Analysis of the Fusion of XC Cells Induced by Homogenates of Murine Leukemia Virus-Infected Cells and by Purified Murine Leukemia Virus

JOURNALOFVIROLOGY, June 1971,p. 753-758 Vol.7,No. 6 Copyright ( 1971 American Society for Microbiology PrintedinU.S.A.

Analysis of the

Fusion

of

XC

Cells Induced by

Homogenates of Murine

Leukemia

Virus-Infected Cells and

by

Pur-ified

Murine Leukemia Virus

GEORGE S. JOHNSON,1 ROBERT M. FRIEDMAN, AND IRA PASTAN

Laboratories of Molecular Biology and Pathology, National Cancer Institute,Bethesda, Maryland20014

Received for publication 22 February 1971

The fusion of XC cells inducedby murine leukemia virus (MuLV)-infected cells is also induced by homogenates prepared from the infected cells and by purified MuLV. The fusion-inducing factor appears to contain a heat-labile

lipoprotein.

No synthesis of specific macromolecules by the XC cells is necessary to obtain fusion. The results suggest that specific components of the viral particle are the

activatorsfor the fusion process andtheymay also be present in the membranes of

infected cells.

Tissueculture cells of the XC cellline, aRous sarcoma virus-induced rat tumor (14), undergo

syncytium formationwhenplacedin contactwith

mouse embryo cell cultures infected with murine

leukemia virus (MuLV;reference 7). This mixed

culture cytopathic effect has been utilized as an

indicatorsystemfordetectingMuLV-infected cell

lines (7) and as arapidmeans oftitratingthese

viruses intissue culture

(13).

The

syncytium

for-mation is thought to be due to cellular fusion

rather than to endomitosis (7), but the

mecha-nism of thefusion and the biochemical stimulus

for syncytium induction are unknown.

MuLV is released from the infected cellsby a

process of

budding

fromthe

plasma

membrane.

It isthuspossiblethat the activators for the fusion

areeithervirus-specific components of the

cellu-lar membraneorthevirusitselfintheprocess of

budding or adhering to the cellular membrane.

In this study, we report that intact, infected

cells are not required but that cellular

homoge-nates also inducethe fusion. Furthermore, high

concentrations of

purified

MuLV

particles

induce

the fusion. The results suggest that components

of theviralparticlewhich mayalsobepresent in

the membranes of theinfected cells are the

acti-vators for the fusion process.

MATERIALS AND METHODS

Tissueculture.The XCcell line (7, 14) andMuLV

(Moloneystrain) (11) wereobtained from J.Hartley

IRecipient of Public Health Service postdoctoralfellowship

no. l-F02-GA35507-01.

and W. Roweof the NationalInstitute ofAllergy and

Infectious Diseases.

Cultures of 15-to17-day-oldNationalInstitutesof

Health (NIH) Swiss mouse embryos were obtained

from Microbiological Associates, Inc. (Bethesda,

Md.). Secondarycultureswereplantedandinoculated

aspreviously described (5, 6). The mouse embryo cells

and the XCcells were grown inEagle's minimal

es-sential medium with 10%fetal bovineserum(Grand

IslandBiologicalCo.,GrandIsland,N.Y.),glutamine

(2mM),penicillin (50,g/ml),streptomycin(50 units/

ml),polymyxin sulfate (100units/ml),and mycostatin

(50 units/ml). Both cell types were grown in 32-oz (0.946 liter) tightly stoppered, glass culture bottles

andincubatedat37 C.

Preparation of fusion factor from infected mouse

embryocells.Cellculturesofmouseembryocellswere

incubated 1 to 2 weeks after inoculation. The cells

were removed from the monolayer by scraping and

were suspended in hypotonic buffer (RSB), [0.01 M

tris(hydroxymethyl)aminomethane(pH 7.4), 0.0015

MMgCl2,0.01MNaCl].Thecellswerepelletedat700

X g, resuspended inRSB, and homogenizedwith 20

strokes of a tight fitting Dounce homogenizer. The

homogenate wascentrifuged for 10 minat 700 X g.

Theresulting700 X g supernatantwascentrifugedat

35,000 X g for15minandthepelletwassuspendedin

RSB.Samples were treatedasdescribed. The resultant

suspensions were recentrifuged at35,000 X gfor 15

min, and the pellets and supematantswereanalyzed

forfusion-inducingactivity.

