceptiblehuma nlIe l.a celltothenons uscc ptlblc mouseLcell.nudsubsequen tlythereceptor gen eW: IScloned(Mendelsohn('I (/1.•1% 6;McndelsohnctClI.,19N9).ThepoIYPCI)liJcencodedbythcrcccpt ur gene is atmu smcmb rnncprote inwith threehomologou sImmunoglobulin -like domains,whichgroupsthemo lec uleintothelnnnuuoglobutlnsuperfamily (Me nd e lsoh netel.,1%'tJ).Theno rma l blolo glcnlfunction
o r
this unide ntified me mberof thcimmunoglobulin superfamilylmsnot yetbeendetermined.1.6.3.Rec ept orfurRhinovlruscs
Itiswellknownthat rhincviruscs initia teinfection ofcellsbynunchlngto nspecificcellularreceptor(HnffetClI.,IWM't:srou&.Hea th,11J70;'1l101Il:lSet
23
til.•ICJ7lJ). However,formany yea rs,ourknowled ge about the nature of
recep torsforrhinoviruscswereverylimited.Weknew thatreceptorsappear tobe gfycopr ct cins sincepretreatmentofcellswithtryps inorConcanavalin Ainhi bitssubse que ntvirataunchment to cells(Slott&Heath, 1970; Lcnberg- llohn,11)75). Furtherstudies011thenature oftherecep torforrhinovirus, whichreq uired the purified receptor. wereham peredbythe scarcityof rcrcpt nrs on cells.llowc vc r,successin genera tingmonoclonalanti-recept or nntihodics pavedthe,':ayforsubsequentstud iesof thisvirusrecepto r,such as recept oriso lation.bioche micalcharactcrizntion,nmlnoacidsequencing,and ("hllJillgthereceptor gene.
Thelin;' success fulprod uctionof monoclonalantibodies10 cellular recept orsfurIhemajor grouprhinoviruscswereachievedbyimmunizing miee withwholehumanllct,ncells(Colonnoelui.,19H6).The specificity ofthe .unl-rcccproramibodicswasprovedby:(I) Abilityof theami-re cept or :lIllihmlksInblockattachme ntandinfectionofHeL1 cellsbymajo rgroup rhmovir uscs lindgroup A ccxsnckicviruses,but notbyoth erviruses, such as minorgrn up rbiuovir uscs,poliovirus,gro up Dcoxsnckievirusesorhe patit isA virus(Colon no('//II.•1lJl{().Theseresult s confirmed result s fro m previou s studiesimlicntingthatthemajorgrouprhinov iruscs sharethe same receptor
24
withgroup A co....sackicvir uses, but not otherplcomnviruscs(Lonbcrg-llolm e(al.,1976a;Abraham&Colonna, 19H4). (2)The nntl.rcccptorantibo dies au ach to a widevarie ty of huma n andchimpanzeecells,bUInotto cellsof othe r species(COIOllllOe(al.,19H6). lt is know n that the majo rgroup rhinoviruses only infecthuma ns andchlmpnnzccs(Dick, It)M!;Pinto&lIuff, ]t)6l).Th us,anii-rece ptor antibodies showed iden ticalhost spcclflcityasthnt displayedby the majorgrouprhlnovhuscs (CU10llll Octat.,11)1\6).
Subsequently,themonoclonalanti-receptorantibodieswereusedtoisolat e therh inovirusreceptor.Acellularrecep tor proteinwithmolecular weightof 90 kDwasisolat ed fromdetergent solubilizedIle La cell membranesbygel Iihratlon, foll owed by monoclonal nmi-rcccp tcr antibody nfflulty chro nmtogrn phy,Thepolyclonalantiserum,prepared inrabbits iuuuunizcd with purified putat ivereceptorprote in,inhibi tedinfectio norsusce ptiblecells bythemajorgroup rbinoviruscsandgroup A coxsnckicviruscs,butuot othe r viruses (Tcmnssinl&Cclonuo,19H6).This resultconflnncd that thel)(J· kD proteinwasthc majorgroup rhinovirus recept or, or atleast thefunct ional comp on ent oftherecep tor.Furtherbiochemic alclmructcrbmlon ofthis 90·
kD polypep tidereveal edtha t ollgosaccharldcs cons titute30%of itsmolecu la r mass.SevenN-linkcdglycosylatlon sites were detectedbypartial re moval of
25
ollgcsacc haridcs fromthepolyp e ptidewith Ncglycana se (fomassinielaL, I(M"',).
