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Neuroblastoma Cell Fusion by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus


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Neuroblastoma Cell Fusion by a Temperature-Sensitive

Mutant of Vesicular Stomatitis Virust



DivisionofBiology, Kansas StateUniversity,Manhattan Kansas66506,1and Departments ofMedicine2

andPathology(Neuropathology),3Northwestern UniversityMedical School and VeteransAdministration Lakeside Hospital, Chicago,Illinois60611

Received forpublication 28 December 1978

Atemperature sensitive mutant of vesicular stomatitis virus which does not

mature properly when grown at


promoted extensive fusion of murine

neuroblastoma cells atthis nonpermissive temperature. Polykaryocytes

appar-ently formedas aresult of fusion from within thecells that requires low doses of

infectious virions for itspromotion and is dependent on viral protein synthesis.

Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after

infection, larger polykaryocytes continued toform, leading to an average of 28

nucleiperpolykaryocyte asaresult of polykaryocytes fusing to each other. Two

neuroblastoma cell lines have been observed to undergo fusion, whereas three

other cell lines (BHK-21, CHO, and 3T3) were incapable of forming

polykary-ocytes,suggesting that nervoussystem-derived cellsareparticularly susceptible

to vesicular stomatitis virus-induced fusion. Although the normal assembly of

theprotein components of this virus is deficient at 39°C, the G glycoprotein was

inserted into the infected cell membranes at this temperature. Two lines of

evidence suggest that the expression of G at the cell surface promotes this

polykaryocyteformation:(i) inhibition of glycosylation, which may be involved in

themigration of theGprotein to thecellularplasma membranes, will inhibit the

cell fusion reaction; (ii) addition ofantiserum, directed toward the purified G

glycoprotein, will also inhibitcellfusion.

Althoughanumber of RNA and DNA viruses

inducepolykaryocyteformation (19, 20, 23),

rel-atively few viral systems have beenanalyzedfor

theviral components thatareessentialfor

cel-lular fusion. The two mostdistinctive types of

virus-induced fusion have been termed fusion

from without (FFWO) and fusion from within

(FFWI)asfirstdescribed byBrattandGallaher

(2). Virus-induced FFWO requires neither viral

replicationnorviralprotein synthesisbecause it

canbeproduced by both infectious and

inacti-vated virions. A large multiplicity of infection

(MOI)is necessary forFFWO,andfusionoccurs

ashort time after addition of virus. In contrast

to FFWO, virus-induced FFWI is associated withintracellularreplicationofvirus in eithera fullyproductiveorrestricted infectiousprocess.


in-fectious virionsand for some virus-directed

pro-tein synthesisand byoptimum fusion at a low

MOI.Interestingly,viralglycoproteins appearto

be important to polykaryocyte formation both inFFWO,as withthe FproteinofSendaivirus

t Contribution no. 79-152-j, Kansas Agricultural Experi-mentStation, Kansas State University, Manhattan, KS 66506.

(8, 18, 26, 27), and in FFWI, as with the B2

glycoprotein ofherpessimplexvirus(15, 24).

Inourlaboratorieswehave beeninvestigating

the effects of intracerebral inoculation of mice

withvesicular stomatitis virus (VSV)

tempera-ture-sensitive(ts)mutants, oneofwhich,tsG31

(III), producesaprolongedclinical disease and

markedstatusspongiosusof thecentralnervous



lesion has beenprimarilyassociated with

"atyp-ical infectious agents" which cause slow viral

diseasessuchaskuru andCreutzfeldt-Jakob

dis-ease of humans and scrapie and transmissible

mink encephalopathy of animals. In


these agents promote polykaryocyte formation

both in vivo(1, 12,13) and in vitro(7, 11).Since

theabilityof these infectiousagentstopromote


centralnervoussystemlesions,it isquite

inter-esting that the VSV mutant,tsG31 (III),isalso

ableto inducepolykaryocyteformation.Thists

mutant has been shown to be a member of

complementation groupIII whose membersare

characterizedbyatsMprotein (9).Inthis initial

reportwe willpresentdataconcerning the fusion


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ments was prepared by infecting BHK-21 cells and was purified bydensity gradient centrifugation (22).

