radioactive posphate virus

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The Incorporation of Radioactive Phosphorus in the Influenza Virus and Its Distribution inSerologically Active Virus FractionsAuthor(s): L. Hoyle, B. Jolles, R. G. MitchellSource: The Journal of Hygiene, Vol. 52, No. 1 (Mar., 1954), pp. 119-127Published by: Cambridge University PressStable URL: http://www.jstor.org/stable/3860798 .

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119 ]

THE INCORPORATION F RADIOACTIVEHOSPHORUSIN '1't1EINFLUENZAVIRUS AND ITS DISTRIBUTIONIN SEROLOGICALLYC'l'lVEVIRUSFRACTIONSBY L. HOYLE, B. JOLLESAND R. G. MITCHELL

PubltcHeatthLaboratory, ortharnyton,md heRadiotherapyepartrnent,GeneralHospttat,NorthamptonGraham& McLelland1949,1950) howed hat whenthe PR8 straUinf influenzavirus A was grown n fertile eggs, into the allantoic sacs of which radioactiveinorganicphosphatehad been introduced, he virus incorporated2p into itsstructure. Graham 1950) howed hat the incorporated2p W&Stobe foundpartlyin the phospholipid ndpartly n the nucleicacid fractionsof the virus.This paperdescribes he production f influenza irus a.belledwith 32p, and astudy of the distribution f the isotope n serologically ctivefractions f the virusproduced y etherdisintegration.

PRODUCTION OF INFLUENZA VIRUS LABFJJ,T,E,nWITH 32pIn the originalworkof Graham& McLelland1949) he virus wasgrown n eggsinoculatedwith less than 1,uc.Of 2p, since t was thought hat largeramountsof32p might be lethal to the embryo. Later Graham,Dempster&Buchner 1952)found hat this was not soand that much arger mountsof 32p couldbe used.Weourselveshave noted no deaths in eggs inoculatedby the aJllantoicr yolk-sacrouteswith 10s500 Hc. 2p.A majordifficulty n the productionOf 2p labelledvirus ies in the sepaUrationof the virus fromcontaminating adioactivephosphate n the infectedalla.ntoicfluids,and muchpreliminary orkwas necessary o determinehe best methodofvirus purification.Preparationswere regula.rly btained n which the non-viralradioactivityamounted o less than 10% of that caxTied y the virus by thefollowing echnique.TheD.S.P. strainof influenza irusA was usedthroughouthework. Radioactivephosphoruswas obtained romthe AtomicEnergyResearchEstablishment t Harwell n the formof calmier-freerthophosphoriccid, his wasneutralizedwith sodium hydroxide,diluted to contain 100 juc. n 0j2 ml. andsterilizedby immersion n boiling-wateror 15 min. The radioactivephosphatesolutionwas introducednto the allantoic ac of sixteen 12-day-oldertileeggs ina dose of 0a2ml., eacheggthus receiving100Hc.of 32p. After 4 hr.incubation t36 C. the eggswere noculated y the allantoic outewith 0a1ml.of a. 1:100dilu-tion of D.S.P. virus-infectedllaJntoicluid. After a further40 hr. incubation heeggs wereopened, he chorio-allantoicnembranesorn throughand allowed obleed nto the allantoic luid.The blood-stained llantoic luidwas collected n areceiver hilledwith ice. From he 160ml. of fluidcollected hered cells carrying


