Biochem. J. (1961) 81, 51

Studies on Protein and Nucleic Acid Metabolism in Virus-InfectedMammalian Cells

4. THE LOCALIZATION OF METABOLIC CHANGES WITHIN SUBCELLULAR FRACTIONSOF KREBS II MOUSE-ASCITES-TUMOUR CELLS INFECTED WITH

ENCEPHIALOMYOCARDITIS VlRUS*

BY E. M. MARTIN AD T. S. WORKNational Institute for Medical Re8earch, MiU Hill, London, N.W. 7

(Received 18 May 1961)

Martin, Malec, Sved & Work (1961 b) showedthat, during a single growth cycle, infection withencephalomyocarditis virus caused no changes inthe total deoxyribonucleic acid, ribonucleic acid orprotein of the host ascites-tumour cell. However,by the use of [6-14C]orotic acid and [14C]valine, itwas shown that there were substantial changes inthe rates of turnover of ribonucleic acid andproteinat different times during the cycle of virus growth.In particular, about 5 hr. after infection there wasa striking increase in the rate of turnover of ribo-nucleic acid, and this increase coincided in timewith the appearance of new virus. However, thisstimulation in turnover appeared to be quanti-tatively greater than could be accounted for byformation of new virus ribonucleic acid.Methods have now been developed for the well-

defined separation of disrupted Krebs II ascites-tumour cells into nuclei, mitochondria, micro-somes and cell sap (Martin, Malec, Coote & Work,1961 a). By the use of these methods we have beenable to investigate the effect of virus infection onthe turnover of the ribonucleic acid and protein ofthe subcellular components of the host cell, andthus to localize more exactly the sites of metabolicchange within the infected cell. This paper de-scribes the result of such a study.

METHODS

The origin and propagation of both the Krebs II mouse-ascites-tumour cells and the encephalomyocarditis virushave been described by Martin et al. (1961 b).

Conditions of infection. Sufficient virus was added tosuspensions of washed ascites-tumour cells in Earle'smedium (2-3 x 107 cells/ml.) to infect all cells (approx.3 plaque-forming units/cell), and the cells were dispensedin 10 ml. portions into incubation flasks. The virus wasallowed to adsorb for 30 min. at room temperature, fol-lowed by 15 min. at 360. Suspensions of control (unin-fected) cells were treated similarly. The cells were thenincubated at 36° as described by Martin et al. (1961 b).

* Part 3: Martin, Malec, Coote & Work (1961 a).

For the study of protein and nucleic acid turnover,14C-labelled precursors were added at appropriate intervalsduring the virus growth cycle to flasks containing infectedand control cells; 30 min. later the flasks were removed anddiluted with an ice-cold solution of the unlabelled precursorin phosphate-buffered saline (Martin et al. 1961 b), and thecells sedimented by centrifuging at 120g for 5 min. Theywere then washed twice with buffered saline and stored inan ice bath until required for disruption.

Disruption of Kreb8 11 cells andfractionation of subcellularcomponents. Cells were prepared for disruption by washingwith calcium- and magnesium-free buffered saline (Martinet al. 1961 b), then with 0-125M-sucrose-0-075M-KCl solu-tion. The cells were disrupted by a combination of doubleosmotic shock and Potter homogenizer, as described byMartin et al. (1961 a). Nuclei, mitochondria, microsomesand cell sap were separated from the tumour-coll homo-genates by differential centrifuging in 0-25M-sucrose-0- 1M-KCI solution (Martin et al. 1961 a). Preparations ofisolated nuclei were usually examined microscopically afterstaining with nigrosin, and were always found to containless than 3% of whole-cell contamination. The inter-mediate fraction that separated between nuclei and mito-chondria, and which represented only a very small per-centage of the cell contents, was discarded. Mitochondriawere washed once with sucrose-KCI solution, but the micro-somal fraction was not further treated after isolation exceptto rinse the surface of the pellet with distilled water.