Only theB-typevirionwasused in theseexperiments. Cell culture lines. Two murine neuroblastoma cell lines wereused in theseexperiments: N-18, obtained from Brian Spooner (Kansas State University) and grownasdescribed elsewhere (9);andN2a, obtained from the American TypeCulture Collection (Rock-ville,Md.) andculturedaspreviously described(16).

Theconditions for thegrowthof BHK-21 cells have beendetailed elsewhere (21).3T3 and Chinese

ham-sterovary (CHO)cellswerepropagated in thesame mediumasthe N2acells. All cell linesweregrownat 37°C in a 5% C02-95% air atmosphere in a

water-jacketed incubator.

Assay forpolykaryocyte formation. Formost

experiments, N-18 cells (106)were seededontoglass coverslips (22 by22 mm)inculture dishes (35 mm)

with 2ml of culture medium and incubated2to3days

until confluent. The appropriate virus, usually at a multiplicity of 10 virions per cell, was allowed to

adsorbtothe cells for30minat25°C;2mlof medium

wasadded,and the cellswerethenincubatedateither 31 or390C.At5-hintervals afterinfection, thecells werefixedby placing thecoverslipsin acetone-meth-anol (2:1)at-20°Cfor10min.The nucleiwerethen stained withMayerhematoxylin stain,and the cells werecounterstained with eosin Y(3).Approximately

1,000 nuclei per coverslip were counted, and their distribution in fused and nonfused cells was deter-mined. From thisinformation,twodifferent indexes of fusionwerecalculated: the percentage of nuclei

pres-entinfused cells and the average number of nuclei per fused cell. In some experiments, D-glucosamine

(SigmaChemicalCo.,St.Louis, Mo.)wasaddedto 1 or10mM inthe tissue culture medium after theviral infection.

Isolation of viral Gglycoprotein. TheVSV gly-coproteinwasisolated fromsucrosegradient-purified

virusbyamodification of the procedures outlined by

Kelleyetal. (10)and Hale et al. (6). Essentially,the viralenvelope proteinsweresolubilized with non-ionic detergent Nonidet P-40at aconcentrationof 1%. The nucleocapsids of the virionswerethenseparated from the membrane proteins G and M by pelleting the nucleocapsid through 20% sucrose at 136,000xgfor

90 min. The viral envelope proteins were then col-lected from the interface of the20%osucrose and the topvolume. NaClwasaddedto 0.4M, and the enve-lope proteins were separated on a Sephadex G-75 column(56by1.1cm) withphosphate-buffered saline with 0.4 M NaCl to elute the two proteins. The G glycoprotein (molecular weighta69,000)eluted in the

voidvolume,whereasthe Mprotein (molecularweight

M 29,000) eluted later. Analysis by sodium dodecyl

neous injection and with boosterinjections every 2

weeks. The antiserumwaspurified bythe method of McMillen andConsigli (17).


Cytopathic effect resulting from

infec-tion by WT VSV or ts G31. While studying

the growth of the WT VSV and a ts mutant

virus, ts G31, we observed thatinfected

neuro-blastoma cells underwentavarietyof

cytopath-ological alterations, including the formation of

polykaryocytes. The N-18 neuroblastoma cells

attach, spreadout onthe surface ofthe culture

flask, and extend neurites which can attain

lengths of5 to 10times that of thecell

perikar-yon(Fig. 1).Uponinfection with WTVSV (MOI

of 10),cellsbecamerounded and manyappeared

to retract orlose their neurites (Fig. 2). At 24 h postinfection, most cells became unattached

from the culture vessel surface and floated in

the medium. This cytopathological effect was

observed at avarietyof temperaturesatwhich

the WT virus has beengrown (31, 37,or


Incontrast to WT VSV, infection with ts G31 (MOI of 10) resulted in quite a different cellular

response whenthecellswere incubated at


thenonpermissive temperature for the

replica-tionof this virus (9). N-18cellsinfected with ts

G31 and incubated at 39°C underwent fusion

within15h(Fig.3). The fusioninitiallyproduced

large polykaryocytes,with almost 10nuclei per

polykaryocyte; but upon further incubation

many polykaryocytes appeared to fuse with

other polykaryocytes. This eventually resulted

in polykaryocytes containing as many as 50 to

100 nuclei. Although the actual site of cellular

fusion has not been observed, cells often have

been observed with long cytoplasmic bridges

between polykaryocytes which resemble the

neuriteextension (Fig. 3). Incubation of ts

G31-infected cellsat


the permissive

tempera-tureforvirusreplication, did not fuse thecells, but rather they became rounded in a manner

similarto the cytopathological effect produced

byWTVSV(Fig. 2).