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120 L. HOYLE,B. JOLLESANDR. G. MITCHFTJT.the a.dsorbed irus were sedimentedby centrifugaJtionnd washed twice with160 ml. of ice-cold aline.The adsorbed irus was then eluted fromthe cells nto16 ml. of salineby 4 hr. incubation t 37 C. The cells werethen sedimented ycentrifugation. hesupernataJntluid,representing tenfoldconcentration fthevirus n the original nfectedallantoic luid,still contained largeamountof non-viral 2p, and furtherpurification as therefore ecessary.The fluidwas adsorbedwith 10% guinea-pig ed cells, the cells werethen separatedby centrifugationand washedsix times with 25 ml. quantitiesof ice-coldsaline. After the finalwashing he cellswere uspendedn 8 ml. of salinecontaining in 10,000CaC12ndwere ncubated or 4 hr. at 37 C. to allowelutionof the virus.The cellswere hensedimented y centrifugationnd removed nd 0a08 /Oodiumazidewas added othe supernatant luid. This constituted he labelledvirus preparation.A 1 ml.sampleof the preparation as adsorbed wice with 10/O uinea-pig ed cells,andthe radioactivityof the originaland adsorbed luids was tested. The red-cellagglutinin nd complement-fixing.ntigenitres werealso measured.

TECHNIQUE OF RADIOACTIVITY MEASUREMENTS1 ml. samplesof the fluids o be testedwereplaced n shallowmetaldishes2a5 m.in diameter, he fluid thus giving a layer 1@6 mm. thick. The disheswere thenplacedbeneath Geiger-Mullerounterof the end-windowype at a distanceof2 cm. Counting eriods anged rom 1 to 5 min. according o the potencyof thepreparation.Under hese conditions 00 uc.Of 2p wouldregister ,650n000ountsin 1 min. (c.p.m.).The background ountaveraged8 c.p.m.RESULTSThe labelled virus preparation ad a red-cellagglutirlin itre of 16,000 and acomplement-fixingntigen itre of 48. Two successiveadsorptionswith 10/O edcells reducedboth titres to zero.The originalprepara,tionegistered 13 c.p.m. nthe Cleigerounter,while he adsorbedluidregistered nly 36 c.p.m. It wa.s here-foreconcludedhat 877 c.p.m.represented2p incorporatedn the virus.When hevirus was eluted from the red cells used in the adsorption he eluate registered845c.p.m.Table1 shows he resultsof fivesuchexperimentsn which he viruswaseffectively abelled.

Table 1. Radioactivityf labelled irms reparsbtzonsefore nd, fterred-cetl dsorptionComplement- Geiger count Geiger count Virus radio-

Red-cell fixingantigen per min. afterred-cell activityPreparation agglutinin titre titre per ml. adsorption (counts/min./ml.)1 8,192 14 610 49 5612 16,384 48 913 36 8773 48,000 48 2,563 66 24974 8,192 12 336 28 3085 25,600 24 732 35 697


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Incorporation f radioacttwehosphorus n the influenzavirus 121'1'HER FRACTIONATIONOF LABF,T,T,F,n IRUS

Hoyle (1950,1952) howed hat influenza irusparticles ouldbe disintegratedytreatmentwith ether with the liberationof separatered-cellagglutinating ndcomplement-fixingsolubleantigen particles f smaller ize. It wassuggestedhatthe viruselementary ody consisted f an aggregate f red-cellagglutinating ndcomplement-fixingarticlesenclosed n a lipid envelope. Ether destroys thisenvelopeand releases he smallerunits. Prolongedhakingwith ether,however,causesdenaturationf the virusproteinwith ossof itsserological roperties.Whenthe ether ractionationechniquewas applied o the labelledviruspreparationstwas found hat the duration f the ethertreatment,and especially he aJmountfshaking,was muchmorecritical hanhad beenfound n previouswork, his beingpossiblydue to the very great purity of the labelledpreparationsesulting romthe doublecycle of adsorption-elutionnd the numerouswashingsnecessaryoremove norganic hosphate.Satisfactoryther ractionation asattained n threeout of five experiments. n one experimenthe ether reatmentproved nadequateand only slight separationof agglutininand solubleantigen occurred,while inanotherexperimenthe ether treatmentwas excessiveand the serological ro-pertiesweredestroyed.The techniqueused in the threesuccessful xperimentswas the following ne.The labelledviruspreparation as shaken or 5 sec.with half its volumeof etherand incubatedor 1 hr. at 37 C.in a closed ube. It was againshaken or 5 sec.and incubated or a furtherhour.The tube was then centrifuged nd the etherlayer removed. A slight depositof denaturedproteinresultedfromthe ethertreatment.Preliminary orkhad shown hat this depositcontained hospholipidwhichcouldbe extractedby ethanol.The denatured roteinwa.s hereforewashedwith saline and extractedovernightwith ethanol.The ethanolextractwas thenadded o the ether,the wholeconstituting he phospholiptdraction.Theresidualdenatured roteinwas dissolved n N/1 sodiumhydroxide nd neutralized;his isreferred o as the denat?l,redrotesnraction.

