JOURNAL OF BACTERIOLOGY, Apr., 1966Copyright ( 1966 American Society for Microbiology

Vol. 91, No. 4Printed in U.S.A.

Application of Cell Fractionation Techniques in theStudy of Cells Infected with Polyoma Virus and

Newcastle Disease VirusHAROLD W. FISHER, HIDEMI MATSUMIYA,1 AND MASANOBU AZUMA

Department of Chemistry, University ofRhode Island, Kingston, Rhode Island

Received for publication 24 December 1965

ABSTRACTFISHER, HAROLD W. (University of Rhode Island, Kingston), HIDEMI MATSU-

MIYA, AND MASANOBU AZUMA. Application of cell fractionation techniques in thestudy of cells infected with polyoma virus and Newcastle disease virus. J. Bacteriol.91:1645-1651. 1966.-Techniques which permitted rigorous separation of nucleiand cytoplasm were applied to the study of the formation of Newcastle diseasevirus (NDV) in an established line of Chinese hamster cells and of polyoma virus(PYV) in mouse embryo fibroblasts. The results obtained for hemagglutinin andplaque-forming titers during virus growth were in agreement with those obtainedby others, using different techniques. These indicated that NDV matures in thecytoplasm, and PYV in the nucleus, of host cells.

The application of cell fractionation techniquesto virus-infected mammalian cells permits a moredetailed biochemical analysis of the maturationprocess of the virus than the other more com-monly used techniques like the fluorescent-antibody technique and electron microscopy. Touse cell fractionation techniques, it is essential toknow whether in fact they give results similar tothe other techniques or whether the fractionationdisrupts the virus components or the cell-virusassociation.The method devised by Fisher and Harris (7)

for the isolation of nuclei from normal animal cellcultures makes possible a direct and quantitativeapproach to the investigation of the virus-infectedcell. This method, which is applicable to a widerange of animal cell cultures (8), permits the re-covery of all the nuclei from a population of cells,and these nuclei are free from adherent cyto-plasm (4).

In the present study, these procedures wereapplied to Newcastle disease virus (NDV), aribonucleic acid (RNA) virus which has beenshown to mature in the cytoplasm, by the fluores-cent-antibody technique (19, 20, 25, 26, 27) and inelectron microscope studies of virus inclusions(1, 3, 14). The fractionation techniques were alsoapplied to polyoma virus (PYV), a deoxyribo-

1 Present address: Department of Bacteriology,Hokkaido University, Sapporo, Japan.

nucleic acid (DNA) virus which has been shownto mature in the nucleus (12, 16, 18, 21, 29, 31).With these nucleus isolation techniques, it has

been shown, in confirmation of earlier work, thatNDV, like other RNA viruses (2, 13), maturesin the cytoplasm and PYV in the nuclei.

MATERIALS AND METHODSVirus. NDV, strain KM, was kindly supplied by

P. W. Chang of the University of Rhode Island, andwas grown on 11-day-old embryonated eggs. Thehemagglutination titer (HAT) of the stock was 1: 2,560hemagglutination units (HAU) per ml and the plaque-forming unit titer (PFU) was 2 X 108 PFU per ml.PYV used for these experiments was kindly

supplied by R. Sheinin of the Ontario Cancer Institute,and was derived from a stock of strain T grown onmouse embryo secondary cultures (23). The HAT andPFU of this strain were 1:2,560 HAU per ml and 1.2X 107 PFU per ml, respectively.

Cell culture and media. For NDV experiments, theestablished cell line of Chinese hamster lung cell,strain CHL-1 (Puck; 10), was cultivated according tothe methods described by Ham and Puck (11). Themedium used was FIO supplemented with 5% fetalcalf serum and 0.01% Panmede (Paynes and Byrne,England) and called FlOPFC(5) (10).