Estimation of virus. Virus was estimated in tumour-cellhomogenates and in the subcellular fractions derived fromthem by both haemagglutinin titration and plaque assay,as described by Martin et al. (1961 b). To release anyadsorbed virus, the nuclear fractions were incubated at 320for 1 hr. with deoxyribonuclease (Worthington BiochemicalCorp., Freehold, N.J., U.S.A.; 0-1 mg./ml.) before virusassay.

Estimation of protein and nucleic acids. Protein, RNAand DNA were isolated from tumour-cell homogenates,mitochondria, microsomes and cell sap and estimated asdescribed by Martin et al. (1961 b). The extraction methodswere not considered ideal for the isolation and estimationof RNA from the nuclear fraction. Therefore the nuclei,free of lipids and material soluble in cold 0-2N-HClO4, weretreated with 0-3N-NaOH at 370 for 18 hr. to hydrolyseRNA, and protein and DNA were precipitated by additionof HC104. The DNA was removed from the acid-insolubleprecipitate by extraction with 0-5N-HCl04 at 700 for30 min. or, when required for the estimation of specific

5;14

METABOLISM IN VIRUS-INFECTED CELLS

Table 1. Specific radioactivities of Krebs ceU protein and nucleic acids after incubation with [4C]valineand [6-14(C]orotic acid and a mixture of both labelled compounds

Flasks containing 108 ascites-tumour cells in 5 ml. of Earle's medium and the indicated "4C-labelled com-pounds were incubated for 30 min. at 360 (Martin, Malec, Sved & Work, 1961 b). Total nucleic acid and proteinextracts were prepared and their radioactivities determined, as described in the text.

"4C-compound added[14C]Valine (0.1 ftc)[6-J4C]Orotic acid (5 ,uc)[140]Valine plus [6-14C]orotic acid[14C]Valine plus [6-14C]orotic acid

Time ofincubation

(min.)3030300

Specific radioactivities

Nucleic acidsProtein (1Lc/mole of(/Amc/g.) nucleic acid P)

276 273 714[

266 6653 2

radioactivity, by extraction with 10% (w/v) NaCl solution(buffered at pH 6-0 with 0-1M-sodium acetate). DNA was

recovered from the hot saline extract by precipitation with2-5 vol. of ethanol.

Mea8urement of radioactivity. Since the amount of workinvolved in cell disruption, in fractionation of the sub-cellular particles and in the separation of RNA and proteinis rather considerable, it was thought best to study theincorporation of radioactive precursors (in this case

[6-14C]orotic acid and [14C]valine) into both RNA andprotein simultaneously. Before this could be done, how-ever, it was necessary to show that cross-contamination ofnucleic acid with protein, or vice versa, would not cause any

significant error in the estimation of specific radioactivity.Ascites-tumour cells were therefore incubated in mediacontaining either [6-14C]orotic acid or [14C]valine or a

mixture of both labelled compounds. After incubationunder the standard conditions, the appropriate 12C carrierwas added, and the protein and nucleic acid were assayedfor radioactivity by the following method. The cells were

resuspended in 0 1 M-DL-vahne solution, HC104 was addedto a concentration of 0-2m, and the acid-insoluble materialtreated with an aqueous solution of trimethylamine (0-5M)containing DL-vahne (0-07M) for 20 min. at 55°. Thematerial did not completely dissolve, but the suspension,became translucent. The suspension was cooled to 0°,HClO4 added to a concentration of 0-2M, and the pre-

cipitated material centrifuged off. The supernatant was

examined for the presence of extracted nucleotides. Onlyabout 3% of the total RNA was extracted during the tri-methylamine treatment. The precipitate was washed twicewith ice-cold 0-2M-HClO4. Total nucleic acidIs and proteinwere then extracted, and their specific radioactivities deter-mined as described by Martin et al. (1961 b).From the results of this experiment (Table 1) it was con-

cluded that there was no contamination of protein by thelabelled orotic acid, but that slight contamination (about4%) of the nucleic acid with labelled valine may occur. Ina further experiment, Krebs cells were incubated for30 min. with a fixed amount of [6-14C]orotic acid (5 ,uc/flaskcontaining 108 cells in 5 ml. of medium) and variousamounts of [14Clvaline (0-02-0-2 ,uc/flask). When the radio-activity of the nucleic acid and protein was measured itwas found that the slight contamination of the nucleicacid extract with valine was directly proportional to thespecific activity ofthe protein fraction. It was thus possibleto apply a suitable correction factor to the measuredspecific activity of nucleic acid to account for any con-

taminating radioactive amino acid present in the extract.In experiments where both labelled precursors were usedsimultaneously, two control incubations were thereforeincluded:

