The Incorporation of Radioactive Phosphorus in the Influenza Virus and Its Distribution in Serologically Active Virus Fractions Author(s):

L. Hoyle, B. Jolles, R. G. Mitchell Source: The Journal of Hygiene, Vol. 52, No. 1 (Mar., 1954), pp. 119-127 Published by: Cambridge University Press Stable URL: http://www.jstor.org/stable/3860798 . Accessed: 21/10/2011 09:26
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119 ]

THE INCORPORATION RADIOACTIVE OF PHOSPHORUS IN '1't1EINFLUENZAVIRUS AND ITS DISTRIBUTION IN SEROLOGICALLY AC'l'lVE VIRUS FRACTIONS
BY L. HOYLE, B. JOLLESAND R. G. MITCHELL PubltcHeatth Laboratory, Northarnyton, theRadiotherapy cmd Departrnent, General Hospttat, Northampton Graham McLelland & (1949,1950)showedthat whenthe PR8 straUin influenza of virus A was grownin fertile eggs, into the allantoicsacs of which radioactive inorganicphosphatehad been introduced,the virus incorporated into its 32p structure.Graham1950)showed ( that the incorporated W&Sto be foundpartly 32p in the phospholipid partlyin the nucleicacid fractions the virus. and of This paperdescribes production influenza the of virusla.belled with 32p, and a studyof the distribution the isotopein serologically of activefractions the virus of produced etherdisintegration. by
PRODUCTION OF INFLUENZA VIRUS LABFJJ,T,E,n WITH
32p

In the originalworkof Graham McLelland & (1949)the viruswas grownin eggs inoculated with less than 1 ,uc.Of32p, sinceit was thoughtthat largeramountsof 32p might be lethal to the embryo. Later Graham, Dempster& Buchner(1952) foundthat this wasnot so andthat muchlarger amountsof 32p couldbe used.We ourselveshave noted no deaths in eggs inoculatedby the aJllantoic yolk-sac or routeswith 10s500 Hc.32p. A majordifficulty the production 32p labelledviruslies in the sepaUration in Of of the virus from contaminating radioactive phosphatein the infectedalla.ntoic fluids,and muchpreliminary workwas necessary determine best methodof to the virus purification.Preparations were regula.rly obtainedin whichthe non-viral radioactivityamountedto less than 10% of that caxTied the virus by the by following technique. D.S.P. strainof influenza The virusA wasusedthroughout the work. Radioactive phosphorus obtainedfromthe AtomicEnergyResearch was Establishment Harwell the formof calmier-free at in orthophosphoric this was acid, neutralized with sodiumhydroxide,diluted to contain 100 juc.in 0j2 ml. and sterilizedby immersion boiling-water 15 min. The radioactivephosphate in for solutionwas introduced the allantoicsac of sixteen 12-day-old into fertileeggsin a dose of 0a2ml., eachegg thus receiving 100Hc.of 32p. After4 hr. incubation at 36C.the eggswereinoculated the allantoic by routewith 0a1ml. of a.1:100dilution of D.S.P. virus-infected allaJntoic fluid. Aftera further40 hr. incubation the eggs wereopened,the chorio-allantoic Inembranes torn throughand allowedto bleedinto the allantoicfluid.The blood-stained allantoicfluidwas collectedin a receiver chilledwith ice. Fromthe 160ml. of fluidcollected red cellscarrying the

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L. HOYLE, JOLLES B. ANDR. G. MITCHFTJT.

the a.dsorbed virus were sedimentedby centrifugaJtion washedtwice with and 160 ml. of ice-coldsaline.The adsorbed viruswas then elutedfromthe cellsinto 16 ml. of salineby 4 hr. incubation 37C. The cells werethen sedimented at by centrifugation. supernataJnt representing tenfoldconcentration The fluid, a ofthe virusin the original infectedallantoicfluid,still contained largeamountof nona viral32p, and further purification therefore was necessary. fluidwas adsorbed The with 10% guinea-pigred cells, the cells werethen separated centrifugation by and washedsix times with 25 ml. quantitiesof ice-coldsaline. After the final washing cellsweresuspended 8 ml. of salinecontaining in 10,000 the in 1 CaC12 and wereincubated 4 hr. at 37C.to allowelutionof the virus.The cellswerethen for sedimented centrifugation removed 0a08 sodiumazidewasaddedto by and and /O the supernatant fluid. This constitutedthe labelledvirus preparation.A 1 ml. sampleof the preparation adsorbed was twicewith 10/O guinea-pig cells,and red the radioactivityof the originaland adsorbedfluids was tested. The red-cell agglutinin and complement-fixing a.ntigen titres werealso measured.
TECHNIQUE OF RADIOACTIVITY MEASUREMENTS