Development ofcelularfusion. The XC cellswere

removed from the substratum bytrypsinization and

were plantedat 1.2 X 101to 1.4 X 101 cells per

60-mm plastic, tissue culture dish (FalconPlastic,Div.

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JOHNSON, FRIEDMAN, AND PASTAN

ofB-D Laboratories, Los Angeles, Calif.). The cell

layer developedtoaconcentration ofapproximately

2.5 X 106cells by 18to24hrat37Cinahumidified

atmosphere containing 5% CO2. The appropriate

sampleof thefusionfactorwasthenadded, andafter

48 hr the cellswerestainedwithhematoxylin.

Cell andmembranefractionation. Acell homogenate

wascentrifugedfor 10 minat700 X g,and the

result-ing supernatants were centrifuged successively at

11,000 X g for 10 min, 35,000 X gfor 10 min, and

90,000 X g for 1 hr. To fractionate cell membranes,

the 700 X g supernatantwas made30% (by weight)

sucrose in RSBby addition of65% sucrose. A

dis-continuous gradient (3) was formed bylayering su-crose solutions in thefollowing order: 3 ml of65%,

7ml of 45%, 7 ml of 40%, 10 ml offusionfactor

sus-pensionin 30% sucrose, 7 ml of 25%, and 4 ml of RSB ontop, Thegradient wascentrifugedat 86,000

X g for 16 hr inaSpincomodel SW 27rotor. Visible

bandsofmaterial werecollected bymeansofaPasteur

pipette.Thefractions were diluted 5- to10-foldin RSB andpelleted at35,000 X g for1 hr.Thepelletswere

suspended in 1 ml of RSB, anddensities were

deter-minedbyweightratiosrelativetowater.Inall

experi-ments, protein concentrations were determined by

the method described by Lowry et al. (9) by using

bovineserumalbuminasthestandard.

PurificationofMuLV.Purified MuLVwasobtained

from Electro-Nucleonics Laboratories (Bethesda,

Md.). It had beenpurifiedbyuseofoneortwo

iso-pycnic sucrose gradients with zonal

ultracentrifuga-tion (16). In thefirst gradient, viruswascollected at

densities of 1.14to 1.16g/ml,whereas, in the second

gradient, virus was collected at 1.15 to 1.16 g/ml. Viral particles from both collections were

concen-tratedto101l to1012virusparticles/ml.The final

con-centrations weredetermined

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.:i

byelectron microscopy.

..gr~~~~~~.

.:'*

. 4;*. wj:' *a::^:.... .:

Thesecond banding removed some nonviral material.

The concentrated, once-banded virus contained 0.8 to

1.0 mg of protein/ml(i.e.,per 101l to 1012 virus

parti-cles), whereas the concentrated, twice-banded virus contained 0.5 to 0.7 mg of protein/ml.

Electron microscopy. The 700 to 35,000 X g pellets

were fixed in glutaraldehyde and osmium tetroxide

(4) andembedded by the method described by Luft

(10). Theelectron micrographswere taken by William

Hall of Electro-Nucleonics Laboratories at X 19,000

magnification by using an Hitachi HU-IlE electron

microscope.

RESULTS

Description of fusion process. When XC cells are added to a monolayer ofmouse embryo cells in-fected with MuLV, syncytium formation occurs. Ourpreliminary experiments showed that similar

syncytium formation also occurs when infected

mousecells are added to XC monolayers. Within

afew hoursafter addition of infected mouse

em-bryo cellsortheir homogenates, but not uninfected

cells oruninfected cell homogenates, to a mono-layer of XC cells, cellular fusion is apparent

(Fig. 1). The multinucleated cells become larger

and more numerous withfurther incubation, and

thefusionprocess is completewithin 48 hr. The

resulting cells can be divided into three general

groups:(i) small multinucleatedcells composed of

2to10nuclei; (ii) largercellscomposed ofabout

10to 30 nuclei, often accompanied by vacuoles

and occasionally nuclear fusion; and (iii)

ex-tremely large

cells composedof anindeterminable

number ofnuclei with nuclear fusion, often

ex-tensive, and marked vacuolization.

..

.-A'4

FIG. 1. XC cellsin the processoffusion. Cells were stained with hematoxylin at 5 hr after addition of the

fusion-inducing factor. An identical sample caused +5 fusion in 48 hr in another culture. X 250.