(1) A flask in which the cells were incubated for the fullperiod with [14C]valine, then [6-"4C]orotic acid added justbefore the carrier. The radioactivity of the nucleic acidsfrom these cells gave a measure of valine contamination,and the extent of this contamination was assumed to beproportional to the specific radioactivity of the proteinfraction.

(2) A flask in which cells were incubated with [6-14C]-orotic acid for the full period, then [L4C]valine added justbefore the carrier. The specific radioactivity of the proteinfrom these cells was used as the zero-time control forestimates of protein radioactivity.

EXPERIMENTAL AND RESULTS

Effect of infection on incorporation of precursorsinto the protein and nucleic acids of subceUularcomponents from tumour cells. A series of infectedand control cultures of Krebs II ascites-tumourcells were incubated in Earle's medium underidentical conditions. Exactly 30 min. beforeremoving each flask from the incubation chamber,a mixture of [14C]valine and [6-14C]orotic acid wasadded. Flasks were removed at hourly intervalsthroughout the major portion of the virus growthcycle, 12C carrier was added, and the cells were

cooled at 00, washed and disrupted by the double-osmotic-shock method (Martin et al. 1961 a).Portions of the whole lysate were saved and theremainder was subjected to differential centrifugingin 0-25M-sucrose-0-1M-KCI. Protein and RNAwere isolated from the nuclear, mitochondrial,microsomal and cell-sap fractions, as describedunder Methods, and their specific radioactivitieswere determined. Specific radioactivity measure-

ments were also made on DNA from the nuclei.The results are summarized in Table 2. Difficultywas experienced in obtaining a representativesample of the nuclear protein by the method used(cf. Martin et al. 1961 a), and no figures for the turn-over rate of protein in the nuclear fraction are

33-2

Ratio ofspecific

activities:Nucleic acid

Protein

2-592-50

Vol. 81 515

E. M. MARTIN AND T. S. WORK

given. As each subcellular fraction was isolatedfrom the infected cells, part was set aside and itsvirus content assayed by the haemagglutinationtechnique. The results of these assays are also givenin Table 2.

Haemagglutinin is to a great extent concentratedin the mitochondrial fraction and appears in themicrosomal fraction only in smaller amounts(Tables 2 and 4). At no time during the infectioncycle were significant amounts of haemagglutininfound in the cell-sap fraction, and the trace ofhaemagglutinin found in the nuclear fraction wasin part due to interference with the haemag-glutinin reaction at low dilutions by DNA fromthe nucleus and in part to slight contaminationwith mitochondria.

The percentage of virus in the mitochondrialfraction of homogenates prepared by osmoticshock varied in successive experiments (67 % oftotal in the experiment reported in Table 2, and95% in the experiment of Table 4), but thisprobably reflects the impossibility of exact dupli-cation of any method of cell rupture rather thanbiological variation.The association of virus with the mitochondria

was unexpected. It is certainly not caused by thesedimentation characteristics of the viral particle,since virus added to the cell homogenate beforefractionation appears almost exclusively in themicrosomal fraction after separation by differentialcentrifuging. As a further check, we have dis-rupted Krebs cells by an alternative method

Table 2. Effect of encephalomyocarditi8-vir?hs infection on the incorporation of [14C]valine and [6-14C]oroticacid into the protein8 and nucleic acide of a8citeB-tumour 8ubcellular fractionm