1 ml. samplesof the fluidsto be testedwereplacedin shallowmetaldishes2a5cm. in diameter,the fluid thus giving a layer 1@6 mm. thick. The disheswere then placedbeneatha Geiger-Muller counterof the end-window type at a distanceof 2 cm. Counting periodsrangedfrom 1 to 5 min. according the potencyof the to preparation. Undertheseconditions ,uc.Of32p wouldregister 100 3,650n000 counts in 1 min. (c.p.m.).The background countaveraged8 c.p.m.
RESULTS

The labelledvirus preparation had a red-cellagglutirlin titre of 16,000and a complement-fixing antigentitre of 48. Two successive adsorptions with 10/O red cellsreduced both titresto zero.The original prepara,tion registered c.p.m.in 913 the Cleiger counter, whilethe adsorbed fluidregistered 36 c.p.m. It wa.s only thereforeconcluded 877c.p.m.represented incorporated the virus.Whenthe that 32p in virus was eluted fromthe red cells used in the adsorption eluate registered the 845c.p.m.Table1 showsthe resultsof fivesuchexperiments whichthe viruswas in effectivelylabelled. Table 1. Radioactivity labelled of virms preparsbtzons and, before after red-cetl adsorption
Complement- Geiger count Geiger count Virus radioRed-cell fixingantigen per min. afterred-cell activity Preparation agglutinin titre titre per ml. adsorption (counts/min./ml.) 1 8,192 14 610 49 561 2 16,384 48 913 36 877 3 48,000 48 2,563 66 2497 4 8,192 12 336 28 308 5 25,600 24 732 35 697

phosphorus the influenzavirus tn of Incorporation radioacttwe

VIRUS '1'HER FRACTIONATIONOF LABF,T,T,F,n

121

by couldbe disintegrated virusparticles Hoyle (1950,1952)showedthat influenza and treatmentwith ether with the liberationof separatered-cellagglutinating that size. It wassuggested ' of 'soluble antigen particles smaller complement-fixing and of of body consisted an aggregate red-cellagglutinating the viruselementary particlesenclosedin a lipid envelope. Ether destroysthis complement-fixing shakingwith ether,however, envelopeand releasesthe smallerunits. Prolonged When properties. withlossof its serological of the virusprotein causesdenaturation it was technique appliedto the labelledviruspreparations the etherfractionation of the of was foundthat the duration the ethertreatment,and especially aJmount work,this being shaking,was muchmorecriticalthan hadbeenfoundin previous resultingfrom possiblydue to the very greatpurityof the labelledpreparations washingsnecessaryto and the doublecycle of adsorption-elution the numerous in was etherfractionation attained three phosphate.Satisfactory remove inorganic proved inadequate the out of fiveexperiments.In oneexperiment ethertreatment while in and only slight separationof agglutininand solubleantigenoccurred, prothe anotherexperiment ether treatmentwas excessiveand the serological pertiesweredestroyed. one. was experiments the following The techniqueused in the threesuccessful was The labelledviruspreparation shakenfor 5 sec. with half its volumeof ether for and incubated 1 hr. at 37C. in a closedtube. It was againshakenfor 5 sec. and and incubatedfor a furtherhour. The tube was then centrifuged the ether layer removed. A slight deposit of denaturedproteinresultedfrom the ether phospholipid workhad shownthat this depositcontained treatment.Preliminary washed proteinwa.s therefore whichcouldbe extractedby ethanol.Thedenatured with ethanol.The ethanolextractwas then with salineand extractedovernight fraction.The residual the addedto the ether,the wholeconstituting phospholiptd this and in denatured proteinwas dissolved N/1 sodiumhydroxide neutralized; is fraction. protesn referred as the denat?l,red to to in was viruspreparaUtion incubated an opentube overnight Theether-treated examination removeall tracesof ether. Afterremovalof a samplefor serological guinea-pig with 20/O was measurements remainder adsorbed the andradioactivity were haemagglutinin the The cells carrying adsorbed red cells, and centrifuged. salineandelutedfor 4 hr.at 37C.intosalinecontaining washed twicewithice-cold 1 in 10,000 CaCl2and 50 Australianunits of crystallinereceptordestroying recoveryof the enzyme(R.D.E.) per ml. The R.D.E. was addedto ensurecomplete and The cells were then removedby centrifugation the superhaema,gglutinin. ayglutinin fraction. the natant fluidconstituted red-cell was suspensionafter red-celladsorption of The supernatant the ether-treated and removalof haemagglutinin, with red cellsto ensurecomplete againadsorbed solbleantigen the complement-fixing after removal of the cells constituted fraction. and fractions then measured the serological was of Theradioactivity the various titres determined. Red-cellagglutininwas measuredby the Salk (1944)test,

