p~~r~';1 sbo..~three vh~le

List of Tables

1. Enzyme assays of fractionated Krebs cells "

2. Io d i n a t i on of Krebscellswith5011Ci12 51 68 3. Io d i n a t i on of Krebscells with 10011Ci12 51 69 4. Io dina t i on of isolatedKrebs plasma membraneswith 5011Ci_ 71

5. Iodinationof Krebs plasma eeebr-anes with different 72 amounts of12 5 1

6. Therole of the glycocalyx in the attachment of EY.C 86 virus to Krebs cells

,.The role of the glycocalyx in the growth of DiC virus_ _ 89 inKre bs cells

8. The effe c t of timeof exposure to vi rusonthe attac hment _93 of38 labelled FJoIC virus to Krebs cells at a concentration of 2.5x10' cells/1Il1

9. The effeet of t i _ of exposure to virus on the attachment_94 of 38 labelled E.."lC virus to Krebs eells at a concentration of 5 x 10' cells/tal

10. Comparisonof the attachment of 38 labelled~Cvirus to_96 Krebs cellsand erythrocytes

II.Compa ri s o n of the attachment of 3H labelledE....Cvirus to_ 9 8

Krebsp.Ia sma membra nesand erythrocytes

12.The effect of trypsin on the attachmentof FJolC vi rusto_ l03 Krebs eells

13. The effect of trypsine reareent of Krebs cells on the_ _lOS infectivity of DIC virus

Xl

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Thework onthe influenzavirusre e e p eoe on re d blood cej Is is probably the \!lOst comple te at th e pres entti llle . Virusre c e p tor subs t ence for infl uen zaviruswasisolatedfro..human eryt hro- eyte s (50) and is ina ctivatedbyre mov i ng carbon ato ms from NANAre s i d u es (112) or byremovin gthewhole're Sid ue (51). Sia licacid eontaining glyc o p ro te i n s .knownasa or Franeis inhibito rs .competewithvirus receptorsubatence for virus part icles thus reducingth e binding of virusto the vir us re c e p to r sub s t ance (37). Host ofthehumanerythr oc y tesu rfaceNANAwa s found tobe pre s e nt inth e vir us rec ep t o r subs t a n c e which wa s ealcu1a t~dtobeof molecularweight31.000(115) . Thisrec e p t ot- substance.servingasthehuman erythrocyt ere c eptorfor influenza typesA and Bvi ruses. ha sbeen identif ied as glyco pho r i n A (54).

Other virus e s suchaspolyomaviru s andmostofthe para .-y xo v irus e s shOW"siul1 la r at tac hment cha ree rerrs.ttc stothe in f l uen za virus e s10 that sialic acId isre quired and attach ment isinhibited byFranei s in hi b i t o r s . Exe ep t i on swithin the paramyxovi ridaeare Senda i viruswh i e h mayutilizea ganglioside receptor (I2 2) and theMorb illiv i rus genuswhe rethe re mo val of si al icacidhas noef f e ct-c nattachment. The simi l ar itybetwe e n influen z a an dpolyomaviru s at tachmentis qu itest rongbec au se infl uenza viru s can us epolyomavirusreceptor si t e s on eryt hrocyt e s (82)but the r e arefewerpolyoma re c e pt or si tes than influenza

- 10-

re c eptorsites (8 2).

Aden ovi ru s7 rece p tors onmon keyeryt h ro cyte shav e be e n solu bi l iz e d an d par tia llypu r i fi ed (a 4). Thisneura minid a se ins e n sit iv erecep tor is of ~4.000molecula rweigh t and quite distin ctfrolll gIy c o ph orfn,ha vi ngno in h ibitoryeffe c t on inf l uen zavi rus attachment.( 84).

Cur rent lymo st advances havebeen madewithvirus e s th a t attachto glyc o pro t ein re cept orsbutthe reis evidenc ethat Sindbisvi rusmay bindtolipid rece p torsoner y t hroc yt es (81).