Kinetics ofcellfusion. In our initial

exper-iments, we examined the effect ofthe MOI on

the formation ofpolykaryocytes. ThebestMOI

for fusion was 10 infectious virions per cell,

which resultedinfusion of 80 to90% ofthe cells,

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FIG. 1. Phase-contrastmicroscopyof uninfectedN-18neuroblastoma cells. These neuroblastoma cellswere platedand grownat37°Cfor3days (x800).

whereas either higherorlower doses resultedin

significantlyless fusion (with approximately35%

of thecells fusedat anMOIofeither 1 or 100).

Using an MOI of10, we have quantitated the

kinetics ofcell fusion by determiningboth the

number of cells that uadergo fusion and the

averagenumberof nucleipresent per polykary-ocyte.The kinetics of fusionwaslinear to 15 h

when 90% of the cells were fused; however, by

quantitating the average number of nuclei per polykaryocyte,itwasclear thatfusion continued

for at least 40 h (Fig. 4), since the average

number ofnuclei perpolykaryocyte continued

toincrease from 10at 15h toalmost30 at 40 h.

Apparently polykaryocytes fused together,

re-sulting inlargerpolykaryocytes,many of which

contained50ormorenuclei. N-18cellsinfected

with ts G31were simultaneously fixed with2%

glutaraldehyde and 2% osmium tetroxide and

postfixed with osmium.Thinsections examined

byelectronmicroscopy confirmed the

multinu-cleatenatureof thepolykaryocytes (Fig. 5).

Fusion of othercell lines.Five different cell

lines have been examined for their ability to

undergopolykaryocyteformation after infection with ts G31 at39°C (Table 1). Onlythose cell

lines derived from the nervous system have

proven capable ofundergoing fusion. It is not

clear atthis time


N2a cells, another

neu-roblastoma cell


didnotpromote fusion as

readily as the N-18


although the neurite

extensions of the N2a cells are less elaborate

when this line is grown in



Three other cell lines,


BHK-21 and





used for




did notshowany


formation upon infection withts G31when

in-cubated at39°C. All ofthecell lineswere able







bycell roundingatthe



Evidence for fusion from within. As

de-scribedin theintroduction, there aretwo basic

types of virus-induced cellular


FFWO and FFWI. The kinetics of fusion


4) were



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5 to 6 logs, the ability of the virus to promote

cellular fusion was lost (Table 2). The fusion

reaction wasalsodependent on protein synthesis

since the addition ofcycloheximide to the

in-fected cells completely abolished the capacity

forfusion. Theproteinsynthesis is presumably

of viralorigin,since the addition ofactinomycin

D to infected cells did not affect the initial

amountof fusiontoany great




Ourdataarethusconsistent with the idea that

a viral component, which isresponsible for


,* ..; ,,.. .

thecytosol rather thanassociating with the cell

membranes, while the Gglycoprotein,however,

accumulates in the cell membranes. Since we felt that the association of G with the cell

mem-branes may beresponsible for the initiation of

fusion, we have begun to study the role this

protein plays in cellular fusion. Our initial

ex-periments have examined the effect of inhibiting

the processing of the G glycoprotein on cell

fusion. Since theglycosylation ofG is involved

inthemigrationof Gtotheplasmamembranes




FIG. 2. Phase-contrast microscopyof N-18 cellsinfectedwithWT VSV. N-18 cells weregrown toconfluency asinFig.1,infectedwith WTVSVatan MOI of10, andincubatedat39°C. Most of the cells are rounded and

have losttheirneuriteextensions by20hafter infection. Thesesame morphological changes are seen with WT VSV-infectedcells incubatedat31°C orwith N-18 cells infected with ts G31 and incubated at 31°C (x800).