122

L. HOYLE, JOLLES R. G. MITCHET.T] B. AND

0 4 ml. of a range of dilutionsof the preparation being mised with 0 4 ml. of 1:250 guinea-pig cells. Readings red weremadeafter 2 hr. at roomtemperature. Complement-fixing antigenwas measured the technique Hoyle (1945),using by of pooledhumanconvalescent serumfromthe A primeinfluenza epidemic 1951. of This serumreactedmainlywith the groupantigenof the virus, the serumcontainingvery little antibodyto the specificantigenof the D.S.P. stra.in influenza of
virus.

Resultsof a typicalexperiment shownin Table2. Theoriginal are labelled virus preparation a Salktitre of 16,384anda complement-fixing had antigentitre of 48. It registered c.p.m.in the Geigercounter. Adsorption 913 with red cellsreduced the serological to zeroandthe Geiger titre countto 36 c.p.m.Therefore c.p.m. 877 represented arirus and 36 c.p.m.contaminating 32p inorganic 32p. Table2. Ether fracttonatzon theD.S.P. strainof of tnJ!?enza viruslabelled 32p with
Material Originallabelled virus preparation Preparationafter two adsorptionswith red cells Ether-treatedsuspension Phospholipid fraction (ethereal extract+ ethanol extract of denaturedprotein) Residual denaturedprotein fraction Red-cell agglutininfraction Complement-fixing soluble antigen fraction Red-cell agglutinin titre 16,384 Nil 32,768 32,768 Nil Complement- Geigercount fixing per min. antigen titre per ml. 48 913 Nil 36 72 8 64 708 160 42 65 646

Ontreatmentwith etherthe agglutinin titre wa,sdoubled the complementand fixing antigen titre was also slightly increased.The ether-treaJted preparation registered708 c.p.m. The phospholipid fractionregistered160 c.p.m. and the denatured protein42 c.p.m. Adsorption the ether-treated of suspension with red cellsresulted almostcomplete in sepaWration of red-cell agglutinin complementand fixingantigen.Thesolubleantigenfractioncontained haemagglutinin. gaJve no It a complement-fixing antigentitre of 64 and registered c.p.m.in the Geiger 646 counter. The red-cellagglutinin fractionhad a haemagglutinin of 32,768. It titre containeda little complement-fixing antigen,giving a titre of 8, and its radioactivitywas 65 c.p.m. It was evidentthat the radioactivity the ether-treated of suspension associatedwith the complement-fixing was solubleantigenand not withthe haemagglutinin, slight radioactivity the haemagglutinin the of fraction beingentirelyaccountable its smallcontentof complement-fixing by antigen.
PHOSPHOLIPID CONTENT OF THE VIRUS

Previous work (Hoyle, 1952)had suggestedthat the viruselementary body was enclosed a lipid envelopederivedfromthe wall of the host cell. Whenvirus by preparations shakenwithethera precipitate denatured are of proteinoccurs the at ether-water interface. Lipid is presentin the ether extract, but this does not

virus 123 phosphorus thein;fuenza in Incorporation radioactive of

represent wholeof the virus lipid since morecan be extractedfromthe dethe with that natured protein precipitate ethanol. It seemsprobable the lipidis present by as a lipoprotein whichis denatured ethertreatment,the lipidbeingpartlydisprotein. In attachedto the denatured solved by the etherand partlyremaining 33 one experiment with labelledvirusthe etherextractregistered c.p.m.whilean proteinregistered127 c.p.m. In three experiethanolextract of the denatured withoutserious loss of mentsin whichetherdisintegration the viruswas achieved for lipidextractsaccounted 18, 23 and 23/O of serological properties combined the to shakingwith etherit is possible of the total virusradioactivity.By prolonged properties, and withtotal lossof serological denature wholeof the virusprotein the of in oneexperiment whichthis wasdonethe radioactivity the etherextractand in proteinrepresented /O the total virus 26 of of an ethanolextractof the denatured of concluded some20-25/O the radioactivity that radioactivity.It was,therefore, is This of 32p labelled viruspreparations dueto phospholipid. resultis in agreement Of virus whoshowed that the radioactivity 32p labelled withthat of Graham1950), ( the was partlydueto nucleicacidandpartlyto phospholipid, nucleicacidfraction beingabout 4 times as active as the lipidfraction.
PROGRESSIVE DENATURATION OF THE VIRUS PROTEIN BY '1'HER