L:I(Cr.studies fromthre e ind epe ndent groupsreveal ed thatthe receptor formajo rgroup rtunoviru scsisth einte r cellular adhesionmolecule-I(ICAM- I). rCAM·' is:1member ofthe immunoglobulinsuperfamilywit hfive immunoglobufln-llkcdomains(D ustinf.'lIII.,19M;Stauntonetal.,1988j Shnmous('I
at.,
IlJXI'i) find serves3S acell adhesion moleculefor thelymphoc yt e Iuncricn-asso chucdnmlgcn-J(LPA - I)(Ma kgoba etaI.,1988).
One!!l"UUjlor inve s tigat ors showe d thatthe urnlnoacid se quence s ofre c e ptor polypeptid es nnd,therCA M·'molecule.and thenucleo tide sequencesof a
d)NA clone oftherecepto r nud theeDNA cloneofIC A M·I were identical
«ircwrtil..IIJS9 ).At the sametime.anothergroupshowedthatthemajor
~l"tlllPrhinoviruscsbind specifica lly tobothpur ifiedIC AM -Imolecules and to!CAM-I moleculesexp ressed on cellstmusfccr ed withtheICAM-l gene (Stu muon('f1/1,.!CJXIJ),Thesameconcl usion wasreach edby anothergroup whichfi rs tisolate dIh~re ceptor for the majorgrouprhino virusbymonoclonal anti-receptorant ibodies(Tcmnssini& Colcnno,19H6).Theirstudiesalso showed tha itheclonedmajor gro up rhinovirusrecept or <.:DNA encod es a prou-inwitha se quenc e nearly Ide nticn1tothnt ofICAM -l(Tomass inieral:
26
19S9a). Thus,it iswellestabli shedthat the receptor for themajor gro llp rhlnovirusesis teAM-I,a member oftheimmunoglo bulin supe rfa mily.
Recently,thebindingdomainoftheICAM·!mo lecul efor the major group rhinovirushasbee nextensivelystudied. Itwasre ported thatthe bindingof these vir uses to ICAM·! wasblocked only byICA M· ! monoc!ona l antibo dies, whic h can blockICAM-l·L FA-1 interaction ,but notbyantlbodlcs to theother epu opesonthe ICAM·I molecule(S ta untonet al.,19HIJ). Thisfinding suggestedthat bindin gsites onICA M· Ifor themajor grouprhinov irusarc proximaloridenticnl(0tha tof LFA·I(Suunuouct
at.,
19MIJ). Furthermore, by employ ing huumn-murlneICA M-lchimeras withsinglenndmult iple amino acid mutagenesis ,aswellasmonoclona lantibodycpitopc ma pp ing. the primarybinding site forthemajorgroup rhlnovlrus csW'I Slocalized to the donwin I of ICAM· 1.Cert ainamino adds locatedonthe Nucrmlunldomnln appea r cri tical forvirus binding(Stauntont:l(II.,11)')0; Lineberger ctal.,19'J{);Register ct1/1.,1991).Howe ver,expressionofdcmnin Iand domain2nppe:lr tobedependentoneach other ,and the presenceof domains3,4 nnd5 signiflcmulyaffec ts the accessibilityof the bind ing siteonthe ICA M·]
molec ulefor themajor group rhlnoviruses(Stauntonctat.,]I)IJO).Aunlysisof thedoma inI ofICA M- ]bycomputermodelling suggestedthat halfofthe N·
27
n-munulofdoma inJcan fitinto the"canyon"on thevirionsurface and internetwithuminoacid slocat edatthewallor floo r ofthe "canyon" (Giranda a(/1" J9IJ(J). Again, thisfindingsuppo rts theinvolv eme ntofthe"ca nyon" in virus- rece ptorinteracti on.Itis expe ctedtha tsuccessinde terminingtheco- crysta lstruc tureofthe domain 1ofteAM·!andthemajor gro uprhinovi rus willprovidemo reinfo rmation aboutthemechanismof virusattachment.