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FIG. 3. N-18 cellsinfectedwithtsG31at39°C. N-18 cellswereinfectedat anMOIof10and incubatedat 39°C for24hafter infection,atwhich timeanumberofpolykaryocyteswereforming (x800).

(14,25),wehave added glucosamine, an

inhibi-torofglycosylation,tocellsinfectedwith ts G31

toprevent the incorporation of G into the cell

surface. Either 1 or 10 mM glucosamine

in-hibited the fusion reactioninitiallyto about 75%

(Fig. 6, compared with the controlsin Fig. 4).

Evenuponfurther incubation up to 40 h

post-infection, the fusion reaction appeared inhibited.

Sinceglucosamine couldhave inhibitedprotein

synthesis (28),wemeasured the effectsof 1 and

10mMglucosamine onbothcellularand

virus-directed translation. Although 10 mM reduced

cellular and viral protein synthesisby

approxi-mately 50% in N-18 cells, the lower dose of 1 mMinhibited neither hostcellnor viralprotein

synthesis. Thisstudyindicates that the process

ofglycosylationis essential for the promotion of

fusion, presumably by its importance for the

normalmigration of theGprotein into the

cel-lularplasma membranes.

As a more direct assay ofthe role of the G

glycoproteinatthecell surface, we have

exam-ined the effects of antiserum specific for G on

fusion. For this experiment,N-18cellswere

in-fected withtsG31 and then incubatedat


At 2hafterinfection,thespecificserum(diluted

1:10) wasaddedtotheinfected cells. TheN-18

cellsfusedupto80±5%(for triplicate samples)

whennorabbitserum wasadded. The addition

of antiserum fromarabbitimmunized with

pu-rified G completely inhibited the formation of

polykaryocytes (nofusion was


Con-trol serumcollected from the rabbit before

im-munizationdidnotinhibit fusion when addedto

theincubation medium of the infectedcells


± 2% of theN-18cellswerefused





The formation of polykaryocytes by ts G31

appeared to be the result of an intracellular

defective maturation andassembly of the viral

polypeptides. ThisFFWIwasinducedby

infec-tiousvirions, and thekinetics of cellular fusion

coincidedwith theperiods of intensive viral

pro-tein synthesis (9). It is interestingto notethat


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20 E z




Hours After Infection

FIG. 4. Kinetics of polykaryocyte formation

in-ducedbytsG31inN-18 cells. N-18 cellswereseeded

onto glass coverslips, incubated 3 days until

con-fluent, andtheninfectedwithtsG31atanMOIof10 and incubatedat39°C.At 5-h intervals after infec-tion, thecoverslipswerefixedand stained and the

amountof polykaryocyteformation wasdetermined

as described in the text. Two different indices of cellularfusionarepresented: (A)Thepercentage of nucleipresent in fused cells, and (B) the average

number ofnucleiperfusedcell. All determinations

aretheaverageoftriplicate samples±therange.

the cellular fusionoccursat390C (the

nonper-missivetemperaturefor viralreplication)sothat

theproduction of infectious virions didnot

ap-pear to be necessary for polykaryocyte forma-tion. Theuse of themetabolic inhibitors

cyclo-heximide andactinomycin D hasdemonstrated

thatsomeviralprotein metabolismwasessential

forcellular fusion. Infact,ourlaterexperiments

havesuggested thatasingle VSVprotein, the G

glycoprotein,mayberesponsible for this FFWI.

Our previous report on ts G31 had

demon-strated that, although the otherproteins of the virus (in particular, the M protein) appear

in-capable of undergoing the normal assembly

process at the nonpermissive temperature, the newly synthesized G protein is almost totally localized inthemembranes of the cell (9). Inhi-bition of glycosylation with D-glucosamine, whichpresumably blocks the normal migration oftheGglycoproteintothe plasma membrane (14), also blocked theprocessof cellfusion (Fig.

6). Although glucosamine may have inhibited

fusion by other mechanisms (28), adecrease in

protein synthesis was not the reason for

de-creased polykaryocyte formation. Finally,

anti-serum directed against G completely abolishes

(15, 24, 26, 27). Presently, the mechanism by

which theseviralglycoproteinsinitiate the

proc-ess ofcell fusion is unclear, aswell aswhether

all these viral glycoproteins act in a similar

fashionatthe cell surface.