fromethertreatproteinprecipitate resulting The radioactivity the denatured of withethanolthe extraction mentis not entirelydueto lipid. Evenafterprolonged in residual precipitate still radioactive.In two experiments whichtherewas no is was 42 and 44 properties residualradioactivity this apparentloss of serological In /O c.p.m.,representing and6*3 of the totalvirusradioactivity. anexperiment 4a9 the in loss properties residual in whichethertreatment resulted 50/O of serological 23 of denatured protein gavea countof 140c.p.m.,representing /O the total. When denatured by the the virusproteinwas totally denatured ethertreatment residual 60 of gave a countof 1504c.p.m.,representing /O proteinafterethanolextraction the total virusradioactivity. is Sincethe non-lipid virusradioactivity almostcertainlydue to nucleicacidit is of denatured protein dueto nucleic is probable the radioactivity the residual that of somemeasure the amountof denaturation of acidandthis radioactivity affords on the virusnucleoprotein whichoccurs ethertreatment.It is of interestthat when denatured ethertreatmentthe wholeof by the virusnucleoprotein completely is with the denatured combined the nucleicacidis not split of; a largepartremains properties in protein.Thusin the experiment whichcompleteloss of serological to the radioactivity amounted 1980c.p.m., resulted fromethertreatment non-lipid of which 1504 c.p.m. was found in the denaturedprotein precipitate,while as 476 c.p.m.was foundin the aqueousphaseafterethertreatment,presumably free nucleicacid.
DISTRIBUTION OF THE VIRUS NUCLEIC ACID

virussuspensions in partdueto contaminating is Theradioactivity ether-treated of but labelledviruspreparation, the majorpart inorganic presentin the original 32p to ( be of the activitymustin viewof the resultsof Graham1950) attributed nucleic

124

L. HOYLE, JOLLES R. G. MITCTTE.T.T] B. AND

acid. About 75/0of the originalvirusradioactivity appears the ether-treated in suspension. Whenthe suspension adsorbed is with red cellsthe majorpart of the activity appearsin the complement-fixing soluble antigen fraction.The small amountof activity presentin the agglutininfractionis exactly relatedto the complement-fixing antigencontent of this fraction.The resultsof three experimentsareshownin Table3. Theyindicatethat the radioactivity is associated with the complement-fixing soluble antigen and not with the haemagglutinin. The haemagglutinin not contain32p and is evidentlynot a does nucleoprotein. Table3. Distribqltion vir?s n?cleic acid radioacttvity of
Corrected Complement- Geiger count Geiger count Haemagglutinin fixing per min. per min. Expt. Material titre antigen titre per ml. per ml. 1 Ether-treated suspension 32,768 72 708 672 Agglutinin fraction 32,768 8 65 65 Soluble antigen fraction Nil 64 646 610 2 Ether-treated suspension 51,200 24 486 451 Agglutinin fraction 51,200 2@5 51 51 Soluble antigen fraction Nil 24 454 418 3 Ether-treated suspension 8,192 7 355 306 Agglutinin fraction 8,192 Nil 2 2 Soluble antigen fraction Nil 7 357 308 Note. (1) The corrected Geiger count is the true virus non-lipid radioactivity obtained by subtracting from the observed radioactivity the activity due to contaminating inorganic 32p presentin the original virus preparation. This activity appears in the ether-treated suspension andin the soluble antigen fraction but not in the haemagglutinin fraction. (2) It will be noted that in each of the three experiments the sum of the solubleantigen fractions is slightly greater than that of radioactivities of the agglutinin and the original ether-treated suspension. Thisis probably a sampling error, but might be due to slight differences in the self-sereening propertiesof the various preparations.