Portions (10 ml.) of control and infected (3 plaque-forming units of virus/cell) suspensions of ascites-tumourcells (3 x 107 cells/ml.) were incubated at 360 for the various times indicated under conditions described byMartin et al. (1961 b). No attempt was made at synchronizing the infectious process. [14C]Valine (0-5 ,uc) and[6-14C]orotic acid (25 jAc) were then added as a solution in 1-0 ml. of buffered saline, and the flasks incubatedfor a further 30 min. The cells were washed, disrupted by osmotic shock and separated into the various sub-cellular fractions. Portions of each fraction were diluted with distilled water, stored overnight at 40 and assayedfor haemagglutinating activity. Protein, RNA and DNA were isolated from the remaining portions and theirspecific radioactivities determined. All estimates of RNA specific activity have been corrected for contaminationwith valine, and of protein specific activity for contamination with orotic acid. The virus haemagglutinintitres are given in total haemagglutinin units/flask (3 x 108 cells), see Martin et al. (1961 b).

Time after inoculation with virus (hr.)

Flask

RNA (,&c/m-moleof RNA P)DNA (,c/m-moleof DNA P)

Virus haemagglutinintitre

Protein (,uc/g.)

RNA (juc/m-moleof RNA P)

Virus haemagglutinintitre

Protein (,uc/g.)

RNA (j,c/m-moleof RNA P)

Virus haemagglutinintitre

Protein (,uc/g.)

RNA (pc/m-moleof RNA P)

Virus haemagglutinintitre

1

ControlInfectedControlInfectedInfected

ControlInfectedControlInfectedInfected

ControlInfectedControlInfectedInfected

ControlInfectedControlInfectedInfected

55-845-30-1000-108

200

1-331-481-221-040

1-161-240-4870-4480

0-840-933-192-680

2 3Nuclear fraction31-4 32-115-9 12-20-088 0-0730-083 0-058

200 200

Mitochondrial fraction1-08 0-931-01 0-910-910 0-7750-780 0-6200 0

Microsomal fraction1-04 1-031-08 0-840-315 0 3080-206 0-1400 0

Cell-sap fraction0-68 0-620-79 0-662-55 1-741-98 1-070 0

4

30-15-60-0740-043

200

0-820-570 5950-665

1100

0-990-730-3100-16535

0-490-431-621-220

5

30-37-80-0660-042

250

0-720-460-5650-655

1100

0-990-520-3240-18970

0-530-301-531-380

6

31-73-20-0540-019

2050

0-660-650-6101-98

36000

0-760-470-3230-545

18000

0-430-241-922-05

40

516 1961

METABOLISM IN VIRUS-INFECTED CELLS

(Dounce homogenizer in 10 mm-MgCl2; see Martinet al. 1961 a), and again found most of the haemag-glutinin in the mitochondrial fraction. Bellett &Burness (1960) have also reported the concentra-tion of haemagglutinin in the mitochondrialfraction of Krebs II cells 45-6 hr. after infectionwith encephalomyocarditis virus.When infected cells were disrupted by ultra-

sonic vibrations as described by Martin et al.(1961 a), the virus was found largely in the micro-somal fraction. In keeping with this observation itwas found that virus could be released from mito-chondria by treatment with ultrasonic vibrations.When these experiments were designed it was

assumed that haemagglutinin could be equatedwith virus. This assumption is supported by ourdemonstration (Faulkner, Martin, Sved, Valentine& Work, 1961) that the ratio of haemagglutinin toinfectivity is the same in crystalline encephalo-myocarditis virus as it is in crude virus preparation.This view is further strengthened by the resultsshown in Table 4, which indicate that haemag-glutinating activity is associated with infectivevirus both in time of appearance during the growthcycle and in the site within the cell of maximumvirus concentration. More precise measurements(E. M. Martin & T. S. Work, unpublished work)show that a slight delay occurs between the forma-tion of viral protein and the appearance of anequivalent haemagglutinating activity, and thisagain suggests that haemagglutinating activity is ameasure of the whole infective virus particlerather than of partially completed forms.