The ether-treatediruspreparaUtionas incubatedn an opentube overnightoremoveall tracesof ether. Afterremovalof a sample or serological xaminationandradioactivitymeasurementsheremainder as adsorbedwith 20 /Ouinea-pigred cells, andcentrifuged.The cells carrying he adsorbedhaemagglutinin erewashed wicewith ce-cold alineandeluted or 4 hr.at 37C. ntosalinecontaining1 in 10,000 CaCl2and 50 Australianunits of crystallinereceptordestroyingenzyme R.D.E.) per ml. TheR.D.E. was added o ensurecomplete ecoveryof thehaema,gglutinin.he cells were then removedby centrifugationnd the super-natant fluid constituted he red-cellyglutininraction.The supernatant f the ether-treateduspensionafter red-celladsorptionwasagainadsorbedwith red cells to ensurecomplete emovalof haemagglutinin,ndafter removalof the cells constituted the complement-fixingolbleantigenfraction.Theradioactivity f the various ractionswas thenmeasured ndthe serologicaltitres determined.Red-cell agglutininwas measuredby the Salk (1944)test,


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122 L. HOYLE, . JOLLESNDR. G. MITCHET.T]0 4 ml. of a rangeof dilutionsof the preparation eingmised with 0 4 ml. of1:250guinea-pigedcells. Readingsweremadeafter2 hr.at room emperature.Complement-fixingntigenwasmeasured y thetechnique f Hoyle(1945),usingpooledhumanconvalescenterum romthe A prime nfluenza pidemic f 1951.Thisserumreactedmainlywith the groupantigenof the virus,the serumcon-tainingverylittleantibody o the specificantigenof the D.S.P.stra.in f influenzavirus.Resultsof a typicalexperiment reshownnTable2.Theoriginalabelled iruspreparationada Salk itreof 16,384anda complement-fixingntigen itreof 48.It registered 13c.p.m. n the Geiger ounter.Adsorptionwithredcellsreducedtheserologicalitreto zeroandthe Geiger ount o 36c.p.m.Therefore77c.p.m.representedrirus2p and36 c.p.m.contaminatingnorganic 2p.

Table2. Etherracttonatzonf theD.S.P. strainoftnJ!?enzairus abelledwith32pRed-cell Complement-Geigercountagglutinin fixing per min.Material titre antigentitre per ml.Original abelledviruspreparation 16,384 48 913Preparationafter two adsorptionswith red Nil Nil 36cellsEther-treated uspension 32,768 72 708Phospholipid fraction (ethereal extract+ - 160ethanolextractof denaturedprotein)Residualdenaturedproteinfraction - - 42Red-cellagglutininfraction 32,768 8 65Complement-fixingolubleantigenfraction Nil 64 646