Ourexperimentshave alsodemonstrated that

the virus-induced cellular fusionwashighly


thetwoneuroblastoma cell lines underwent

po-lykaryocyte formation, with N-18 cells fusing muchmoreextensivelythan N2a cells. Theonly morphological difference between these

neuro-blastoma cell lines wasthe greater elaboration


and length of the neurite outgrowths of N-18

TABLE 1. Fusion induced in various cell lines by ts G31 at390C0

Cellline % Nuclei in fusedcells

N-18 80.0±5%

N2a 10±1.5%


BHK-21 0

3T3 0

aThe various


lines, propagatedonglass


slipswereinfected when confluent withtsG31atan

MOI = 10and incubatedat390C. Fusionwas deter-mined at 15 h after infection and is presented as the percentage of thenuclei that were present in fused

ceUls± standard deviation. Continued incubation (to

40h)didnotresult in greater fusion.

TABLE 2. Evidenceforfusionfromwithin' Virus Additiontocells %Nuclei in

tsG31 82.0±3.0

tsG31 inactivated 0

with UVlight

tsG31 +Cyclohexi- 0

mide at100


tsG31 +Actinomycin 67.0 ± 2.0

Dat 5,ug/ml



wereinfected withtsG31at anMOI of 10, or thesame amountofvirusafterUVinactivation,

and the cellswereincubatedat39°C. Cycloheximide oractinomycin D wasaddeddirectly after the cells

were infected, and fusion wasassayed at 15 hafter infection. Thepercentage ofthenuclei thatwere

pres-ent infused cells ± standard deviation for triplicate cultures ispresented.

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FIG. 5. Electron micrograph of N-18 cells infected with ts G31 at 39°C. N-18 cells were infected at an MOI

of10,incubatedat39°C, and then fixed at 24 h after infection for electron microscopic observation(x3,300).








45-0. S.5



0 10 20 30 400 10 20 30 40

HoursAfter Infction




z 20


20 %.

cells. The presence of these neurites may be

responsiblefor the initial fusion reaction and the

cytoplasmicbridgesbetweenfusingcells(Fig. 3).

Alternatively, moresubtle differences of thecell

surface cytoskeletonarchitectureormembrane

biosynthesis may underlie the differential

sen-sitivity of cell linestocellular fusion.

Polykaryocyte formation whichwas seenwith

theseneuroblastoma cellsmay be restrictedto

nervoussystem-derivedcellsinfected withsome

tsVSVmutantsand may be


apart of the

unique cytopathological effect of ts VSV on

FIG. 6. Kineticsofpolykaryocyteformation in the presenceofglucosamine. N-18 cells were infected with

tsG31asindicated inFig. 4, except glucosaminewas

addedtotheincubation media to either 1 mM(0)or

10mM(0). Theinfectedcells were then incubated at

39°C,andtheamountof fusionwasquantitated as described in the text. The determinations are the averageof duplicate samples+the range.

A. B


VOL. 30,1979


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[image:7.505.43.438.74.412.2] [image:7.505.41.443.449.659.2]

sueculture underlies the rnechanisms associated

with theseultrastructuralchangesthatoccurin

vivo in the mousespinalcord. Moreimportantly,

it may besignificantthattsG31 infectionresults

in cellular ultrastructure changes and a status spongiosus that are similarto slow viral

infec-tions such as kuru and Creutzfeldt-Jakob

dis-eases (4, 5, 21). Perhaps, the mechanisms that

lead to thecentralnervoussystemspongioform

changes and polykaryocyteformation willprove

tohave acommonbasis.


We thank Sevinc Akinc for her technical assistance. Thisstudy wassupported byPublic HealthServicegrant

NS13045from theNational InstitutesofHealth,theKenyon Giese Memorial Grantfor Cancer(BC-232)from the American CancerSociety,andMRS7319 from the Veterans Administra-tion.S.G.R. isaClinicalInvestigatorof the Veterans Admin-istration.


1. Beck, E.,I. J.Bak,J.F.Christ,D.C.Gajdusek,C. J.

Gibbs, Jr.,and R.Hassler.1975.Experimental kuru in thespidermonkey. Brain 98:595-612.