For the growth of PYV, primary mouse embryocell cultures were prepared according to the method ofDulbecco and Vogt as modified by Youngner (32),with the use of 0.25% trypsin (trypsin 1: 300) andmedium CMRL-1066 (23) with 5% fetal calf serum(1066-FC5). Briefly, mouse embryos from 15th to

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18th-day pregnant Swiss-Webster mice were washedwith Saline F (11), eviscerated and decapitated, finelyminced with scissors, and treated with trypsin solutionfor 7-min intervals at 37 C. The pooled cell sus-pensions from 10 trypsinizations were filtered sterilelythrough gauze, and the cells were washed with salineby use of low-speed centrifugation. The washed cellsresuspended in 1066FC(5) were plated at 106 cells permilliliter on 90-mm glass petri dishes or 60-mm plasticpetri dishes. The secondary mouse embryo cellcultures were prepared by trypsinization of 3- to 4-day incubated primary mouse embryo cells.

Infection of cells and sampling. For NDV experi-ments, CHL-1 cells were infected in suspension bydispersing monolayer cultures in exponential growthwith 0.05% trypsin and washing with Saline F bycentrifugation. These cells were resuspended inFIOPFC(5) at a concentration of 5 X 105 cells permilliliter, and were inoculated with NDV at a multi-plicity of 10 PFU per cell; they were then incubatedat 37 C for 90 min. The cells were washed twice withSaline F and resuspended in fresh medium, and 5 mlof the infected cell suspension was plated and incu-bated at 37 C in a 5% C02-flushed incubator in 60-mmplastic petri dishes. In the NDV experiments, cellswere harvested at 0, 8, 12, 16, 24, 32, 40, 48, and 72hr. Two dishes were removed, and the cell sheets werelifted with a Teflon scraper and were transferred to atest tube for fractionation.

For PYV experiments, mouse embryo cell culturesin exponential growth were washed once with SalineF, and infected with PYV at a multiplicity of 100PFU per cell. After 90 min of adsorption at 37 C, thecells were washed free from unadsorbed virus withSaline F and incubated with 5 ml of fresh medium ina C02-flushed incubator at 37 C. In the PYV experi-ments, cells were harvested at 0, 12, 24, 36, 48, 72, and120 hr, lifted with a Teflon scraper, and transferred toa test tube.

Cell fractionation. Cell fractionation was carriedout according to the method of Fisher and Harris (7)with some modifications.

For NDV-infected CHL-1 cells 5 ml of the pooledwhole cultures was centrifuged at 220 X g for 10 min,and the supernatant fluid was harvested (henceforthcalled the culture fluid fraction). The cell pelletportion was washed once with Saline F, taken up in2.5 ml of fresh 0.1% Tween 80 (polyoxyethylenesorbitan; Sigma Chemical Co., St. Louis, Mo.) solu-tion (v/v, in distilled water), stirred for 15 min at roomtemperature with a Vortex Junior mixer, and thendiluted with 2.5 ml of Saline F with two times concen-trated salts. This suspension was mixed thoroughlyand centrifuged at 220 X g for 5 min. The supernatantfraction containing the cytoplasmic contents washarvested and transferred into a new test tube (thecytoplasm fraction). The pellet was washed once andresuspended in 5 ml of Saline F (the nuclear fraction).

For PYV-infected mouse embryo cells, fractiona-tion was carried out as above except the cell sus-pension was incubated for 2 min at room temperaturein 2.5 ml of 0.1% Tween 80 solution and then stirredfor 2 min at room temperature with a Vortex Juniormixer.

Before titration of virus, each fraction was sub-jected to freezing and thawing three times, or to sonicvibration for release of the virus components.Enzyme assay. The activity of glucose-6-phosphate

dehydrogenase was used to check the contaminationof the nuclear fraction by cytoplasm, and was carriedout according to the method of Kornberg (15). Theenzyme activity was determined spectrophoto-metrically by reduction of nicotinamide adeninedinucleotide phosphate (NADP) in a reaction mixtureof 0.5 ml of 0.05 M tris(hydroxymethyl)aminomethane(Tris) buffer (pH 7.4), 0.1 ml of 0.3 M MgCl2, 0.1 ml of0.0015 M NADP, 0.1 ml of 0.025 M glucose-6-phos-phate, 0.1 ml of enzyme, and distilled water to 3.0 ml.The increase in absorbancy at 340 m,u at 25 C wasrecorded at 1-min intervals for 10 min. One unit ofenzyme was that amount which caused the reductionof 1 ,umole of NADP (2.07 optical density units) perminute.