Ontreatmentwithetherthe agglutininitrewa,sdoubled ndthe complement-fixing antigentitre was also slightly increased.The ether-treaJtedreparationregistered708 c.p.m. The phospholipidractionregistered160 c.p.m.and thedenatured rotein42 c.p.m. Adsorption f the ether-treateduspensionwithredcellsresultednalmostcompleteepaWrationfred-cell gglutinin ndcomplement-fixingantigen.Thesolubleantigen raction ontained ohaemagglutinin.t gaJvea complement-fixingntigentitre of 64 andregistered 46 c.p.m.in the Geigercounter.Thered-cellagglutininractionhad a haemagglutininitreof 32,768. Itcontaineda little complement-fixingntigen,givinga titre of 8, and its radio-activitywas 65 c.p.m. It wasevidentthat the radioactivity f the ether-treatedsuspensionwas associatedwith the complement-fixingolubleantigenand notwiththe haemagglutinin,he slightradioactivity f the haemagglutininractionbeingentirelyaccountable y its smallcontentof complement-fixingntigen.PHOSPHOLIPIDCONTENTOF THE VIRUSPreviouswork(Hoyle,1952)hadsuggested hat the viruselementarybodywasenclosed y a lipidenvelopederived romthe wallof the host cell.Whenviruspreparationsreshakenwithethera precipitate fdenatured roteinoccursat theether-waternterface. Lipidis present n the etherextract,but this does not


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Incorporationf radioactivehosphorusn the n;fuenza irus 123represent he whole of the virus lipid since more can be extracted rom the de-naturedproteinprecipitatewithethanol. It seemsprobablehatthe lipid s presentas a lipoproteinwhich s denatured y ether reatment, he lipid beingpartlydis-solved by the ether and partlyremaining ttached o the denatured rotein. Inone experimentwith labelledvirusthe etherextractregistered 3 c.p.m.whileanethanol extract of the denaturedproteinregistered127 c.p.m. In three experi-ments n whichetherdisintegration f the viruswas achievedwithoutserious ossof serological ropertieshe combinedipidextractsaccounted or 18, 23 and 23 /Oof the total virusradioactivity.By prolonged hakingwithether t is possible odenature he wholeof the virusproteinwith total loss of serological roperties, ndin one experimentn which his was done he radioactivity fthe etherextractandof an ethanolextractof the denatured rotein epresented 6/O f the total virusradioactivity.It was,therefore,oncludedhat some20-25 /O f the radioactivityof 32p labelled iruspreparationss dueto phospholipid. hisresult s in agreementwith that of Graham1950),whoshowed hat the radioactivityOf 2p labelled iruswas partlydueto nucleicacidand partly o phospholipid,henucleicacidfractionbeingabout 4 times as activeas the lipid fraction.

PROGRESSIVE DENATURATIONOF THE VIRUS PROTEIN BY '1'HERThe radioactivity f the denatured roteinprecipitate esulting romether reat-ment s not entirelydueto lipid. Even afterprolonged xtractionwith ethanol heresidualprecipitates still radioactive.In two experimentsn which herewas noapparent oss of serological roperties his residual adioactivitywas 42 and 44c.p.m.,representinga9 nd 6*3 /Of the totalvirusradioactivity. n anexperimentin whichether reatment esultedn 50 /Ooss of serological ropertieshe residualdenatured rotein avea countof 140c.p.m.,representing 3/O f the total. Whenthe virusproteinwas totallydenatured y ether reatment he residual enaturedproteinafterethanolextraction ave a countof 1504c.p.m.,representing 0/O fthe total virusradioactivity.

Since he non-lipid irusradioactivitys almostcertainlydue to nucleicacid itis probablehat the radioactivity f the residual enatured roteins dueto nucleicacid and this radioactivity ffords omemeasure f the amountof denaturation fthe virusnucleoprotein hichoccurs n ether reatment.It isof interest hat whenthe virusnucleoproteins completelydenatured y ethertreatment he wholeofthe nucleicacid s not splitof; a largepartremains ombinedwith the denaturedprotein.Thus in the experimentn which complete oss of serological ropertiesresulted romether reatmenthe non-lipid adioactivity mountedo 1980c.p.m.,of which 1504 c.p.m. was found in the denaturedproteinprecipitate,while476 c.p.m. was found n the aqueousphaseafter ethertreatment,presumably sfree nucleicacid.