There are large differences in the rates of in-corporation of orotic acid and valine into thedifferent subcellular fractions of normal (control)Krebs II cells (Table 2). The rates of valine in-corporation into the proteins of nuclei, mito-chondria, microsomes and cell sap are in the pro-portions 0-28:1-0: 1-2:0-67, whereas the rates oforotic acid incorporation into the ribonucleic acidsof the same fractions are in the proportions50:1 0:0-5:3 0. By using the data of Martin et al.(1961a) for the distribution of protein and RNAamong these fractions, it can be calculated thatturnover ofRNA in the nucleus accounts for 88% ofthe total cell RNA turnover, whereas mitochondria,microsomes and cell sap contribute 24, 33 and 39%respectively to the total turnover of protein.

Infection with encephalomyocarditis virus causedan initial slight stimulation of valine incorporationinto the proteins of all fractions. This stimulationwas observed with whole-cell preparations (Martinet al. 1961 b). The effect was most marked in thecell sap, thus supporting the suggestion that thisinitial stimulation in protein synthesis may repre-sent the synthesis of new enzymes necessary forviral replication.

There then follows a period of general inhibitionof protein turnover. The inhibition continued pro-gressively during the course of the experiment inall fractions except the mitochondria, whichshowed marked increase in turnover rate from 5 hr.after infection. It is reasonable to suppose thatthis stimulation of protein turnover is associatedwith the appearance of virus in this fraction, and itis possible that it represents the incorporation ofvaline into the viral protein, as 6-6-5 hr. afterinfection is the period of maximal viral-proteinsynthesis under the growth conditions used in thepresent series of experiments.

Quantitative changes in ribonucleic acid in nuclei andmitochondria during virus infection

Martin et al. (1961b) showed that there was nosignificant change in the overall composition ofinfected Krebs cells as compared with normalcontrols. The marked fall in RNA turnover withinnuclei of infected cells and the threefold increase inrate of turnover of RNA in the mitochondria ofinfected cells towards the end of the infectiouscycle prompted the thought that there might havebeen quite substantial changes in the overall com-position of subcellular fractions, but that, bychance, these had balanced one another and soproduced the apparent overall constancy of com-position observed earlier (Martin et al. 1961 b).This view was strengthened by the results of theexperiment described in Table 2, when it wasobserved that the net recovery of RNA from themitochondrial fraction of cells in the later stages ofinfection was far higher than their correspondingcontrols, whereas the RNA to DNA ratio in in-fected nuclei appeared to fall. Accordingly, anexperiment was set up to settle this point.Krebs cells were harvested, washed and sus-

pended in Earle's medium in the usual way (Martinet al. 1961 b). The cell suspension was infected withencephalomyocarditis virus. All flasks were left atroom temperature for 30 min. and then incubatedat 360 under the standard conditions; one controland one infected flask were removed at 2 hr.,another pair at 4 hr. and the last pair at 6-5 hr.The cells were collected, washed and disrupted bythe double-osmotic-shock method (Martin et al.1961 a). The nuclear and mitochondrial fractionswere isolated in the usual way, but the microsomefraction was not separated from the cell sap. Thenuclear fractions were analysed for RNA and forDNA (see Methods), and the mitochondrialfractions were analysed for protein and for RNA(Table 3). In addition, the cytoplasmic fractionswere assayed both for haemagglutinin and forviable virus (plaque count) (Table 4). The results ofthis experiment show that infection produces asubstantial (39 %) increase in the amount of RNA

Vol. 81 517

E. M. MARTIN AND T. S. WORK

Table 3. Effect of encephalomyocarditis-viru8 infection on the net amount8 of nuclear and mitochondrialribonucleic acids of Krebs II cells

Suspensions of ascites-tumour cells (2 x 107 cells/ml.; 10 ml./flask) were incubated with encephalomyocarditisvirus (3 plaque-forming units of virus/cell) for 2, 4 and 6-5 hr., together with uninfected controls. The flasks wereremoved, and the cells washed and disrupted by the double-osmotic-shock method. The lysates were fractionatedto yield nuclear, mitochondrial and microsome-plus-cell-sap fractions (Martin, Malec, Coote & Work, 1961a).The mitochondrial fraction was analysed for protein and RNA content, and DNA and RNA estimates werecarried out on the nuclear fraction. AR results have been calculated as the total amount of each constituentfor 108 cells, assuming the distribution of protein and DNA to be that given in Table 5 [Martin et al. (1961a)].The total nuclear-plus-cytoplasmic RNA content/108 cels was 230 ug. of RNA phosphorus.

