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Fate of Infecting Simian Virus 40-Deoxyribonucleic Acid in Nonpermissive Cells: Integration into Host Deoxyribonucleic Acid


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Copyright(© 1972 American Society for Microbiology Prinitedin U.S.A.

Fate of Infecting Simian

Virus 40-Deoxyribonucleic



Nonpermissive Cells:







Inistitut fiir Virusforsclhung, Deutsches Krebsforschunigszem'trimn, 69 Heidelberg, Germaniy

Receivedforpublication25April 1972

Nonpermissive 3T3 cellswere infected with purified superhelical simian virus 40

(SV40) deoxyribonucleic acid I (DNA I). Onehourafter infection, approximately

60%ofthe intracellularSV40DNAwasconvertedtorelaxed forms. One dayafter infection, all intracellular SV40 DNA was present as slow-sedimenting material,

and noSV40 DNA I wasdetectable. At 2days after infection there appeared viral

DNA sequences cosedimenting with cellular DNA during alkaline velocity

cen-trifugation. Furthermore, byboth alkalineequilibrium gradient centrifugation and by DNA-ribonucleic acid hybridization analysis, covalent linkage of viral DNA sequences to cellular DNA was demonstrated. Integration of SV40 DNA into

cellular DNA didnot appearto require DNAsynthesis, although DNA synthesis

followedby mitotic division of the cells enhanced theamount of viral DNA

inte-grated. Based on data obtained by two different methods, it was calculated that

1,100 to 1,200 SV40 DNA equivalents must be integrated per cell by 48 hr after


There have been many lines of evidence that demonstrate the


of the simian virus40 (SV40) viral genome intransformed cells. These

cells have characteristic new nuclear and surface antigenic




rescuable viral



display specific

sequences of hybridizable intracellular viral


acid (DNA)







addition, examination of transformed cells has shown that viral DNA appearstobeattachedto

the cellular DNA


alkali-stable covalent link-ages



it has been shown that

infection of primarycells with SV40 virions leads

tosuch covalent


earlyafter infection and that


is not


on cellular DNAsynthesis


Itis of

interest, therefore,


examine such early events in terms of the con-formational

changes undergone by




It has been shown that isolated SV40 DNA loses both


and the



cells upon


of its



(1, 17).

Inaddition to evidence for thecovalent

integra-tion of the viral DNA, it has been established that transformed cell lines do not contain


supercoiled forms of this DNA

(10, 18).

It is reasonable, therefore,to assume aconversion of the circular superhelical SV40 DNA molecule

to an open integrated duplex during the initial steps of transformation. The considerable im-portance of the process of integration for the

mechanism of viral oncogenesis warrants a

detailed analysis ofthe early structural changes

undergone by infecting viral DNA that lead to the finalassociation oftheviral genomewith the

hostcell DNA.

We report here a kinetic analysis of the

con-formational changes of SV40 donor DNA upon

infection of nonpermissive 3T3 cells. These cells permit transformation rather than productive

infectionbecause thereis little, if any viral DNA-replication and virus maturation (15). Cells were infected with freeDNAinstead of with virions to allow a direct kinetic analysis of the fate of the

DNA without complications due to events

associated with the uncoating of the virus.

Evidence will be presented to show that the

infecting supercoiled SV40 DNA becomes

covalently linked to cellular 3T3 DNA within

2 days after infection. Further proof for the

integration of SV40 DNA into cellular DNA

is based on sedimentation velocity and equi-librium density analyses at alkaline pH. These methods of analysis show that viral DNA

sequences indeed reside in molecules displaying

thecharacteristics of cellular DNA.


on November 10, 2019 by guest




Wehave previously reported that cell division enhances association of SV40 DNA with the

nucleus of the dividing cell, although such

asso-ciation can also be observed to a smaller extent

inresting cells(12). From the data presented here, it appears that this integration process is not

dependent on cellular DNA synthesis, although it seems to be enhanced during mitotic division

ofthe infected cells.


Virus and cells. Production of SV40 and culture conditions forCV-1 and3T3 cells were as previously

described (4).The 3T3cellswerekindlyprovidedby


Preparation of radioactively labeled SV40 DNA.

Confluent CV-1 cultures in large, roller production

vessels (Bellco Glass Inc., Vineland, N.Y.) were in-fectedat amultiplicityof1 plaque-formingunit/cell.