RADIOACTIVITY OF THE SOLUBLE ANTIGEN

The majorpart of the non-lipid radioactivity the virusappears the soluble of in antigen fraction,and it seemsprobable that the antigenis a nucleoprotein. However, was thoughtpossiblethat the effectof ether it treatmentmightbe to split nucleic fromthe virusprotein, that the radioactivity acid and of the soluble antigen fraction mightbe due to freenucleicacid whichwas not actually combined with the antigen.The following experiments indicatethat this is not so and that the antigen itself radioactive. is Previous workhad shownthat the antigencouldbe precipitated half by saturation ammonium with sulphate. Considerable technicaldifficulty experienced was in precipitating antigenfromthe radioactive the preparations a resultof their as great purity. Additionof ammonium sulphateresultedin the appearance only of a faint opalescence and it proved extremelydifficultto centrifugedown the precipitate the result that great losses of antigenoccurred. with However,the experiment done and the slight precipitateobtainedwa.s was washedwith half

Incorworatzon radtoacttwe of zphosphorus the snffuenza sn v?,rqts125

saturatedammonium sulphateand redissolved water. Serological radioin and activity measurements gave the following result:
Originalsoluble antigen fraction Complement-fixing antigen titre, 64 Geigercount, 610 c.p.m. Redissolvedammoniumsulphate precipitate Complement-fixing antigen titre, 14 Geigercount, 101 c.p.m.

It appeared that the antigenwas in fact radioactive.In a secondexperiment the antigenwas subjected purification six serialprecipitations to by with ammonium sulphate. The final producthad a complement-fixing antigen titre of 3 0 and registered c.p.m.in the Geigercounter. 21 Solubleantigencan also be precipitated with lanthanum acetateand partially recoveredfrom the precipitateby extractionwith sodiumphosphate. A radioactive solubleantigen fractionwas precipitated with an equal volume of 1% lanthanumacetate,the precipitate washedwith waterand extractedwith 2e5/0 disodium phosphate solutionwith the following result:
Originalsoluble antigen fraction Phosphateextract of lanthanumacetate precipitate Complement-fixing antigen titre, 32 Geigercount, 244 c.p.m. Complement-fixing antigen titre, 10 Geigercount, 87 c.p.m.

Theseexperiments indicatethaUt radioactivity the solubleantigenfraction the of is due to the antigen,and that the antigenmust therefore contain32p.
DISCUSSION

Thisworkshowsthat wheninfluenza virusis grownin fertileeggsin whichradioactivephosphate beenintroduced, virusincorporates into its structure. has the 32p Thisconfirms original the workof Graham McLelland & (1949). It hadbeenhoped that sufficient mightbe introduced the virusto enablestudiesto be made 32p into of the fate of radioactive virusintroduced a primary as inoculum fertileeggs. in The amountOf32p incorporated the virusis, however,probably smallfor in too this purpose. labelled The viruspreparations obtained this workhadan average in haemagglutinin titre of 21,000,and an averageradioactivityof 820 c.p.m./ml. Sincethe maximum amountof viruswhichcan be inducedto enterthe cellsof the chorio-allantoic membranein growth cycle experimentsis from 500 to 1000 haemagglutiiin unitsthe radioactivity sucha primary of inoculum wouldbe only 20-40c.p.m.Thisamount probably is inadequate reliable for results.It is, however, possiblethat the virus might be more effectivelylabelledby the use of larger amountsOf32p, andalsothat the sensitiveness the counting of technique couldbe increased abovethat attainedin the presentwork. In our experiments Hc.Of32p registered 100 3,650,000c.p.m. This quantityof 32p should actually emit 3,700,000p particlesper second, hence the counting efficiency only about 1e7 By the use of driedpreparations was /O. closelyapplied to the window the Geiger of counter, Graham McLelland & (1950)attaineda much higher countingefficiency. However,our virus prepara.tions were much more effectively labelled thanthoseof Graham McLelland calculated in their & who that

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L. HOYLE, JOLLES R. G$. B. AND MITCHET]TJ