IIisinterestingthntseve raleth e rviruses also usetheimmu noglobulin superfamilyproteinsastheirreceptor or as nnecessarycomponentofthe ir recept ors. These include CD4for the human immunodeficiency virus (Dalgleish ('I III., I~H4; Klntzmanu et al., 1984), an unident ifie d inuuuno glo buliusup erfamily prot einforpoliovirus (Mendelso hnf!lal.,1989), andMI lecla ssI pro teinis an integralcom pon entof the re ceptorfor simian virus-10(Bre au1.'1al..•19Y2). Theblclogtcalslgnff'icanceof the re cep torsfor these virusesheing me mbers of theiuuuunoglobu llnsupe rfamilyremains unclear.IsIhis pheno menonacoincid ence , or n reflectio nofauniqueroleof there ce pto rsinvirus -cellinte ra ction ?Onehypoth es is suggeststhat the use
or
lCAM·1asthe rhinovirus rece pto raidsthespreadof viruswith inthe host (Staunt onct11/.•[1)1':9). '111issuggestionwa sbasedallthe facttha t ICAM- l surfaceexpression ishighlyinducedbylymphoklnc sandmonok ines, whichcan2M
be stimulatedbyvim!infection (Stauntone ul.,19H9).
1.6.4. Recept orsforOther Picomaviruscs
In contrastto the relat ively wellcharacte rized rece ptorsforpoliovirusand the majorgro uprhinovi rus,on ly limited informat ionabout receptorsfor other piccmaviruscs is nvnilnblc. Aputativereceptor fo r groupBcoxsac klcvirus waspurifiedbytaking ndvantn gc ofthefact thatthevirus- recept orcomplex is stableafterdetergent ext ra ction(Kmh&Crowell, 19H5;Mnpolcs ctal., 1( 85).The purified receptorpolypeptidewith molecularweight nho utS f kD wasused as an immun ogen10producepolyclounl and monocl onalantibod ies (Hsuet(/1.,1981'1).Both types of antibodiesprevente d infection ofJleLa ce lls byallsix scrotypcsofgroupDcoxsackicvhus cs(lls uetul. ,J9HH).Aminoacid sequencingof the rec e ptor polypeptid eand isolationofthe cDNA encoding thepolypeptide havenot yetbeenrepor ted.
The stat usof stud ieson thereceptor for minorgrou p rhinoviruscsis at:I very similarstagens forthe group 13coxsnc kicvir uscs. A putati ve vir us receptorwithmolecula rweigh t about450 kDwas dete ctedafte rthereceptor 's activitywas enriche d bylectin affinitycbromntogruphyfollowed byge l permea tionand anion exchange chronmtogrnphy(Mlsclmket ul.,IlJHH),
29
However, neithertheaminoacid seq uence of the receptor polyp e ptide norits eDNAseq uence hasbeenreported. Thesea rch for the foot-and-mouth diseasevirus receptoris just beginningto yieldresults. It has been cstahlishc dthat manycellsconta in variousadhesion proteins,int egrins,which recognizeandinter act withmany extracellularligan dscon t aining Arg-G ly-Asp sequences (Ruosluhti&Plcrschbnchcr,19tH ).Theinh ib ition ofattach ment oftoot-and -mouthdiseasevirusLa itstarget cellsbypeptldescontainingArg- Gly-Aspsequencessugges tstha tone or moreintegrtus arecomp onentsoftile receptorfor thisvirus(Foxaal.,1989).
1.6.5.Receptor for EMC Virus
StudiesOilrhcrecep torforEMC vir uscallbe tracedbackfou rde cades.