Theviruswasadsorbed for2hr; the cellswerewashed with Hanks solution and were then maintained in mediumcontaining either500MCi of3H-thymidine/50

mlor50,Ci of'4C-thymidine/50 ml for4 to 5days. Labeled SV40 DNA was selectively extracted (7), phenolized (14), subjected for 1 hr at 37 C to ribo-nuclease treatment (10Ag ofpancreatic ribonuclease [previously heated for15minat90C] perml),and

re-extracted with phenol. After removal of the phenol

(14), the DNAwasprecipitatedwithtwovolumesof undenatured ethanol, resuspended in 0.1 X SSC (standard saline citratebuffer),andfurtherpurifiedby

bandingtheDNA inacesiumchloride-ethidium bro-mide (CsCl-EtBr) equilibriumgradient (Spinco rotor

40,38,000rev/min, 64 hr,20C, 150 ,ug ofEtBr/ml in 0.02 M tris(hydroxymethyl)aminomethane [Tris],

0.002M ethylenediaminetetraaceticacid [EDTA],pH

7.0). The peak ofhigher density was subjected to a

velocity sedimentation through an alkaline sucrose

gradient as described below.The peak fraction sedi-menting at 53S (SV40 DNA I) was isolated,

neu-tralized, and dialyzed against 0.1 X SSC. The

puri-fied, labeled SV40 DNAwasstored at -70 C(specific

activity ofthe3H-SV40 DNAIwas39,100countsper min per ,g; 14C-SV40 DNA I, 9,800counts per min per,ug).

Infection of cells withSV40 DNA I. With the

ex-ception of theexperimentdescribed in Fig. 1 (for de-tails,seelegendtoFig. 1),thefollowing procedurewas

employed. Confluent monolayers of3T3cellsin milk dilution bottles (2.5 X 106


i.e., per 44

cm2) were washed with phosphate-buffered salt

solu-tion and infected with 3H-SV40 DNA I (2.3 X 105

countsperminper ,ugofDNA and 150/ug of

diethyl-aminoethyl[DEAE]-dextranin0.5ml ofHanks solu-tion). Mock infectionwasperformed by replacing the volumecomprised by the SV40 DNA I with 0.1 X

SSC. Thecultureswereincubatedfor 30minat 37 C

with occasional agitation.Then themedium contain-ngthenonadsorbed DNAwasremoved. The cultures

were washed with 15 ml of Hanks solution, and a

deoxyribonuclease treatment was performed. The

re-action mixture added to a milk dilution bottle



ofbovine pancreaticdeoxyribonuclease (Mann, New York) in 1 ml of Hanks solutionwith 0.005 M MgSO4. The cultures were reacted with deoxyribonucleaseat 37 Cfor10min and thenwashed twice with 15ml of Hankssolution followed by addi-tion of15 ml of fresh mediumsupplemented with un-labeled thymidine (5


'ml). To part of the cultures, inaddition, 15Mugof arabinofuranosylcytosine (ara-C) (Serva, Heidelberg) wasadded. Three hours postin-fection the cultures weretrypsinized and reseededat a 1:2 dilution into new vessels in the media supple-mented with unlabeled thymidine or ara-C, or both, asindicated. The number ofcells per flask that had re-adhered by7 hrpostinfection was6X 105 to 7 X 105. Isolation ofDNA from infected cells. At various times after infection, cell extracts were prepared bya

modification of the method described by Doerfler (2). Thecells were washed with Hanks solution (30 ml) and lysed by addition of2mlof 0.5'S sodium dodecyl sul-fate in 0.1 M Tris, 0.02 Ni EDTA (pH 7.2) per flask. After 10 min at room temperature, the lysates were treatedfor20min at 37 C with 300 ,ug of PronaseB

(Serva, Heidelberg) per ml, which had been preincu-batedat37Cfor90 mintoinactivate nuclease3.

The lysates were extracted twice with redistilled phenol saturated with 1 MTris, pH 7.5.After removal of phenol and incubation with ribonuclease as de-scribed above, the purification procedure was com-pleted byafinal phenol extraction and reduction of thevolume of the DNA solution by Sephadex as


Preparation of labeled cellular DNA. 3T3 cells (6 X 105) were seeded intomilkdilution bottles. The medium was supplemented 5 hr later with 1 ,Ci of

3H-thymidinepermlfor another48hr.Then the DNA

wasisolatedasdescribed above.