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MURINE LEUKEMIA VIRUS

Thesize andnumberofthemultinucleatedcells

is dependent upon the amount of fusion factor added. Hence, afusionindex system based on the number of multinucleated cells and the number of nuclei per cell can beutilizedas a method of

quan-titation. Inthis indexsystem,fusionconsisting of

asmallnumber ofmultinucleated cellsand a few

nuclei percell is given a + 1 rating, whereas

fu-sion involving roughly one-half of the cells is

rated +3. Inthe fusionrated +5, essentially all

theXC cells areinvolved in the fusion, and

nu-merous, extremely large,bizarre cells are appar-ent.

Typical

resultsareshowninFig.2and 3. The presence of excessive nuclear fusionprevents the

utilization ofamoreprecise quantitative method

suchasthatbasedontheexactnumberof nuclei

per

cell.

Distribution of thefusionactivity.The

distribu-tion of the fusion-stimulating activity in the

in-fectedcell homogenates is showninTable1. The

activity is detected in all oftheparticulate

frac-tions butnotinthe 90,000 X g supernatant. The

11,000 to35,000Xgfraction ismostactive. This

fractionrepresents at leasta fivefold purification

of the fusion factor overthewhole homogenate.

Various treatments of the homogenate were

utilized in attempts to solubilize the fusion

fac-tors. The treatments used were sonic treatment

for 2min at 0 C

(model

W140D, Sonifier Cell

Disruptor, 50 w power), rapid freezing and

thawing in RSB or in RSB

plus

1.7 M

KCI,

and

storage ofthehomogenate in RSB plus 0.001 M

ethylenediaminetetraacetic acid overnight at 4 C.

None of these treatments significantly alters the

distribution, although they decrease the total

amount of fusion caused by a given sample of the homogenate.

Thedensity ofthecytoplasmicmembrane

frac-tionscontaining thefusion-inducing activitywas

determined by discontinuous sucrose gradient

centrifugation (Table 2). In the gradient system

applied, the most active components band at a

density of1.15 g/ccwith an additional band of

activity of 1.18g/cc. Thereare no visible bands

other than those indicated.

Stability studies. The stability of the

fusion-stimulating

factors to treatment by various en-zymes wastested.Samples of the700 X g super-natant or 700 to 35,000 X gfraction (200to 300

,ug

of

protein)

wereincubated for 15to30minat

37 C inthepresence ofoneofthefollowing en-zymes: ribonuclease (0.5 ,ug/ml),

deoxyribonu-clease (25

,ug/ml), trypsin (5

to 200,g/ml), or

neuraminidase (50

,g/ml).

Thetrypsintreatment

at concentrations as low as 5

,ug/ml completely

destroys the fusion-stimulating activity, whereas

the other enzymes have no effect. Incubation

overnight

at0 Cwith thelipase,

steapsin (0.07%;

Nutritional

Biochemicals

Corp.,

Cleveland,

Ohio), also destroys the

activity.

Treatment of thehomogenates with ether

fol-lowed byremoval of the ether by

bubbling

with

nitrogen

completely

destroys the

activity.*

The activity is stable

during

10 to 15 min of

K

I.*

.1

.S,.

*s :%

....,X+

Y

.as...x

i

_.

*. vs #:

,

.

.,Q¢S i

$ s

d

i i.t

,,

".s:

9I ,

..F. .C

FIG. 2. Control XC celi( culture. X 250.

VOL.7,1971

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It

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:ea % ...;:..

;*-%>s<

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I

4-~~~~~~~~~~~, ..

FIG. 3. Syncytium induced by homogenates

of MuLV-infected

mouse

embryo

cells. Pictures show

typical

examplesofthefusionindexsystem

employed

inthis

study:

(A) +2 fusion

rating;

(B)

+3

fusion

rating;

(C)

+5

fusionrating. X 250.

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MURINE LEUKEMIA VIRUS

TABLE 1. Distributionz offusion-inducingactivity

in mouseembryo cell homogenates

Fraction_Fraction Amt of

_{~~tested (ug)}

protein Fusionindex

Whole cell homogenate.. 510 +3

700X g Pellet... 350 +2

700 XgPellet... 1,050 +4

700to11,000 Xg...350 +5

11,000to35,000 X g .... 120 +4

35,000to90,000 X g... 240 +2

90,000 X gSupernatant 500 0

TABLE 2. Densityof cell homogetiatefractions contiainingfusion-inducing activity

Density (g,'cc) Amtofp(otein Fusion index

1.099 97 0

1.100 97 +1

1.146 125 +3

1.181 230 +2

incubation at 37 or 44 C but is completely

in-activated at 50 C. About 50%0 of the activity is

destroyed during6 hrofincubation at37 C.