Thebind in gof ra d i oac t i vel ylabelledSin d b is virusto lipo s o mal modelmembranesmad efromamix t u r e ofery t hrocyt ephospho li p ids wasgre a~lydimi ni shed when eith e r chole stero l orph osphat id yle- tha n olami ne we r e cnttee d, The au thors suggest that these two coepco enesare importan t in abi l ayer configuratio nfor speci f i c virusbinding (al ).

Having brieflydiscussed some ofth e moreimportan tachi ev e - eenteinstudieson theattachmen t of virusesto cel ls,the fo l lowingsecti o nisa moredetail ed re v i e w ofth e current knowledgecon cernin gpic o rna viral attachment.

ii) The attach mentof pi c ornavirus e s to cel ls

me

vi rus ,whic hisus edin this st udy is apicornav irus.

Thereisconsid era bl eknowledge co n ce r n i ngtheattachmen tof pic ornaviruse sto cel lsbut li t t l e iskn o wn abo u tat ta c h me n t at themolecu l a r le v el.

Many of the Pico rnav l r l da e at tachto asmal l ran g e ofhost

- 11-

Materials and Methods

Gr owt h andHarvest i ng of Kre b s asci tes turnour cel ls

Phosphat e buffered saline. (PBS) . (29)va s prepa re dfromsolu tions A andB. Solut ionA contained'Ogxect, 19 KCI. 5.75g Na

2KPO, and19 KH

2PO,in , Htre s ofdistil ledwat er. SolutionB con ta ined 0.5 gof dried Ca CI

2• 0.5 gMgC1

26"2 0and dist i l led wa te r toa volume of1 litr e. These so l ut ions weresteril iz e d separa te ly by autoclaving at 121·Cfo r 20 minute s. Aftersteri lization th e solut ionswerecombined and 500.000unitsofpenicillinand 1 mega- unit of streptomycinwereadded. The pH which shou ldbe pH7.3.

va s ch ec ke d ....ith aBeckman digitalpHmeter . Calc i um and magnesium fre e PBS (Ca++-Hg++free PRS).was preparedidentical ly to PBS exc ept th atso l ut io nBwas olll1ttedand so l u tion A wasaadeup to 5litre s. Trypan bluewasa 0.1%solution inPBS.filteredthrough Wha t lD8.nnumber 1 pa per.

Krebsasci te s tumourcel ls we r e grown ingenetically hetero genous.

albinom.iceofapproximately30ginweigh t. Either0. 1ml of a wa shed cel l sus p e ns i on or 0.2..1ofa fro ze n cell sus p ens i o nea c h containin g108

cells/ml was in j e cted intra perit on e allyintoeach mous e. Thecel ls wereharve ste d ase p t ical l y into CA++-Mg++free PBS

·7to 8daysaft erinjection. The cel ls we r ewashe d once In ca++-Mg++- fr eePBS by sed ime n t i n gat 500 to 1000rpmfo r 5min utes and re su sp e n din g them inPBS. The cel ls'were wa sh ed atlea st twice IIIOrein PBSuntilall the erythro cyt e swer ere mov ed. Wash ed.

- 24-

pack e d cellswe r ere su s pended in an equalvol ume of PBSanda sampl e was count ed bydilut in gfirs tl y, 10 fold inPBS an dsecon d ly , a furt h er10fold in0.1%tryp an blue. This sample wa s coun te d ina hae moc yt o me t er. Theperc e nta ge of"de ad" , trypanblue stai n i ng,cel ls wa s calc ulatedand the co nc e ntra tionof viablecel lsadj uste d to lr:ft cel ls!:ml. Up to10%of thece lls sta i n i n g withtrypan bl uewas con side r e d acceptab le foruse .

Growthof EMC virus (99 )

The Earlessal inegrowth med i um con tain ed : 100 mlof lOXcon ce n tr a t i on of Earle'ssaline.

50 mlof44%sodium bicarbonategassed with carbon dio x i de for about ha l f an ho u r immediately prio rto use.