However, verylittleinformation is availablein termsofthevir u s receptoron nucleatedcells.Mostof our know ledgeaboutthe receptorfor EMCvirushas h~~ 11nchlcvcdby studying EMC· erythroc ytcinteractions .
1.6 ..'i.1.GlycophonnA -tbc Receptorfor I3MC VirusonEryth rocytes As111l1llyothervir uses,EMC virusattachesto erythrocytes of many species (A ngel .'\:Burness.11)77),however,the biolog icalsign ificance ofsuc han
311
interactionremains unclear. Onereasonable explanation isthor EMC- erythrocytebin di ngmayaidthehostto gelridof th einvadingvirus1II0rc effectively,bypresentingthevirustoimmunocompeten tcelts: (McOi nlockt"
al.,1980). Earlyobservations showedthathcmngglutinatloncausedby EMC virus was inhibitedby pre-treatmentof eryt hrocyte s with neuraminidase (Angel&Burness.1977;Enegrcn&Burness,1977;Burness&Pardoe .IlJRI).
Thisfin d ingrevealedthe involvementofsialicacidsinvirus nunchmcnt.
Glycophorin Is nfnmilyofglycoprotclns. Curreurly, the yarc named glycophorin1\,glycophorin B, glycophortnCand glycopho rinD in lunnau erythrocytes (D nhr,I!JH6 ).Thefirstthreemembe rsofthefamilynoruntlly compriseaboutH6%,7.5%an d 1.5%of totalslntoglycoprotcin,respe ctively, whereas, the glycophori n 0 C.1Ilonlybedete ctedill purifiedglyco phorin prepamtions(Da br,19H6~ 111estudies whichledto theconclusionIhnt glyeophorin1\istherece ptorforEMCvirusto ok advantageof theexistence ofsevera l erythrocytevariantslackingoneormorespeciesorglyccphcrln (Dahr.19M).Thus,EIl(n-)cellscompletelylnck gtycophortn A,but contain normalamountsorothe rglycopho rinsfindglycoliplds(Taliano ,Il)HO;Anstcc, 19H!); whereas, 5·s-V
+
erythrocytesla ckglycophori nIl,but nrcucrmalill ot her asp ects(Dahrauf.,197M).'(liefindingthatEMC viruswasuna bleto31
attachto En(;I·) erythrocyt es,butattached105-s-U+erythrocytesat normal levels(Allaway& Burnes s ,1986;Allawayr:l01.,1986),toget he rwiththe pre vious coucluston that theEMCvir usreceptor isasialo glycopro tein (Vc rliudc&Dcllnnn ,1949;Angel&Burness,1977), revealedthatglycophorin 1\wasthe receptorfor EMCvirus on human erythrocytes (Allaway&
Humcss.[1)1-:(1).
l.ft.5.2 Site ofAtta chmentofEMC Virus011Glycophor inA A detailedcxamtnntlouofthest ructureofglycophorinsand thesegments cleavedbyprorcnse s allowe dinve s tigators todeterminethebinding site for 1:f\·1Cvirus onglycophoriuA(Allaway&Burness, 19&1;Allaway
a
al.,1986).Ulyccphorin!I.contains131aminoacidsorwhich 70 areexposedonthecell surfucc withone N-linkcdI1l1d 15ide nticalQ-linkedsinlc-o ligcsaccharideside chains(Tomita('fat.,1978; Dahr,1986),Glycoph orinBisextremelysim ilar In gfycoph o rinA instru c ture. TIIC structures
o r
glycophorinA and glycopho rlnnarcidcntlcnlin the regionor
amino acids I10 26 and very sim ilarill tlrcreg ion;6to72,butthe segment 27to 55 isabsentin~lyn)phllrillIt Ther efore,thetact thaL EMCvirusdoesnotbindtoEIl(n-) cryuuucytcs.burbindsto S-s-U + eryth rocytes:11normalleve ls strongly
32
sugges ts tha tthebindingsite forEMCvirlis(1lIglycophortnA mustbelocated between<Iminoacids 27 to 55, theregionthaIdistinguishesglyc oph oriuA from glyccpborlu B(Allaway&Burness,IlJH6;Allaway (.(at.,19H6).