FusioninducingabilityofpurifiedMuLV. Since

MuLVis released fromthecellby budding from

the cellular membrane. it seems possible that

virusparticlesintheprocessofbuddingorloosely

adheringto thecellmembrane maybepresentin

thecellularhomogenate andthus be the stimulus

forsyncytiuminduction. To check for sucha

pos-sibility we tested purified virusfor

fusion-induc-ing abilityand checked for thepresence of viral

particles in the homogenate. Extensive fusion is

observed with the addition of large amounts

(more than 109 viral particles per dish) ofboth

once-andtwice-banded virus (Table 3);however,

negligible fusionisobserved withsmallersamples

of the virus. The resulting multinucleated cells

developwithin afew hours after addition of the

virus and are identical in appearance to those

shown in Fig. 3. The twice banded virus is

ap-proximately five times more effective per

milli-gramofprotein than themostefficientparticulate

fraction from infected mouse embryo cells

(Table 1);25,ugofvirus protein is required for a

+4 fusion, whereas 120 ,ug of protein from the

particulate fraction is required for similar levels

of fusion. Itappears that thefusion is caused by

thevirus andnotbyacellularcontaminantinthe

virusfraction sinceapproximately equalamounts

offusionare obtainedbyusinganequal number

of particles of once-purified or twice-purified

virus(seeMaterials andMethods). Also,the

pres-enceofacellular contaminant bandingat a

den-sityof1.15 to 1.16g/mlin sufficientamountsto

induce the fusion is unlikely.

Electron microscopywasemployedto estimate the number of viral particles in the cellular

ho-mogenate.Numerous viral particles are found in

the 700 to 35,000 x g fraction of the infected

mouseembryo cellhomogenate. As many as 109

to1010particlesarepresentinsamples whichcause

extensive fusion. Since this is inrange of the

re-sults shown in Table 3 for purified virus, it is in-deedpossible that the fusion caused by the cellu-larhomogenates is due solelytothe viralparticles

and not to specific components in the cellular

membranes, but additional investigations are

necessaryforanunequivocal answer tothis

ques-tion.

Is therearequirement for cellular metabolic

ac-tivityfor fusion? XC cellcultures wereincubated for 6 hr at 37 C with 1010 viral particles (once banded) and one of the following compounds: actinomycin D (0.5 ,g/ml), puromycin (20 ,ug/ ml), cycloheximide (10

Ag/ml),

rifamycin (120 ,ug/ml), or rat serum interferon (60 units in 0.1 ml rat serum). None of the added compounds in-hibited or activated fusion, suggesting that spe-cificsynthesis of macromoleculesorviralparticles by the XC cells is notrequired forsyncytium for-mation.

DISCUSSION

XC cells undergo syncytium formation when placed in contactwith cellsinfectedwith MuLV.

The syncytiumformation isthoughttobedueto

fusion rather than byendomitosis (7). The results of this study show that intact, infected cells are not required, but that homogenates of the in-fected cells and also purified viral particles will induce XC cell fusion.

MuLV is released from the cells bya budding

process.Thus, viral specificproductsmay remain as part of the cell membrane andinducethe fu-sion,orviralparticlesin the process ofbuddingor

looselyadheringtothecellsurface afterbudding

may be responsible for the fusion.Virus-specific

antigens are found in infected cell membranes

(12).However, thelargenumberof viralparticles

observed in the infected cellhomogenatessuggest

TABLE 3. Fusiont induced by murine leukemia virus

Fusion index No. of virusparticles

Oncebanded Twicebanded

1 X 109 +1 +2

2 X 109 +2 +3

4 X 109 +4 +4

10 X 109 +5 +5

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JOHNSON, FRIEDMAN, AND PASTAN

that the viralparticles may be solely responsible

for the fusion. Further investigation of the

ho-mogenates is necessary for an unequivocal

an-swer.

Cellular fusion induced by various strains of

Newcastle disease virus (NDV) is ofone oftwo

types, fusion from withinorfusion fromwithout

(2). SincethefusioninducedbyMuLV

(Moloney

strain)is insensitive toinhibitors of

macromolec-ular synthesis, the process would be considered

fusionfrom without.However, it ispossiblethat the fusion of XC cells induced by cells infected

with other strains of MuLV (7) may be caused bya fusion from within process.