1('1111.1of inactivated ho r s e serum .

St eri ledistilledwatertoavo l ume of I li tre.

0. 1%trypa nbl ue in PBS,fil tered throughWhatmannumber one pa per, was us e dfor countingcel ls.

Me thod

The Earle's sa l in e growth mediumwas wa r me d to 37°C. Krebscells , ata con centration of10 8 ce1ls/mlwe rein f e cte d wi thvirusat amultiplicityof infect ionof~. The multiplic ityof infec t ion isthetotal numberofpla qu efo r mi n guni t s of vi r usper total numb er of viab lecel ls. After maintainingthe virus -cel lmixt u re at 4°C for 30 minutes the cellsuspe nsion wa s dilutedto107cells/

111.1wit hthe pr e vi ou sly warme dEarle s salinemed i um and dist ributed in Erlernme y erfla s k s suc h thatthecu ltureoccupied 10%of the flask 's

- 25-

prepared solutionCwa sadd ed . After st a nd i n gat room t.eepe rnrure for tenednueee,0. 1IIl1of solu t ionDwa sadded and the colo ur allowe dto develop for 30 minutes befor ere a d ing at 660nlI.va v e-.

len g t hs. Standardsolu tions ,EandaIofate r blank we r e assay ed inthe same wayto give ast a ndard curve fromwhichunknown values co uld be re ad.

Siali c aciddeteminat ion

The th iob arb i t u r i c acid method of Warren (U9 )was use dtodet e rmi ne sial icacidcontent.

Materials 0. 2M sulphuricacid

SolutionAwas 0.2Msodium- m- periodatein 9Mphosphoricacid.

Solu t ionBwas10% sodium arsen ite in 0.5Hsod ium sulph ateand O.lH sulphuric acid.

SolutionCconsistedof 0.6%th i o ba'rbi t ur ic aci d in0.5Hsodium.

sulphate . Cy clohe xanone Met hod.

Thismethoddetectsonlyfree si alicacidwh ichvasrele as ed from 0.1-.nl of plasma membran e suspe nsionbyhydr olyz ingwithanequa l vol umeof 0.2M"2S0 4 at 800

Cfor 1 hou r . To eachsample.0.1ml of sol utionAwa sadded and the sampleswereshak e n and al lowedto standatroOtll temperature for20 minutesbeforeadding 1.0ml of soluti on". The yel low-browncolour fomed disa ppearedaf t e r shakin gand2. 5mlof solutionCwasadded. The samp les were mixed

- 38-

Thesa mpleswere centri f ug edto remove anyprecipitateand the ph os ph ate con ce n trati onin thesupernatan t fluid dete rmined . The act i vity of th e enzymewa s defined in te rms of nu mb er ofVmol esof phosphate rele a sed per hour per mg ofprote in.

5' Nuc leo tidase

Themeth o dofBodans kyand Schwartz (103)was use d.

Mate r i a ls

The rea ctionmixtu reconta inedthefo llowi ng subs t anc es . 10 pI of O.lMKC1

50'Ill of O.l HMgCl

100'Ill of50 WIadenosine monophosphate(AMP) in O.lMtri a no pH8.6

0.5511I1ofO.I Mtris HCl pHS . 4 10%TCA•

.Reage n tsfor pho spho rousdetermtna tlon

Theabove mixt u re wa s fre s h l y prepa redan d0.1 1II1ofmelDbrane suspens ionwas added. The mi x tu rewas inc u ba t e d at 37^0Cfor15 minutes. atwhic hti llle . there a c tionwasstopped by th e additionof1..1of cold TCA. Any precipi tate wa sremoved by centrifugat ionprior to pho s ph oro u s determi na t ionof theeaep'lee, The enzymeactivitywas expr essedasthenumb erofumoLe e of phosphorous rel eased pe rho u rper mgof protein.

Glucose-6-phosphatase(11 3 )

O.IMcitra tebufferpH6.5was madeby aci dificationofsodi um ci t rat ewit h1Mcitricacid.