Th issuggestio n wa s also support edbythree othe rexpe r iments.Thefirst exper ime ntshowed thnt treat mentoferyth rocytes withchy motrypsin, which releases nrnino acids1to 34 from gtycophorin A,has littleor110effect on virusattachm cur, whe reas trypsintreatm en t, whichrelea sesnmiuu acid residues1to 39illglycophorinA,close 10themembrane , cnuscd adropof morethan50%in virusattac hment(Allaway&Burness,Il)H(j;Allawaya
at.,
1996).The second experimen t usedmonoclonalnmi-glycophorinAantibodies directe d ngains tvarious regio nsoftheextracellulardomainof glyccphoriuA (Anstcc 8: Edwards,19H2;Ridgewclletat.,ICJH3;Gnrduc rct/II.,JCJHlJ). It showed thatthemon ocl onal antibody Bllriblockedthe nmluo acidregion 34 to39preventin g nuach mcntofvirus, wherea santibod iesblockingtheregion s 10 to 30and50 to70 hadno effecton virus nunclun cm (Pa rdoe&Burness, 19H6).The thir dexperi mentused Wr(b·)erythrocytesillwhich glycopbo r in Aishighly modifiedbetweenamine acids 57and70. EMCvirusbindsto Wrib-) erythrocytesequally well com pared tonormalcryrhr o cyrcs(Pardoe&
Burness, JlJR6).Over all,the siteof attnclnn cut ofEMCvir usonglycopho ri n
33
Amust lieilltheregion of aminoacids34 to50, and35 to39 maybe particulnny important forEMCvirus binding( Allawayetof.•1986; Pardoe&
Burness,JI)X(t.).
1.6.5.3.Role ofSialic Acid in theAttachment ofEMC Virus toErythrocytes Itiswell knownthat receptorsfor many nnunmalla nvir uses,suchas iuflucnzuvirus,reovirus,and EMCvirus,requiresialicncidresldues(Burness, 11)x 1).Sialic ad dsarc afamily ofabout30 acidicsugars (Reu ter&Schauer , 19Mi'll,that arcallderived fromneuraminicacid(.5-amino-3,5-dideoxy-O- glyccro-Dcgnlncrononuloso nic acid), wit hdifferences in theirsubs titution patternsontheaminonnd hydroxylgroups (Schau er,1987).TIleamin ogroup uf ucum miulc acidC:lIIhe subst ituted eitherbyallacet yl or glycolylresidue, whereasUIICor scvcm lhydroxylgroupsare oltc nmethylated or esterifiedwith
;I(·c tyt. tncryl,sulfateorphosphategroups(Schauer.1982;Corfield&Schauer, IW;2).Sialic acidsare usuallypresentonterminalsuga rs of ollgosaccharldes inglycoprotclnsantignnglfosid es,but they might alsobind tointernal cnrbohydmtcs (Sc ha uer.191'12;Schauer,19K7). Nocetylneuramtnicacid and N-g lyl'olyllCUr:llllillicacidarc two ofthemostcommon neuraminicacidsin manyunlmals.Noticeably.1ll0U SCerythrocytes contain27%9·Q· N-diacetyl
34
neura minic acids (Reuterct
at .,
1988). thatare rclatlvely rcslstnnt (0neura minidas e (Vihrio Clwlerue)digestion (Schauer&Pnlllard, 19( 8).The biologicalroleof sialicacidshas attrac ted3101 ofattention inthe last1tI years. Sialic acidspa rtlclpnte in manyblologlcnland pathologicalprocesses.
Theyhave been found to beinvolvedin constitutingreceptorcom plexes for vario us peptid ehormones,toxins, virusesandmycoplasmaspecies,mai ntain ing activityof glycoprotei nenzymesbyintluencingthe glyco protei nconforma tio n nndspecifying bloodtypesand cell malignan cies(Scha uer,11)85).