The factors responsible for the syncytium

for-mationareheat

labile,

sensitiveto

trypsin, lipase,

andether but insensitive to

ribonuclease,

deoxy-ribonuclease, or neuraminidase. This suggests

thatproteins arerequiredfor the fusion process,

but viral ribonucleic acid (RNA)orother nucleic

acid species such as

products

of the

RNA-de-pendent

deoxyribonucleic

acid

polymerase

(1, 15)

are not. The

sensitivity

to

lipase

and ether

indi-cates that

lipids

or

lipoproteins

may be

impor-tant. However, the

possibilities

that the ether

treatment may inactivate an

important protein

and the lipase may be contaminated with

pro-teases cannot be overlooked. The

integrity

of the

phospholipids of the viral membrane has been

showntobe

required

for fusion of cells induced

by

NDV(8).

ACKNOWLEDGMENTS

WearegratefultoJ.Hartleyand W. Roweforsupplyingthe XC cellsand theMuLV and for many helpful suggestions, T. Pincus andM. Harrisonforpreparingthehomogenatesfor the electronmicrographs,and W. Hall ofElectro-Nucleonics Labo-ratoriesfortakingthemicrographs.

LITERATURE CITED

1. Baltimore, D. 1970. RNA-dependent DNA polymerase in virions of RNAtumorviruses.Nature(London) 226:1209-1211.

2. Bratt, M. A., and W. R. Gallaher.1969. Preliminary analysis ofthe requirements for fusionfromwithin and fusion from without by Newcastle disease virus. Proc. Nat. Acad. Sci. U.S.A. 64:536-543.

3.Caliguiri, L.A., and I. Tamm.1969.Membranousstructures associated with translationandtranscription of poliovirus RNA.Science166:885-886.

4. Chambers,R.W.,M.C. Bowling,and P. M. Grimley. 1968. Glutaraldehyde fixation in routine histopathology. Arch. Pathol.85:18-30.

5. Hartley, J.W., W. P. Rowe, W. I. Capps, and R. J. Huebner. 1965. Complement fixation and tissue culture assays for mouseleukemia viruses. Proc.Nat. Acad. Sci. U.S.A. 53: 931-938.

6. Hartley, J.W., W. R. Rowe, W. I. Capps, and R. J. Huebner. 1969.Isolationofnaturallyoccurring viruses of the murine leukemiavirus group in tissue culture. J. Virol. 3:126-132. 7. Klement,V., W. P. Rowe, J. W. Hartley, and W. E. Pugh. 1969.Mixed culture cytopathogenicity: a new testforgrowth of murine leukemia viruses in tissue culture. Proc. Nat. Acad. Sci.U.S.A.63:753-758.

8. Kohn, A. 1965. Polykai-yocytosis induced by Newcastle disease virus inmonolayers ofanimalcells. Virology 26: 228-245.

9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Proteinmeasurement with the Folinphenol

reagent.J.Biol. Chem. 193:265-275.

10. Luft, J. H. 1961. Improvements in epoxy resin embedding methods.J.Biophys.Biochem.Cytol.9:409-414. 11. Moloney, J. B. 1960. Biological studies on a

lymphoid-leukemia virus extractedfrom sarcoma 37. I. Originand introductory investigations. J. Nat. Cancer Inst. 24:933-951.

12. Old,L. J.,E. A. Boyse, G. Geering, and H. F. Oettgen. 1968. Serological approachesto the study of cancer in animals and inman.Cancer Res.28:1288-1299.

13. Rowe, W. P., W. E.Pugh, and J. W. Hartley. 1970. Plaque assaytechniquesfor murine leukemia viruses. Virology 42: 1136-1139.

14.Svoboda,J.1960. Presence of chickentumorvirus in sarcoma of theadultratinoculated afterbirth with Rous sarcoma tissue. Nature (London) 186:980-981.

15.Temin,H.M.,andS. Mizutani.1970. RNA-dependentDNA polymerase in virions of Rous sarcoma virus. Nature (London) 226:1211-1213.

16.Toplin, I. 1967. Purificationof the Moloney and Rauscher murine leukemia viruses by use of zonal centrifugation systems.Appl. Microbiol. 15:582-589.

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