DISTRIBUTION OF THE VIRUS NUCLEICACIDTheradioactivity f ether-treatedirus uspensionss in partdueto contaminatinginorganic2p present n the originalabelledviruspreparation,ut the majorpartof the activitymust n viewof the resultsof Graham1950)beattributed o nucleic


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124 L. HOYLE, . JOLLESNDR. G. MITCTTE.T.T]acid. About75/0of the originalvirusradioactivity ppearsn the ether-treatedsuspension.When he suspensions adsorbedwithredcellsthe majorpartof theactivity appears n the complement-fixingolubleantigenfraction.The smallamountof activity present n the agglutinin raction s exactly relatedto thecomplement-fixingntigencontentof this fraction.Theresultsof threeexperi-mentsareshownnTable3.They ndicate hattheradioactivitys associatedwiththe complement-fixingolubleantigenand not with the haemagglutinin. hehaemagglutininoesnot contain 2p andis evidentlynot a nucleoprotein.

Table3. Distribqltion f vir?s n?cleicacid radioacttvityCorrectedComplement- Geiger count Geiger countHaemagglutinin fixing per min. per min.Expt. Material titre antigen titre per ml. per ml.1 Ether-treated suspension 32,768 72 708 672Agglutinin fraction 32,768 8 65 65Soluble antigen fraction Nil 64 646 6102 Ether-treated suspension 51,200 24 486 451Agglutinin fraction 51,200 2@5 51 51Soluble antigen fraction Nil 24 454 4183 Ether-treated suspension 8,192 7 355 306Agglutinin fraction 8,192 Nil 2 2Soluble antigen fraction Nil 7 357 308

Note. (1) The corrected Geiger count is the true virus non-lipid radioactivity obtained bysubtractingfrom the observed radioactivity the activity due to contaminating inorganic 32ppresent n the original virus preparation. This activity appears in the ether-treated suspensionand n the soluble antigen fraction but not in the haemagglutinin fraction. (2) It will be notedhat in each of the three experiments the sum of the radioactivities of the agglutinin andsolubleantigen fractions is slightly greater than that of the original ether-treated suspension.his s probably a sampling error, but might be due to slight differences in the self-sereeningpropertiesof the various preparations.

RADIOACTIVITY OF THE SOLUBLE ANTIGENThemajorpartof the non-lipid adioactivity f the virusappearsn the solubleantigenraction,andit seemsprobablehat the antigens a nucleoprotein.How-ever,t wasthoughtpossible hat the effectof ethertreatmentmightbe to splitnucleiccid rom hevirusprotein, nd hattheradioactivityfthesoluble ntigenfractionightbe dueto freenucleicacidwhichwasnot actuallycombinedwiththentigen.Thefollowing xperimentsndicate hat this is not so andthat theantigens itselfradioactive.Previousworkhadshown hatthe antigencouldbeprecipitated y halfsatura-tion ithammoniumulphate.Considerableechnicaldifficultywasexperiencedinrecipitatinghe antigen romthe radioactive reparationss a resultof theirgreaturity. Additionof ammoniumulphate esultedn the appearancef onlyaaint opalescence nd it provedextremelydifficultto centrifugedown theprecipitateith the resultthat greatlossesof antigenoccurred.However, heexperimentas doneandthe slightprecipitateobtainedwa.swashedwith half


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Incorworatzonf radtoacttwephosphorusn the snffuenza ?,rqts125saturatedammonium ulphateand redissolved n water. Serological nd radio-activity measurementsave the following esult:

Original oluble antigen fraction Complement-fixing ntigen titre, 64Geigercount, 610 c.p.m.Redissolvedammonium ulphate precipitate Complement-fixing ntigen titre, 14Geigercount, 101 c.p.m.It appeared hat the antigenwas in fact radioactive.In a secondexperimentheantigenwas subjected o purification y six serialprecipitations ith ammoniumsulphate. The final producthad a complement-fixingntigen titre of 3 0 andregistered 1 c.p.m. n the Geiger ounter.Solubleantigencan also be precipitatedwith lanthanum cetate and partiallyrecovered rom the precipitateby extractionwith sodiumphosphate. A radio-active soluble antigen fractionwas precipitatedwith an equal volume of 1%lanthanumacetate,the precipitatewashedwith waterand extractedwith 2e5 /0disodiumphosphate olutionwith the following esult:

Original oluble antigen fraction Complement-fixing ntigen titre, 32Geigercount, 244 c.p.m.Phosphate extract of lanthanumacetate Complement-fixing ntigen titre, 10precipitate Geigercount, 87 c.p.m.Theseexperimentsndicate haUthe radioactivity f the solubleantigen raction sdue to the antigen,and that the antigenmust therefore ontain32p.

DISCUSSIONThisworkshows hat when nfluenza irus s grown n fertileeggs n whichradio-activephosphate as been ntroduced,he virus ncorporates2p into its structure.Thisconfirmshe originalworkof Graham&McLelland1949). It hadbeenhopedthat sufficient2p mightbe introducednto the virus o enable tudies o be madeof the fate of radioactive irus introduced s a primary noculum n fertileeggs.The amountOf 2p incorporatedn the virus is, however,probably oo small forthis purpose.The abelledviruspreparationsbtained n this workhad an averagehaemagglutininitre of 21,000,and an averageradioactivityof 820 c.p.m./ml.Since he maximum mountof viruswhichcan be induced o enter he cellsof thechorio-allantoicmembrane n growth cycle experiments s from 500 to 1000haemagglutiiin nits the radioactivity f such a primary noculumwouldbe only20-40 c.p.m.Thisamount s probablynadequateorreliable esults. t is, however,possiblethat the virus might be more effectively abelledby the use of largeramountsOf 2p, and also that the sensitiveness f the counting echnique ouldbeincreased bovethat attained n the presentwork.In our experiments 00Hc.Of 2p registered ,650,000 .p.m. This quantityof32p should actually emit 3,700,000p particlesper second, hence the countingefficiencywas only about 1e7 /O.By the use of driedpreparationsloselyappliedto the windowof the Geiger ounter,Graham&McLelland1950)attained muchhigher countingefficiency. However,our virus prepara.tions ere much moreeffectively abelled hanthoseof Graham&McLelland hocalculated hat in their


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126 L. HOYLE, . JOLLESNDR. G$.MITCHET]TJpreparationsherewasonlyoneatomof 32p in 66,500virusparticles.Thefollowingcalculationndicates he presencen ourpreparationsf oneatomof 32p in about670virusparticles.100Hc.Of 2p containing @61012tomsregistered ,650,000c.p.m.by ourtechnique.Hence1ml.of ouraverageviruspreparationegistering820c.p.m.contained1482x 106atomsOf 2p. TheaJverageaemagglutininitreofourpreparations as 21,000. Since15millionredcellswereusedin the haemag-glutinationest andsince t requires -4virusparticles ercellto givefullaggluti-nation, hepreparationmusthavecontained bout1012articles erml.Thisgivesa ratioof oneatomOf 2p to about670virusparticles.Ourpreparationshereforecontainedabout 100timesas much32p AS thoseof Graham&McLelland. hisdifferences dueto the use-inourworkof a doseOf 2p peregg500timesas largeas that usedby them.