The role ofsialic acids inthe nllnchmc nlofEMC virus tohuma n erythrocyteshasbee n extensivelyst ud ied in Dr.A.T.II. Burness'slaborat o ry ntthisinstit ution . Ascomponentsofvirusreceptors,sialicadds canhavetwo possiblerules, ndirectrole or anindi rect role.Being auintegral partof the virusbindingsite on therecepto r,sialicacidscan playa directroleillvir us anncluucutto therece ptor. Altern a t ively,negativelycharged sialic acids couldplayanindirect role byinteractla g wit hpositivelycharged basicamin o acidresiduesinthe receptorpolype pti detoholdthe virusbindingsitein the corre ctconfiguratio n (Tuvakkol&Buruesa,!l)'Jll}.To examine thepossib ility of <In ind irectrole of sialicadds in thebindingofEMCvirusto glycopho rln A, thepositively chargedlysine residuesandarginineresidues inglycopho r in
35
1\were blockedbyacetylation withace t icanhydrideor reac t ion with buumcdicnc. Neitherofthesetreatments affectedbindingofEMCvirusto itsrecept or on humanerythrocytes, suggestin gthatpositivecharge s onbasic ununo acids nrc no t requiredfo r EMC virus attachme ntto human eryth rocyt es. InCOIII,....5I,bloc kingofcarbo xylgrou ps onsia lic acidresidues hyum khuion C:IUSCU n 96%inhibition of EMC virusattachment.
lunhcrmorc,removalofthethree-carbonlongpolyh ydroxy side chainillsialic ru'ids,bymild pcriodntc-borohydridctreatm e nt,hadno effecton EMCvirus nuacluucm. Overall,thestud ies suggesttbntthccarboxylgroups , not the polyhydn»xy sidechainofsialicodds,playadirec trole inEMC vir us uunchmcnr.and thatpositively charg edlysine andargin ineresidues inthe polypeptidearenotinvolvedintheimemctlcnwithnegativelycharged sialic
;tdlls to nmintaln theinte grity
o r
the vir us attac hment siteon human erythrocyte s(Tm';lkkolS:.Burn ess ,I 990).1.7.Purpos e
or
Research1.7,1.Genera lPurpose
o r
Vir usReceptor StudiesVirusesinducevarious humandiseases,fromthe annoyingcommoncold Itlthe lethal Ill'pat ilisIIandAIDS. Un fo rtuna t ely,vaccines, theonly powerfulweaponuvnilnblc in (heba ttlengal nstvlmldisea ses,can notprotect
36
hum a ns fro mmany viral infections. Forexample.itis almostimpossible to devel op a vaccine against thecommoncold. 111cdisease can actuallybe
ind ucedbyalmostZOO diffe re nt vir uses, rhinovirus cs alone has over lOll differentscrotypcs. However, 10init ia te aninfectio n, the virus must nunch to specificreceptors on a host cell.The attac hmentbetween the vir usfindits
cctluln rreceptornot onlygrantsthe virus physical accessto a susce pt iblecell.
but alsocontr ibutesto thesubsequentstages of vimIreplica tion(Crowell&
Lnudnu,19 H3),suchasviruspcuctrn rion(Pustnn&Willingham, 19 HI) and uncoatlng(Crowell&Sink, 1!J7X). Thespecific attachmentstep su ppliesn perfect target to interceptthe viral infectioncycle.To developagents,which will inhibitvirus initia lbind ing to cells,Information on bot hthe VAl'nud ce ll receptorat the molecular levelis essential. IIInddl tlcn,the study
or
vir us receptors is importnm tounde rst an dthe precise mechanisms of viral replicationandtissue tropism. Fina lly,thestudy of vir usreceptors may event uallyle ad tothe ident ificnticnofthe normalphyslological func tionsof thes e cell su rfacemolecules.1.7.2. Purpo se of theProje ct
Thereceptor for EMC virusall human erythrocytesis glycophcrlnA
37
(Allaway«:1,11.,19M).However,sincetheeryth rocyte doesnot supportviral replication ,thecellisnotregarded asa hostnuclemed cell forthevirus.