Alkaline equilibrium centrifugation. Conditions for alkaline CsCl-EtBr equilibrium gradient centrifuga-tion were asfollows. The DNA was dissolved in 4 ml ofaCsClsolution containing 0.02 M Tris and 0.002M

EDTA(pH 12.5) with 150


ofEtBr/mlandenough CsCl to give an initial density of 1.6


The centrifugation was carried out in a Spinco SW50.1 rotor for 64 hrat20 C and39,000rev/min.

Alkaline sucrose gradient. Alkaline sucrose solu-tions were made up in solution containing 0.3 M NaOH, 0.7MNaCl, and0.001 MEDTA. A5to20%

gradient was prepared with a cushion of 0.2 ml


sucroseonthe bottom.Centrifugation was carried out

in anSW50.1 rotor at 46,000


for 90minat 20

C. The gradients were analyzed as described in the


Preparation ofin vitro complementary SV40 ribo-nucleic acid. The preparation ofSV40 complementary ribonucleic acid (cRNA) was performed with Escher-ichiacoli RNA polymerase and SV40 DNA I essen-tially bytheprocedure described by Westphal and Dul-becco.3H-cytidinetriphosphate and 3H-guanosine tri-phosphate wereused asthelabeled precursors inthe reaction mixture. The specific activity of the cRNA

was2.4X 106 counts per min per mg.The cRNA con-sisted ofmoleculeswhose averagesingle-stranded rise

was 1 X 106daltons. Atleast 25% ofthe molecules

had asize of 1.5 X 106daltons or more (A. H. Fried

and F.Sokol, J. Gen.



426 J. VIROL.

on November 10, 2019 by guest



DNA-RNAhybridization. This hybridization tech-nique was employed to estimate the amount ofSV40 DNA in various sucrose gradient fractions. The pro-cedure used including the ribonuclease treatment of the hybrid was identical to that described by Westphal and Dulbecco (18). The SV40 DNA to be analyzed was denatured by heating for 30 min at 100 C in

0.01 X SSCfollowed by rapid cooling in an ice bath. It was then immobilized as described (18). In all ex-periments, the hybridization mixture contained of 30,000 counts of input 3H-cRNA per min in 1 ml of solution per tube. The percentage of input3H-cRNA counts thatremained onablankfilter was 0.05%. In calibration experiments with purified DNA, the amount of3H-cRNA bound to SV40DNA was pro-portional to the amount of DNA immobilized on the filters in the range between 8 X 10-5 to 1 X 10-2Mg of DNA.To1 X 10-21g of SV40DNA 3,800 counts/ min of'H-cRNA were bound. In the experiments in-volving the analysis of gradient fractions, the amount ofSV40 DNA per filterwill be seen to lie always in

this range of DNAconcentration.

Immunofluorescence. Immunofluorescence was per-formedaspreviouslydescribed (13). The anti-Tserum

was thegift of J.vander Noordaa.


Adsorption and penetration of isolated SV40 DNA I. The study of conformational changes

undergone by SV40 DNA during the initial stages of transformation may involve complica-tions ifvirions are employed instead ofpurified circular


SV40 DNA I. In addition

to problems related to the


of uncoating

events, SV40 pools contain


with linear cellular DNAinstead of



DNA (9, 16). It was necessaryin these studies,

therefore, to employ well

defined, purified

viral DNAratherthan virions.

The kinetics ofadsorption ofviralDNA onto

3T3 cells canbeseeninFig.1. Cellswereexposed to purified 14C-SV40DNA Ifor various periods of


deoxyribonuclease treated,

and then

assayed for cell-associated,


radioactivity. Deoxyribonuclease treatment was

used to ensuresynchrony ofevents after

adsorp-tionbyelimination ofthebackground ofinitially

attached butnonpenetrated DNA. For upto 60-min exposuretime,therewas alinear


between time of


and the amount of


protected from extracellular deoxyribonuclease digestion (Fig. 1). Asthe


exposure of

the cells to DNA with DEAE-dextran seemedto

exert sometoxicinfluenceoncellsthatweretobe maintained further, the period of adsorption

chosen intheexperimentstobedescribed did not

exceed 30 min.

InductionofT-antigen by SV40DNA I. Itwas

important to demonstrate the




N 8

x 6/


10 20 30 40 50 60 70

M N.