Virus titration. A plaque assay for NDV has beendescribed in several papers (9, 17, 28). Some modifica-tions were used in the present work. Chick embryocell cultures, 3 to 4 days old, on 60-mm plastic petridishes were used for this titration. The nutrient agarused as an overlay during the development of plaquescontained 0.9% Noble agar (Difco) and FIOPFC(5).After 2 days of incubation at 37 C in 5% CO2 in air,the plates were overlaid with more nutrient agarcontaining neutral red to give a final concentration ofneutral red of 1:40,000. The plates were incubatedfor 3 days after inoculation, and then plaques werecounted.

In the hemagglutination assay of NDV, the patternmethod (22) was employed. Twofold dilutions of thevirus were made with phosphate-buffered saline(PBS). Hemagglutination was observed with NDVby use of a 17% chicken red blood cell suspension, andthe results were read after incubation for 1 hr at 4 C.The end-point readings were confined to completeagglutination.A plaque assay for PYV has been described in

several papers (5, 23, 30). In this study, the plaqueassay for PYV was carried out with 3-day-old culturesof secondary mouse embryo cells on 60-mm plasticpetri dishes, by use of nutrient agar containing 0.9%Noble agar (Difco) and FIOPFC(5). After 7 days ofincubation at 37 C in 5% CO2 in air, the plates wereoverlaid with nutrient agar with neutral red as de-scribed above, and the incubation was continued for10 to 12 days after inoculation.The hemagglutination assay of PYV was carried

out with a 0.5% guinea pig red blood cell suspension(6). The results with PYV were read after incubationfor at least 18 hr at 4 C (24). Again, the end-pointreadings were confined to complete agglutination.

RESULTS

Fractionation of the cells. The isolation ofnuclei from CHL-1 and mouse embryo cells bycontrolled stirring in the presence of low concen-trations of nonionic detergent was comparable tothe quantitative results previously described (7, 8).Figure 1 shows that all of the nuclei were re-

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VOL. 91, 1966 FRACTIONATION OF CELLS INFECTED WITH POLYOMA AND NDV

FIG. 1. (A) Mouse embryo cells before niucleus isolationi, sliglhtly flattenled between a slide anld a cover slip.Phlase conitrast. X 800. (B) Nuclei isolatedfrom mouse embryo cells accordinig to Materials and Methodis. Sameconidition.s as in (A).

covered free from microscopically visible ad-herent cytoplasm. In Table 1 the results of thesepar.tion of pure nuclei from mouse embryosecondary cell cultures are given. The resultsshow that the efficiency was improved with anincubation time prior to stirring and that therecovery was decreased with long stirring times.In Table 2 the results illustrating an enzymaticindex of purity of nuclear and cytoplasmic frac-tions are presented. The results show none of thecytoplasmic enzyme in the nuclear fraction.

Studies with NDV-infected cells. Fractionationdata relating to Chinese hamster lung cells in-fected with NDV are shown in Fig. 2 and 3.Figure 2 shows the HAU titration of the culturefluid, cytoplasm, and nucleus fractions of NDV-infected CHL-1 cells over a 60-hr incubationperiod. It was observed that no hemagglutinatingactivity above the lower limit of detectability at6 to 8 HAU per ml was found in the nucleusfractions throughout the entire 60-hr incubationperiod. At first no hemagglutinating activity wasdetected in the cytoplasmic fraction; however,at 12 to 15 hr after infection, an increase in thetiter was then noticed in the cytoplasmic fraction.This reached maximal activity between 20 and30 hr after infection and subsequently decreasedas the titer in the culture fluid fraction becamemaximal.The results obtained from infectivity titration

on the fractions of NDV-infected CHL-1 cellsare shown in Figure 3. Significant titers of theinfectious virus particles were not present in thenucleus fraction throughout the incubation