- 41-

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Figure2.Ele c tronlIIicrograph of Kr e b s pf ee ea membrane s. Cl umps ofmetllbrane~,wholeandfragment edmembran e s can beseeninthisphoto- graph whi c h istnagn ified41,500 times.

repres e n t s 1'11I

-57-

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Figure 5. 50S-PAGE of Krebs cell fractions. The gels, stained with coomassie blue and electrophoresed for 2 hours, are ofthe foll<ming ; A- cell homogenate with the w hole cells and nucle i removed, B- crude plasma membranes, C- mitochondria, 0- microsomes, E- soluble cellmaterial and F- purified plasma membranes.

61

possiblydue toaggregationof lowermole cularweight components. Since . however , the sameamo un t of pro"teinvasplac ed oneachgel anyprot einconcentra t ed in on efractionwould have a largerpe ak in that profi leand maybe mis singin the prof i le ofan ot he r fracti on whereitsconc entrat ion islowincompar ison to ot he r compon ents.

The ele ctro phore sis profi le of pla sma me mbr an e s an dpart ic- ularl y of puri fied pla s ma membranes showe d dist inct lydiff eren t patterns comparedwithot her cel l fr ac tion s . Compo ne nts pre sentin othe r fra ction s wereve ryfaint in the purif ied pla s mame mbrane sam- pleindic atingthat th ere vasl it tle contaminat i onvithth e s e other fractions.

d) U. SDS-PAGE of purif ied plas ma membranes

Pur ifie d plasmamembra nes ,el ect rophoresedon gels for two hours, gave characte r is t i c,consistent lyobserved patt erns of bandswhether coomassieblue stain for detectingproteinsor periodicaci dSchiff's (PAS) rea gen t for glycoproteins wereus e d.

For comparison purpo se s human eryt hrocyt emembraneswereocca s iona lly analys edonparalle l gels.

Aftertwohour elect rophoresis (fig. 7, 8) at le a st 20 coomas siebl ue st a i ned band swere obse rved, three ofwhi chwere hea vily st a ined. Incon trast, on lyonebandwa s obs ervedaf ter PAS sta i n ing. Sinceth i s single P.ASstainedcomponent migratedave ry short wa yinto the geldur in gthe twoho ur per i od, electr op ho resis wasals ocarri ed out for eight ee nhours aga instain ing withboth ccceassre blueand PAS stains.

A faintba nd wa snow see n in the high molecular weigh t re gi cm - 63-

A

Figure 7 . SDS-PAGE ri purif ied plasma membranes.

The gels. stained with coomassie blueand electrophoresed for2 hou rs. are ofthe following;

A- human erythrocyte membranes andB- Krebs cell plasma membra nes .

64

Coomassie blue rofiles

KREBS PlA SMA MEM- BRAM:S

ERYTHROCYTE MEMBRANES

Periodic acidSchiff' sprofiles

KREB'S PlASMA MEMBRAM:S

ERYTH ROCYTE MEMBRAM:S

4 0

OISTANCE FROM ORIGINtern) Figure 8. SOS-PAGEscansof pur if ied membranes. Plasma membranes preparedfromhuman erythrocytes and Krebscells

we reelectrophoresed for2 hoursandstained with coomassie

bl ueto detect proteinsand PASreagentto detect glycoproteins.

The scans ofthese gels areshown.

65

COOMASS IE BLUE PROFILE

PER IODI C ACID SCHIFF'S PROFILE

0 4 8 DISTAN CE F ROM ORIGIN (em)

Figure 9 . SDS-PAGE o f purifi ed Krebs plasma membranes.

Thes e scans are of gels elec lrophoresed for 18 hours and stainedas shown.