About20-25/O f the 32p in the virusappears o be presentas phospholipid,a resultconfirminghatofGraham1950).Although oattempthasbeenmade nthis work o determine irectly he na,ture f the non-lipid2p, it is undoubtedlylinked o thevirusproteinand nviewoftheresultsofGraham1950) herecanbelittledoubt hat it is presentasnucleicacid.When heviruspreparations treatedwithethermostof the non-lipid 2p remainsn the ether-treateduspension, uta varyingamounts found nthedenatured roteinprecipitatewhichresults romethertrea,tment. heresultssuggest hat shakingwithethercauses progressivedenaturationfvirusprotein, helipoprotein eingattacked irst,andthenucleo-proteinater. It wouldbe expected hat the lipoproteinwouldbe denaturedirstif it is presentas a surfaceenvelope.Prolongedhakingwithethercauses otaldenaturation f the virusproteinwith loss of all serological roperties,but bycarefulcontrolof the extentof the ethertreatment t is possible o denatureheviruslipoprotein nd disintegratehe particlewithoutserious oss of serologicalproperties.Whenthis is donemostof the non-lipid 2p iS found n the aqueousphaseof the ether-treateduspension.Adsorption f theether-treateduspensionwithredcellsresults n moreorlesscomplete eparationfred-cellagglutinin ndcomplement-fixingolubleantigen. Solubleantigenfractionscan be producedwhichcontainnohaemagglutinint all. Haemagglutininractionsusuallycontainsmallamountsof complement-fixingntigen.Thenon-lipid2p iS almostentirelyfoundnthesoluble ntigen raction.Suchradioactivitys is found nthehaemag-glutinin raction s exactlyrelated o the complement-fixingntigen itre of thefraction.Thewholeof the non-lipid 2p Of he virusappearso be present n thecomplement-fixingolubleantigen, he haemagglutininpparently ontaining ophosphorus.Hoyle(1952)brought videnceo suggest hatthesolublea,ntigenwasa nucleo-protein,whilethe haemagglutinin as an enzymicaUllyctiveproteinwhichwasprobably ucleicacidfree.Thepresent esultsconfirmhis conclusion, ndaffordstrongevidence n favourof the hypothesis hat the complement-fixingolubleantigens the fundamentaleplicating ucleoproteinf the influenza irus.


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Incorporatzonf radtoacttrehosphorus >nhe tnflfuenzatrus 127SUMMARY1. When heD.S.P. strainof influenza irusA is grownn eggs ntowhich100Hc.Ofradioactivenorganic hosphatehasbeen ntroducedhe virus ncorporates2p

into its structure.2. Some 20-25% of the virus 32p iS found in the virus phospholipid;heremainders combinedwith the virusproteinandis probably resentn the virusnucleicacid.3. When the virusis disintegrated y ether treatmentwith the liberationofseparate ed-cellagglutinating ndcomplement-fixingsolubleantigen'particlesthe non-lipid2p is found o be associatedwiththesolubleantigen raction ndnotwiththe haemagglutinin.4. It is suggestedhat the complement-fixingolubleantigen s a nucleoproteinwhilethe haemagglutinins a phosphorus-freerotein.RE >'SHENCES

GRAHAM, . F. (1950). The chemical analysis of purified influenza virus A (PR8 strain)containing radioactive phosphorus. Canad.J. Res. 28, 186.GRAHAM, . F., DEMPSTER,G. & BUCHNER,B. (1952). The toxicity of p32 for normal andinfluenza virus-infected embryos. J. Bact.63, 426.GRAHAM, . F. & MCLELLAND,. (1949). Uptake of radioactive phosphorus by influenzavirus. Nature,Lond., 163, 949.GRAHAM,. F. & MCLELLAND,. (1950). The uptake of radioactive phosphorus by influenzavirus A (PR8 strain). Canad.J. Res. 28, 121.HOYLE,L. (1945). An analysis of the complement-fixation reaction in influenza. J. Hyg.,Camb.,44, 170.HOE, L. (1950). The multiplication of influenza viruses in the fertile egg. J. Hyg.,Camb., 8,277.HoYLE, L. (1952). Structure of the influenza virus. J. Hyg., Catnb.,50, 229.SALK,J. E. (1944). A simplified procedure for titrating haemagglutinating capacity of influenza virus and the corresponding antibody. J. IrBmunol. 9, 87.

(MS. receivedor publication 14. x. 53)

radioactive posphate virus

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