TABLE 1. Separationi of nuclei from mouse enmbryocells*

Incubation Stirring Separation of ntucleiCell Incubation StirringCisi time time

Efficiencyt Recoveryl

,lin )71iin

2.5 X 106 0 2 86.7 1000 5 90.0 72

l.0>X1071 2 2 97.2 1002 3 98.7 985 2 96.7 965 3 98.9 985 5 98.9 85

* Secondary mouse embryo cell cultures werefractionated after different intervals of incubationand stirring in 0.1% Tween 80 as described inMaterials and Methods. The numbers of nucleifree from cytoplasm and the unseparated cellswere counted under a phase-contrast microscope.

number of separated nucleitotal nuclei and cells

X 100.number of separated nuclei

initial number of cellsX 100.

period, whereas the titer in the cytoplasmic frac-tion preceded the rise in titer in the culture fluidfraction and was quantitatively consistent withmaturation in the cytoplasm and release into theculture fluid fraction.

In Table 3, the experiments carried out to test

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FISHER, MATSUMIYA, AND AZUMA

TABLE 2. Glucose-6-phosphate dehydrogenaseactivity in mouse embryo cell fractions*

Activity per Total unitsFraction Vol 0.1 ml of per 1.2 x 108

sample cells

ml units

Nuclei.............. 1 0.0002 0.002Cytoplasmic ........ 4 0.0198 0.792

* Secondary mouse embryo cell cultures werecounted and fractionated, and the activity ofglucose-6-phosphate dehydrogenase in the frac-tions was determined as described in the Materialsand Methods.

10001

. Super..

1,'Cytoplasm

0-

/ o o ---ONuclei(3_ _ __ _ _ _--------------___

at 12 hr and then increased to that of the culturefluid after 24 hr. The data indicate, therefore,that the virus released into the culture fluid frac-tion was complete, whereas it is possible that

7Super

/ Cytoplasm

i5E S \

CD

00

0

0

0 10 20 30 40 50 60TIME(hrs)

FIG. 3. Time course of appearance of infectivity inthe fractions of ND V-infected cells. Conditions as inFig. 2, except that fractions were titratedfor plaque-forming activity. Symbols: 0, nucleus fraction; *,

cytoplasmic fraction; O, supernatant culture fluid.

0 le 20 30 40 50 60TIME(hrs)

FIG. 2. Time course of appearance of HAU in thefractions of NDV-infected cells. CHL-J cells were

infected with NDV as described in Materials andMethods. At intervals from 0 to 72 hr, the cultures were

harvested and nuclei were separatedfrom the cytoplasmfraction by the procedure described in Materials andMethods. The fractions were frozen and thawed threetimes and titrated for hemagglutinating activity.Symbols: 0, nucleus fraction; 0, cytoplasmic fraction;O, supernatant culture fluid.

for the presence of incomplete NDV are pre-sented. The ratio of PFU to HAU, which may beused as a measure of complete xirus comparedwith incomplete virus, remained constant in theculture fluid fraction throughout the growthperiod and equal to that of the original egg-growninoculum, suggesting that only completed virusparticles were released from the cells. However,in the cytoplasm the ratio dropped to a low value

TABLE 3. Specific infectivity of NDV and PYVduring growth*

Logio PFU:HAU*Period of

__Virusincubation Cultuid Nuclei Cytoplasm

hr

NDV Inoculum 5.012 5.0 t 3.716 5.2 t 4.424 4.8 -t 4.6

PYV Inoculum 3.712 3.6 2.6 3.524 3.2 3.2 3.636 3.3 3.7 3.5

* Cells infected and fractionated as describedin the Materials and Methods were titrated forhemagglutination and infectivity on each sample.The logio of the ratio of PFU to HAU are pre-sented at different incubation periods.

t No detectable HAU.

oa

laEC

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VOL. 91, 1966 FRACTIONATION OF CELLS INFECTED WITH POLYOMA AND NDV

incomplete virus was present in the cytoplasmicfraction at 12 to 16 hr.