Period of infection (hr.)

Cell fraction FlaskMitochondria Infected

ControlDifference

Nuclei InfectedControlDifference

Net change in RNA P (% of totalceH RNA P)

2 4 6-RNA P (ig./108 cels)

20-622-5-1-925-825-1+0-7-0-5

25-720-7+5-024-329-8-5.5-0-2

30-822-2+8-627-932-4-4.5

Table 4. Distribution of viral haemagglutinin and infective virus among cytoplsmic fractions frominfected Kreb8 II ascites-tumour cells

Portions of the mitochondrial and mixed microsome-plus-cell-sap fractions from the experiment describedin Table 3 were examined for their virus content by haemagglutinin titration and infective particle (plaque)count assay. Results are expressed as haemagglutinin units or plaque-forming units/108 cells.

Period of infection (hr.)

Cell fractionMitochondria

Microsomes pluscell sap

Virus assay methodHaemagglutinin10-6 x Infective particlesHaemagglutinin10-6 x Infective particles

in the mitochondrial fraction 6-5 hr. after infectionand that this is almost balanced by a correspondingdecrease in the amount of nuclear RNA so that theoverall change is negligible (1-8 %). In a secondsimilar experiment the percentage increase inmitochondrial RNA in infected cells at 6 hr. waseven greater than that shown in Table 3.

DISCUSSION

Infection caused profound alterations in theRNA metabolism of the tumour cell. Since therewas a slow fall in the rate of metabolism of thecontrol cells throughout the course of the incuba-tion, the results for the infected cells obtained inthe experiment described in Table 2 have beenexpressed as a percentage of the correspondingcontrol and plotted in Fig. 1, together with thefigures for virus haemagglutinin titre in the mito-chondrial fraction. It is evident that coincidentwith the appearance of virus in the mitochondrial

fraction there is an enormous increase (320 %) inthe rate of orotic acid incorporation into the RNAof this fraction. At the same time there is a similar,though smaller, increase in the rate of turnover ofRNA in the microsomal fraction. A large increasein total cell RNA turnover at about the time ofsynthesis of complete virus was reported by Martinet al. (1961 b). The present results indicate that thisis accounted for largely by the increase in turnoverof the RNA of the mitochondrial and microsomalfractions.

It has been proposed (Martin & Work, 1961) thatthe synthesis of RNA in ascites-tumour cells takesplace entirely in the nucleus. This contention issupported by the results given in Table 2, whichshow a constant relationship between the rates oforotic acid incorporation into the ribonucleic acidsof the nuclear and cytoplasmic fractions, in bothnormal cells and cells up to 4 hr. after infectionwith encephalomyocarditis virus. However, in cellsinfected for a greater period, the rate of cyto-

*5

201-75

120-4

415015-5121-0

6-512300

65067522

5;18 1961

r4A

-I

METABOLISM IN VIRUS-INFECTED CELLS

plasmic RNA synthesis, calculated from the ratiosof specific activities of the uridylic acid in RNAand the acid-soluble pool (Martin et al. 1961 b), farexceeds that which would be expected from thenuclear RNA turnover rate, assuming that thenucleo-cytoplasmic relationship had remainedunaltered. Therefore it may be argued that thisdifference represents the synthesis of viral RNA.However, when the amount of this anomalous,newly synthesized cytoplasmic RNA is comparedwith the amount of virus-associated RNA ex-