Therefore,whetherglyco phorin A isa genuinerecep torforEMCvir us on hostcells remainsunclear. TIleK562cell,ahuman erythrcleu kemlccelltine allowsus to studythisproblem. Thesecellslikeerythrocytesexpress gtycophori u/\ 011their surface (Gahmberget
at .,
1983) and they are susceptible10EMCvirus infection(Pa rdoe<:1ai.,191.)0). However, recent iuformntiousuggeststhatglycophorinAisnotthereceptorforEMCvir uson Kfl52celts.'111Csuggestionisbased on thedatashowi ng that transfecttcn of K:-'112cellswith nmi-scuscglycophcrln1\eDNA.to block synthesi s of glyclljlho ri nA.orsntum tingthecellwithnnti-gtycoph orlnA antibodies . does notaffectvirusattachmen tto.andInfectio n of 1<562cells(Hamid&Grewal, unpublishedresults).Thus,thequestion remains, ir glycophorinAisnot the receptorfo rE~1Cvir\lsonK562cells, whatisIh e receptorforEMC viru s on K5h:!cells'! Therefore,this projectwasdesigne d10gathermore information ..hulIlthenatureorEMC recept orson humannucleatedcells,1.7.3.Experimen ta lstrategy
lu thcbeginn ing, ol\lyhumancrythrolcukcmicK562cellsandthe K5620
3H
clonemu tantce lllin e,whichre sistsEMCviru 5 infection,possiblybecauseof
:I;deficie ncyinEMC virusrecept ors (Pardoeet uf.•199(J).wereinvolvedillthe project. We initiallyconsidered produci ngmOIl()c101l~1 anti-receptor antibodiesas th emnin approachtoisolatethereceptoruntilitwasfoun dthat the colorimetrichybridoma screeningnSS<IY usi ng3-(4,.'i-dilllclhylthiazol-Z-yl)- 2,5-diphenylteLrazoliulllbromide(Mr f)andp-iol!onitrotclrazolitull violet, whichwas used successfullyillscreeningmono clonalanti-receptorantibodies forthepo liovir us (Shepleycfal.,I~HS),wnsnorsuitable forK5fi2celts, which growill suspension.Thiswa sbCC.1tISCSCVCr;I !washes arc requiredin this screeningassay .which caused asigniflcnutlo ss ufcells.Conscq ue ntly,I le La cells, nhuman cervicalndcnocarcincnmcelltine,whichgrows in rnouol aycrs, wereintroducedintotheproje cttoallow fur(he productio nofmonoclonal ami-rece p tornmibodics. Eventually,thenntl-rcccp rormonoclonalantibodies we redevelopedbyothersoutside thisln bornto ry, bUIJlela ccllstudieswere continu edillthis projectforcomparison.
Sinceanti-receptor monoclo nal nmibodlcs arcusually gcnc nucd:,tnvery low freq uencywhenwholecellsnrcusedforhnnumlzmlon ,itwa snec e ssary totryot hermethodstoIsolatethevirus recep to r.The successillisolatingthe EMC vir us receptor onmouseInsulinonmcellsbyEMCvlrus-Sc pharosc
39
affinity chrcmarogruphy(Baldeh&Burness,manuscriptsub mitt ed), motivated usIIItrythesa me techniqueinthisproject.
The principle of thisaffinity chromatography techniquewas based on two fimlillgs fro mstudies ofinterac tions betweenglycophor inA and EMCvirus.
Thefirstfinding was thatthe bondbetweenglycophor inA·EMC virusis a wcukio nichOI1<1 whichcan be ea silybro ke nby0.2MNael(Allaway&
Huruc ss,[1):)7). The secondfindingwasthntglycophorinAbinds to EMC virusOilthe affinitycolumninthe presenc e of 6 mM sodium deoxycholate
(I)( )C)(Haldch. II)H7).Thisaffinitychromatographytechniquewas shown to
be suita bleforthe purific ation ofEMCvirlisrece ptors from humancelllines,
;llId WOIS adopted illtheproje ct.