2. Bratt, M.A., and W. R. Gallaher. 1969.Preliminary

analysisoftherequirements for fusion from within and fusion from withoutbyNewcastle's Disease Virus. Proc. Natl. Acad. Sci. U. S. A. 64:536-543.

3. Clark, G.,R. E.Coalson,and R. E.Nordquist.1973.

Staining proceduresused by theBiological Stain Com-mission, p. 35-36. The Williams and Wilkins Company,


4. DalCanto,M.C.,S. G.Rabinowitz, and T. C. John-son. 1976.Anultrastructuralstudyof centralnervous systemdiseaseproduced by wild-typeand temperature-sensitive mutants of vesicular stomatitis virus. Lab. Invest. 35:185-196.

5. DalCanto,M.C., S.G. Rabinowitz, and T. C. John-son. 1976.Status spongiosusresulting from intracerebal infection of mice with temperature-sensitive mutants of vesicular stomatitis virus. Br. J. Exp. Pathol. 57:321-330.

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7.Harter, D.H., and P. W. Choppin. 1967. Cell-fusing activity of visna virus particles. Virology 31:279-290. 8. Homma, M. 1975. Host-inducedmodificationof Sendai virus, p.685-697. In B. W. J. Mahy and R. D. Barry (ed.), Negative strand viruses, vol. 2. Academic Press, London.

9. Hughes,J.V., T. C. Johnson, S. G.Rabinowitz,and M.C. Dal Canto. 1979. Growth and maturation of a vesicularstomatitis virustemperature-sensitive mutant

U.S.A. 75:2969-2971.

12. Klatzo,I., D.C. Gajdusek,and V. Zigas. 1959. Pathol-ogyof kuru. Lab. Invest. 8:799-847.

13. Lampert, P., J. Hooks,C. J. Gibbs, Jr., and D. C.

Gajdusek. 1971.Alteredplasmamembranes in exper-imental scrapie. Acta Neuropathol. 19:81-93. 14. Leavitt, R., S. Schlesinger, and S. Kornfeld. 1977.

Impaired intracellularmigration and altered solubility ofnonglycosylated glycoproteinsofvesicular stomatitis virusandSindbis virus. J. Biol. Chem. 252:9018-9023. 15. Manservigi, R.,P.G.Spear,and A.Buchan.1977.Cell

fusion inducedbyherpessimplexvirus ispromoted and

suppressed bydifferent viralglycoproteins.Proc.Natl. Acad. Sci. U. S. A. 74:3913-3917.

16. Mathews,R. A.,T.C.Johnson, and J. E. Hudson. 1976.Synthesis andtumover of plasma-membrane pro-teinsand glycoproteins in a neuroblastoma cell line. Biochem. J. 154:57-64.

17. McMillen, J.,and R. A.Consigli.1977.Immunological reactivity of antisera tosodiumdodecyl sulfate-derived

polypeptidesofpolyomavirions. J. Virol. 21:1113-1120. 18.Ozawa,M.,A.Asano,and Y.Okada.1976.Importance

ofinterpeptidedisulfide bond ina viralglycoprotein

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19. Poste,G. 1970. Virus-induced polykaryocytosis and the mechanism ofcellfusion. Adv. Virus Res. 16:303-357. 20. Poste,G.1972.Mechanisms ofvirus-inducedcell fusion.

Int. Rev.Cytol. 33:157-252.

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mutantsof vesicular stomatitis virus. Infect. Immun. 13: 1242-1249.

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Canto.1977.Theuncoupled relationshipbetween the

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multipli-cation ofenvelopedviruses by glucosamine. Virology


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FIG. 1.plated Phase-contrast microscopy ofuninfected N-18 neuroblastoma cells. These neuroblastoma cells were and grown at 37°C for 3 days (x800).
FIG. 2.haveasWT in Phase-contrast microscopy ofN-18 cells infected with WT VSV. N-18 cells weregrown to confluency Fig
FIG. 3.39°C N-18 cells infected with ts G31 at 39°C. N-18 cells were infected at an MOI of 10 and incubated at for 24 h after infection, at which time a number ofpolykaryocytes were forming (x800).
TABLE 1. Fusion induced in various cell lines by tsG31 at 390C0


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