pected to be formed during the same 30 min.period, as estimated from the increase in haemag-glutinin titre by the data of Faulkner et al. (1961),the virus-associated RNA represented only 5-8%of the total RNA formed. Hence, the stimulationin mitochondrial and microsomal RNA turnover inthe later stages of infection cannot be accounted forin terms of synthesis of virus-associated RNA.From the beginning of infection the rate of

precursor incorporation into the RNA of thenucleus was progressively inhibited (Fig. 1). Asnearly 90% of the total cell RNA turnover takesplace in the nucleus, and as it is probable thatmost or all of the cell's RNA is synthesized at thissite, it is likely that the marked inhibition of RNAturnover in the whole cell (Martin et al. 1961 b) and

+0r

-

.

00

4)

c;60

00

4)

cz

4.

0

cB

0q.,d3

0 2 3 4 5Time after infection (hr.)

Fig. 1. Effect of viral infection on rate of [6-L4C]oroticacid incorporation into the RNA of nuclei, mitochondriaand microsomes. Estimates of specific radioactivities ofthe RNA from the nuclei, mitochondria and microsomes ofvirus-infected Krebs II cells have been plotted as a ratioof the estimates of specific radioactivity of control un-infected cells. Experimental details are given in Table 2.*, Nuclear RNA; 0, mitochondrial RNA; /\, microsomalRNA; 0, haemagglutinin titre (units/108 cells) of themitochondrial fraction.

the slight inhibition of turnover in the cytoplasmicconstituents in the eclipse phase (Table 2; Fig. 1)can be ascribed to the effect of infection on nuclearRNA synthesis. Although this inhibition is doubt-less a consequence of viral infection, there is littleevidence to suggest that it is concerned with thereplication of viral constituents.

Sanders, Huppert & Hoskins (1958) and Sanders(1960) have shown that the ability of encephalo-myocarditis virus to kill Krebs II cells is inde-pendent of its power to multiply within them, and asimilar separation between cell-killing propertiesand viral replication has also been observed inHeLa cells infected with poliomyelitis virus(Ackermann, Rabson & Kurtz, 1954). Hence it ispossible that the inhibition of nuclear RNAsynthesis is associated with the process, caused byinfection but unrelated to the replication of viralRNA or protein, which leads to the death of thecell.Huppert & Sanders (1958) showed that in-

fective RNA could be extracted by cold aqueousphenol from Krebs II ascites-tumour cells that hadbeen infected with encephalomyocarditis virus,although no RNA could be obtained from the virusparticles by this treatment. Bellett & Burness(1960) have used the osmotic-shock method ofMartin et al. (1961a) to localize the formation ofinfective RNA, and found it to be almost entirelyconfined to the nucleus during the first 4-5 hr. afterinfection, thus giving a direct demonstration thatthe nucleus is the site of infective RNA synthesisin this system.

Bellett & Burness (1960) found that the titre ofinfective RNA in the nucleus decreased sharplyafter 4-5 hr., and that this loss was accompaniedby a rise in the infectivity of the RNA from themitochondrial fraction. These results suggested anucleo-cytoplasmic transfer of the viral RNA. Thepresent results (Table 3) strongly support the ideaof a transfer of RNA from nucleus to cytoplasm,but the amount of RNA transferred is muchgreater than would be required for the formation ofvirus particles. It may well be, however, that thecell produces considerably more virus-specificRNA than it can incorporate into virus. Theappearance of free infective RNA in the culturemedium at the end of the virus growth cyclesuggests that this does in fact occur (Huppert &Sanders, 1958).Although the present results demonstrate un-

equivocally that virus-protein synthesis takesplace in the cytoplasm of the Krebs tumour cell,they are less definitive with regard to the site ofRNA synthesis. We have emphasized elsewhere(Work, 1960; Martin & Work, 1961) that biologicalreplication always requires the simultaneouspresence of DNA and RNA and that neither DNA

Vol. 81 519

520 E. M. MARTIN AND T. S. WORK 1961

nor RNA virus is capable of replication except inan environment that can supply the missing com-ponents. Such a requirement could well explainthe apparent formation of infective RNA withinthe nucleus of the ascites-tumour cell and thetransfer of this to the cytoplasm before synthesis ofcomplete virus can begin.