Aspanorthelong term objectiveof the receptorstudyfor EMCvirus on luuunncelllines, someprelimin aryphysicaland biochemicalcharacterizations of the rece ptorwerecarriedout. Theseexperiment sincluded thefollowing:
(I ) dctcrminnfion orthenumberofEMCvirusbind ing sitesandthevirus- receptor equilibriumdlssoclnrlon constant(Kd) inHeL1cells. Similar expe riments had t-e endoneusingK562cells before thisprojectwas started
(Pardoe .'\:. Burness.unpublished results);(2) effectsof protea sesand
ucuruminidasc011the uu ncluuentof EMCvirus tointact cells,membra ne [uvpur.uious :H1ddet erg entsolubilized membranes ofK562and HeLacells;
411
(3) effectoflectlns ontheattachmentof EMCvirusto K562 andllc l.n cells, (4) biosynthesisof EMC virusrecepto ractivity011K562 nnd Hcl,n cells alter trypsintreatment.
Cha pter2 Materialsand Melh ods
2.1.Growthand Purification ofEMC Virus
The K2 strulnof EMC virus was used inthis project. Viruswas grownin swirlingculturesofKrebsascitestumor cells(Burnessetal.,1974). 3H_
labeled EMCvirus\W1Sproducedbyadding 3H-lnbeledleucine(Dupont, Boston ,MA, USA) tothecult u remediumdurin gvirus growth.The vir uswas purllcdhythemethodofZlolnandScrnbn(1974) andkindlyprovided by Ms.
PardoeFrom Dr.A.T.JI.Burness'slabora toryofthisinstitution.111e number ofvhul particleswas est imatedasdescribedpreviously (Burness&Clothier, 11)70). In11typical preparationof31-1_lnbclc d virus,specific activityranged fromHOOtoI(JOlJ cplll/~gprotein(i.e.,from1.14 x10,8to1.43 x to·gepm/virus puniclc}.
2.2. Cells 2.21.K%2 Cells
K5(11 cells archuman crythrolcukcmiccells,whichwere originallyisolated Iromnchronic myelogenousleukemiapatientin 1975 (Lazzio&Lczzloetal., lIn )).Becausethecells expressglycophorlnA (Gnlunberger
ut .,
1979),they were often usedtostudyglycophorin biosynthesis(Gahmbergef al.,1980;42
Jokinen1:/at.,19H5; Silveretot.,19H7;Morrow&Rubin,1(87). The expression ofgtycophori u A011K562 cellsalso provide sngood opportunity 10invest igatetherole ofglycophorinAasn receptorfor EMCvirusill nucleate d cells. Recentl y,11 K562cc!l linc, D clone,which isresistant to EMC virusinfectionwas isolat e dFroma pcrslstnnt lyinfectedculture of K562cells (Pardoeetal..1i)t)(J).Itwasproposedthat theresistan ceofinfectionwas due tolack ofreceptorsforEMC virus in the mutantcells, asthe virus bindsvery poorlytothese cells(Pardo ectal.,li)t)(J).
Bot hpare ntalK5fl2 cellsand their0clonemutant cellsweregrownill suspensioninRPMI1040 medium(secAppe nd ix)sup ple me n te d with10%
fe tal bovineserum, IOUUofpcnlctllluperml,and100 ugofstre ptomycinpCI' ml at37(lCill1Ihumidified 5% CO2Inculuu or. RPMI 164(1,fetal bovine seru m and muibiotics wereobtained from GIBCnBRL,GrandIslan d NY, USA. K561cellswe re grown in 75cm2 cellcultureflasks (CostarCo., Cambridge , MA, USA). Thecellswe re harve stedbyccutrlfugntio n whentheir densiti es reached about 1xWficelis/mi.The number of cellsand their viabilitywere measuredbyusinga hemocytometerafter mixingau equal volumeofcellsuspensio nwith 0.1%trypanblue inphosphate-buffered saline, 1'1-17.4(PBS,sec Appendix ). Cellswere washed withcold PBSthree times beforeusc inexper iments.