Studies with polyoma-infected cells. The cellfractionation techniques were applied to cellsinfected with PYV, which is thought to mature inthe nucleus. The results presented in Fig. 4 forthe development of hemagglutinin as a functionof incubation time show that this specific proteinfirst increased in the nuclei, reaching a maximumbetween 40 and 60 hr, and subsequently droppedto less than the titer found in the culture fluidfraction. The titer in the cytoplasmic fraction,however, was less than that found in the culturefluid fraction at all times during the incubationperiod.The results obtained from infectivity titration

on the fractions of polyoma-infected mouseembryo cells are shown in Fig. 5. The infectiousvirus particle titer was found to rise first in thenucleus fraction and, after a lag of approximately20 hr, was found to rise in the culture fluid frac-tion, until at 40 hr of incubation a decrease in thenucleus fraction was found. The titer in the cyto-plasmic fraction remained at 5 to 10% of thenucleus fraction at each time.

U.

Nuclei

Cytoplasm

0 20 40 60 80 100 120TIME(hrs)

FIG. 5. Time course of appearance of infectivity inthe fractions of PYV-infected cells. Conditions as inFig. 4, except that fractions were titrated for plaque-forming activity. Symbols: 0, nucleus fraction; *,cytoplasmic fraction; O, supernatant culture fluid.

0 20 40 60 80 1aTIME(hrs)

FIG. 4. Time course of appearance of I

fractions of PYV-infected cells. Mouse etwere infected with PYV as described in MeMethods. Other conditions as in Fig. 2, 0

harvesting was at intervals from 0 to 120 hi0, nucleus fraction; 0, cytoplasmic fr,supernatant culture fluid.

The results for PYV in Table 3 show that if theCytoplasm test for incomplete virus by use of the ratio of

PFU to HAU was applied to polyoma-infectedNuclei cells a low value was obtained in the nucleus

fraction after 12 hr of incubation, whereas theratio in the cytoplasm and in the culture fluidremained high. This suggests that incompletevirus may be present in the nucleus fraction.

DIscussIoNThe results of the present study of the intra-

cellular site of NDV multiplication by direct cellfractionation confirm that maturation of thisvirus is in the cytoplasm. The hemagglutinin andinfectivity determined on cell fractions during thegrowth cycle in this study confirm the results ob-tained by fluorescent-antibody test of NDV-specific antigen and by electron microscopy ofvirus inclusions. Therefore, this cell fractionation

D0 120 technique can be used for further biochemicalstudies of the site of synthesis of viral com-

YA intheponents.YAU in the P The results in Table 3 suggest that NDV-aterials and infected CHL-1 cells release infectious virionsexcept that into the culture fluid. Wilcox (28) reported thatr. Symbols: NDV-infected L cells produce a high percentageaction; o, of incomplete virus particles. The results in Fig.

2 and 3 show that at no time was the hemagglu-

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FISHER, MATSUMIYA, AND AZUMA

tinin or infectivity demonstrated in the nuclei,whereas the titer in the cytoplasm preceded, andwas quantitatively consistent with, maturation inthe cytoplasm and release into the culture fluidfraction.The results of the present study of the intra-

cellular site of PYV multiplication by direct cellfractionation also are consistent with the earlierreports that maturation of this virus is in thenucleus. Hence, this cell fractionation techniquealso can be applied to further biochemical studiesof the site of synthesis of viral components ofthis DNA virus.The method of direct cell fractionation permits

the proposed examination of the virus and cellularnucleic acids and enzymes on fractions whichhave been quantitatively analyzed by microbio-logical methods. Studies in the nucleic acids andenzymes of polyoma-infected cells are now inprogress.

ACKNOWLEDGMENTSWe are grateful to R. Sheinin for her stimulating

suggestions and advice and to T. Cooper, F. Parlin,B. Nehman, and R. Rhodes for technical assistance.

This investigation was supported by Public HealthService Research Grant CA-07787-01 from theNational Cancer Institute and by American CancerSociety Grant E-336.

Contribution from the Biophysics Laboratories,University of Rhode Island, Kingston.

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