SUMMARY

1. The effect of infection with encephalomyo-carditis virus on the rate of incorporation of 14C0labelled precursors into the protein and ribonucleicacid of the subcellular components of Krebs IIascites-tumour cells has been investigated.

2. The nucleus, which was the major site ofribonucleic acid synthesis in the cell, containednegligible amounts of virus. Infection caused amarked progressive inhibition of orotic acid in-corporation into nuclear ribonucleic acid, and alsosome net loss of ribonucleic acid from the nucleus.It is suggested that this disruption of nuclearribonucleic acid metabolism is related to the cell-killing properties of the virus.

3. Most of the virus sedimented with the mito-chondrial fraction. The amount of mitochondrialribonucleic acid increased progressively duringinfection by an amount approximately equivalentto that lost from the nucleus. Incorporation oforotic acid into mitochondrial ribonucleic acid wasslightly inhibited for the first 3 hr. after infection;thereafter it was stimulated, reaching 320% of thecontrol at 6 hr.

4. Less virus appeared in the microsomal frac-tion. The pattern of incorporation into microsomal

ribonucleic acid was similar to that of the mito-chondria, but less pronounced. In both fractions,the increase in incorporation rate was apparentlyrelated to the amount of virus present, but esti-mates showed that only 5-8% of this newlysynthesized ribonucleic acid could be ascribed toviral ribonucleic acid formation.

5. In all cytoplasmic fractions, infection causedan initial slight stimulation of valine incorporationinto protein, which was most marked in the cell-sapfraction. This was followed by a period of moderateinhibition, until appreciable amounts of virus hadaccumulated intracellularly, when incorporationinto mitochondrial protein was again elevated.

REFERENCES

Ackermann, W. W., Rabson, A. & Kurtz, H. (1954). J.exp. Med. 100, 437.

Bellett, A. J. D. & Burness, A. T. H. (1960). Biochem. J.77, 17P.

Faulkner, P., Martin, E. M., Sved, S., Valentine, R. C. &Work, T. S. (1961). Bioch-em. J. 80, 597.

Huppert, J. & Sanders, F. K. (1958). C.R. Acad. Sci.,Paris, 248, 2067.

Martin, E. M., Malec, J., Coote, J. L. & Work, T. S.(1961a). Biochem. J. 80, 606.

Martin, E. M., Malec, J., Sved, S. & Work, T. S. (1961 b).Biochem. J. 80, 585.

Martin, E. M. & Work, T. S. (1961). Proc. 5th int. Congr.Biochem., Mo8cow, 2.

Sanders, F. K. (1960). Nature, Lond., 185, 802.Sanders, F. K., Huppert, J. & Hoskins, J. M. (1958).Symp. Soc. exp. Biol. 12, 123.

Work, T. S. (1960). In Developing Cell Systems and theirControl, p. 205. Ed. by Rudnick, D. New York: RonaldPress Co.

Biochem. J. (1961) 81, 520

A Study of the Kinetics of the Fibrillar Adenosine Triphosphataseof Rabbit Skeletal Muscle

BY J. R. BENDALLLow Temperature Re8earch Station, Cambridge

(Received 24 February 1961)

One of the most puzzling features of the kineticsof the adenosine-triphosphatase activity of acto-myosin and of the myofibrils in which it is con-tained is the so-called explosive phase of hydrolysiswhich occurs immediately after addition of sub-strate and which is followed under certain specialconditions by a 'linear' phase of lower, but con-stant, velocity. These features were originally

studied by Weber & Hasselbach (1954) in myo-fibrillar preparations at low ionic strengths(< 0.15), but later Tonomura & Kitagawa (1957)showed that they were also characteristic of thehydrolysis of adenosine triphosphate by myosin Bin the presence of Ca2+ ions, at high ionic strength(> 05). Tonomura & Kitagawa (1960) haveextended their observations on myosin B to include