66

of the gel with two darker bands veryclosetogether inth e lower mol e c u larweightregionof the gel (fig. 9). The PAS stained gel run for18 hourshad a very faint band nearthe origin and a more intens e band witha sho u l de r.near the bot teaor lower Plolecular weight regionof the gel,(fig. 9). Onthetop of the gel there wa sPAS staining mate r i a l whic hhad not beenable to enter th e gel probablydue toaggrega t io n.

e) Kre b scell su rface lab ellin gwith1251.

i) Iod i n a t i on of whole cells with50\.lei 12 51.

Anattemptwasmade to int roduceiodine,1251,intocel l surface components to aid in their identificationand to give a con v e n ien t too l for furtheran alys i s . Labellinswas performedin the presen ce ofgl u c ose oxidaseandla c t o pe r o x ida se. Afterin c uba t i ng Kr e bs cells with1251 for 0.5, 1 and 2 hourstheywerewashed an d then fra ct i on ate d. Of thetotalinco r-porat e d cpm llIOSt was found in themi tocho n d ria l-mic rosoma l -so lub l e cell mate r ia l fraction wi thonly 0.028% after 0.5and 1hour'sin c ub ati on and 0.035%af t e r 2 hour s ofthetotalradi oa ctivit yad ded becomin gincor-porat e dinto the plas mamembr an e (tabl e 2). Of the radio a ctivitybound to cells only 1.6%, 2.7%and 4.5% of thera di o a c t i v i tywas bound to the plasma membrane after incubation for 0.5, Iand 2 hours respectively.

Labe llingfor long ertime periods increasedthe amountof radioactiv- ity incorporated intoplas ma memb r a n e s onlysligh t l y.

11) Iod ina tionofKre b scells with 100 uCi 1251.

In an attemptto impro v etheamountof radio activityin purified plasSla IIletllbranes , labellingofwho le cellswas repeate d

- 67-

A B c

Figure 14. 50S-PAGE of Krebs glycocalyx fractions. The gels, stained with coomassie blueand electrophoresed for 2 hours, are of the following; A- crudeglycocalyx. B-cell coat fraction and C- cellcoat particle.

80

par tic lesto Krebscells. Onlya smal l propo rt ionof a givenE!iC vi russuspensioneceprtse e infec t iousparticles (13). Inexperimen ts per tain ingtoth e attachmen tof ra dio a c ti v e l y labelled virusto cellsth e infectious vir ions~reon lya small groupandi fth e y behave diffe rentlyduringattachment it would be easked by th e largenueb er of non-infectious viri o n s present . Byloo k i n g at the effect of glycocalyx removalonvirus growtl)effect ivelyonlythe at tac hmentofin f e c t i ou s virionsis being measured.

EMCvi r u s ....as grown in suspension cu lt uresof10⁸Krebscells with and ....ith outa gLy coc ajyx, Viru s growth ....as monitore d byHA tit r ati o n an dby ce l lki lling.as indicatedby th eupt a k e oftr y pa n blue. A contro l consisted of108uninfectedKrebscellswith an intactglycoc8lyx.

Therewas very little diffe rencein the percentageof cells :.ta ine d and no difference in the HA titre bet....een the infected cells with and without the glycocalyx. (table7). The re raoveL of th e glycocalyx, therefore. has no effecton the growth of EXC virusin Krebs cells.

d) In hi bi t i o nofhaemagdutinationby Krebs glycocalyxfracti ora.

Ifa glycocalyxcomponent....as a receptor fo r E:1C virusi t ....ould be exp e c t e dto interfe rewit h F_'1C virushaemaggl utinationby com- per fn gwitherythrocytestobind viru s. The fo110....ing frac t ions , obtainedduringglycocal)'xpr e pa ra t ion...ere testedfor inhibitio n ofha e ma ggl u ti n a t i o n; the initialcel lsupernatant 0,000g).the 48,000g pellet, the crudeglycocalyx (48,000g supcrnarcnt fluid), th e cel l coat fractionand cell coat particle.

- 90-

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628-632.

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Human (lILA-A ,HLA-B)and curine (H2K.H2D) histocOQpatability antigensarecell surfacere c e p t o r s for Semliki Forest virus.

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