preparations therewasonlyoneatomof 32p in 66,500virusparticles. following The calculation indicatesthe presence ourpreparations one atomof 32p in about in of 670 virusparticles. 100Hc.Of32p containing x 1012 6@6 atomsregistered 3,650,000 c.p.m.by ourtechnique.Hence 1 ml. of ouraverageviruspreparation registering 820 c.p.m.contained1482x 106 atomsOf32p. The aJverage haemagglutinin of titre our preparations 21,000. Since 15 millionred cellswereusedin the haemagwas glutination test and sinceit requires virusparticles cellto givefullaggluti3-4 per nation,the preparation musthavecontained about1012 particles ml. Thisgives per a ratioof one atom Of32p to about670virusparticles.Ourpreparations therefore containedabout 100 times as much 32p AS those of Graham McLelland. & This difference due to the use-inourworkof a doseOf32p peregg 500timesas large is as that used by them. About 20-25/O the 32p in the virus appearsto be presentas phospholipid, of a resultconfirming that of Graham (1950). Although attempthasbeenmadein no this workto determine directlythe na,ture the non-lipid it is undoubtedly of 32p, linkedto the virusproteinandin viewof the resultsof Graham (1950) therecanbe little doubtthat it is presentas nucleicacid.Whenthe viruspreparation treated is with ethermost of the non-lipid32p remainsin the ether-treated suspension, but a varyingamountis foundin the denatured proteinprecipitate whichresultsfrom ethertrea,tment. resultssuggestthat shakingwith ethercausesa progressive The denaturation virusprotein,the lipoprotein of beingattackedfirst,andthe nucleoproteinlater. It wouldbe expectedthat the lipoprotein wouldbe denatured first if it is presentas a surfaceenvelope. Prolonged shakingwith ether causestotal denaturation the virus proteinwith loss of all serological of properties, by but carefulcontrolof the extent of the ethertreatmentit is possibleto denature the virus lipoprotein disintegrate particlewithoutseriousloss of serological and the properties. Whenthis is done most of the non-lipid32p iS foundin the aqueous phaseof the ether-treated suspension.Adsorption the ether-treated of suspension with red cellsresultsin moreor less complete separation red-cell of agglutinin and complement-fixing soluble antigen. Solubleantigen fractionscan be produced whichcontainno haemagglutinin all. Haemagglutinin at fractions usuallycontain smallamountsof complement-fixing antigen.The non-lipid iS almostentirely 32p foundin the soluble antigenfraction.Suchradioactivity is foundin the haemagas glutininfractionis exactly relatedto the complement-fixing antigentitre of the fraction. The wholeof the non-lipid32p Ofthe virusappears be presentin the to complement-fixing solubleantigen,the haemagglutinin apparentlycontaining no phosphorus. Hoyle (1952)brought evidence suggestthat the solublea,ntigen a nucleoto was protein,whilethe haemagglutinin an enzymicaUlly was active proteinwhichwas probably nucleicacidfree.The presentresultsconfirm conclusion, afford this and strongevidencein favourof the hypothesisthat the complement-fixing soluble antigen the fundamental is replicating nucleoprotein the influenza of virus.

vtrus 127 ?>n phosphorus the tnflfuenza of Incorporatzon radtoacttre

SUMMARY

in virusA is grown eggsintowhich100Hc. 1. Whenthe D.S.P. strainof influenza 32p the has phosphate beenintroduced virusincorporates inorganic Ofradioactive into its structure. the 2. Some 20-25% of the virus 32p iS found in the virus phospholipid; presentin the virus with the virusproteinand is probably is remainder combined nucleicacid. by 3. Whenthe virus is disintegrated ether treatmentwith the liberationof 'solubleantigen'particles and separatered-cellagglutinating complement-fixing and antigenfraction not withthe soluble 32p the non-lipid is foundto be associated with the haemagglutinin. solubleantigenis a nucleoprotein that 4. It is suggested the complement-fixing protein. is whilethe haemagglutinin a phosphorus-free
RE >'SHENCES A. GRAHAM, F. (1950). The chemical analysis of purified influenza virus A (PR8 strain) containing radioactive phosphorus. Canad.J. Res. 28, 186. B. G. A. GRAHAM, F., DEMPSTER, & BUCHNER, (1952). The toxicity of p32 for normal and influenza virus-infected embryos. J. Bact. 63, 426. L. A. GRAHAM, F. & MCLELLAND, (1949). Uptake of radioactive phosphorus by influenza virus. Nature,Lond., 163, 949. L. A. GRAHAM, F. & MCLELLAND, (1950). The uptake of radioactive phosphorus by influenza virus 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. 48, HOE, L. ( 1950). The multiplication of influenza viruses in the fertile egg. J. Hyg., Camb., 277. 50, HoYLE, L. (1952). Structure of the influenza virus. J. Hyg., Catnb., 229. SALK,J. E. (1944). A simplified procedure for titrating haemagglutinating capacity of in 49, fluenza virus and the corresponding antibody. J. IrBmunol. 87.

for (MS. received publication 14. x. 53)