FIG. 1. Uptake of'4C-labeled SV40 DNA I into

3T3 cells. Co,ifluient montolayers of 3T3 cells in 6-cmn

plasticpetridisheswerewashed with


salinie and inifected wit/ '4C-SV40 DNA I (39,200

counts per miii per 4 ug ofDNA, 80 Mg of

DEAE-dextrant in 0.1 ml of Haiiks soluitioni). Thzeadsorption

was carried out at 37 C. At the timnes indicated, the

cultures were treated with deoxyribonuclease as

i,idi-cated in Materials anid Methods, except that 80 ug

ofdeoxyribontulclease wasusedperdish. Tleii the cells were washed twice With 5ml ofHaniks solutioni,

har-vested bytrypsintization, antd washedaniothertwo times

bypelletinig anidresuspenidinig in Haiiks solutioni. Tlhe

resuspen2ded cells were precipitated by additiolz of

trichloroaceticacid(togive afinzalconicenltration of 5%) keptoverniighltat 4C,anidcollectedotn membranefilter

pads (Millipore Corp.). After beiiig washed with 5%

trichloroacetic acid, the filters were dried, antd


of the penetratedSV40DNAin theinfectedcells after termination ofthe period of adsorption by treatment with deoxyribonuclease. Therefore, susceptible CV-1 cells (which are permissive and respond more efficiently to infection by viral

DNA than 3T3 cells in terms of synthesis of T-antigen [1]) have been used to examine the interrelationship between time of adsorption of SV40 DNA I, deoxyribonuclease treatment and the capacity of the infected cells to synthesize

T-antigen (Fig. 2). Treatment of the cells with

deoxyribonuclease up to 10 min after infection almost completely abolished their


to synthesize T-antigen, as determined 3 days after infection. After this lag period,however, alinear

relationship was observed between time ofDNA adsorption and the percent


cells scored 72 hr post-infection. It is shown by

these data that termination of adsorption of

SV40 DNA I by removal of the nonadsorbed

viralDNAwithdeoxyribonuclease did not affect

the biological activity of the viral DNA which was protected from the action of the enzyme by

itsclose contactwith, oritspenetrationinto, the 427

on November 10, 2019 by guest





u 8 P







z <2

10 20 30 40

M N.

FIG.2.Relationiship betweeniuptake of SV40 DNA I

anidproduction ofT-anitigeni.


CV-J cellsoni

cover slips in Leighton tubes were washed with I ml

ofphosphate-buffered salinie anzd infected with SV40

DNA I (12jig of DNA, 80 jigofDEAE-dextran in

0.2mlof Hankssolution). Attimes inidicatedthe cull-tures were washed and treatedwithdeoxyribonuclease

as described. Theni the cells were washedagain, and fresh medium wasadded. At 72 hrafter infection, the

cells werefixed anid stained for immunofluorescence.

host cell. In the following studies,


postadsorption events were synchronized by

removing nonadsorbed DNA with


FateofSV40DNA in 3T3cells:integrationinto

cellular DNA. Alkaline sucrose


were employed for the study of the intracellular fate of donor SV40 DNA I in 3T3 cells. These cells were infected with purified form I DNA for 30


deoxyribonuclease treated, trypsinized, and

thenmaintained eitherinthe presenceorabsence

of ara-C.The DNAwasthenextractedfromthese cultures at various times



analyzed on alkaline sucrose


It can be seen (Fig.


that as early as 1 hr



supercoiled viralDNA

has beenpartiallyconvertedtoslowersedimenting forms, probably representing open circular and linear structures.By24hr(Fig. 3c) postinfection,

all the viral DNA appeared in this slower

sedi-menting form II region of the gradient. At 48

hrpostinfection (Fig.3d), bywhich time the cells maintained without ara-C had undergone one division (12), a peak of radioactivity appeared

cosedimentingwith cellular DNA. At 72hr (Fig. 3e), this fast-sedimenting component was still present although in slightly reduced amount. If

theinfectedcells weremaintained in the presence of an inhibitor ofDNA synthesis, the



X30 I

x 20





10 20 30 10 20 30



FIG. 3.Alkalinie velocitysedimenitationt ofDNA

iso-lated from unintfected anid inifected cells.

Sedimenita-tionipatterli ofS V40 DNA I (a) as determined in a

recontstituitiont experiment.


cells which had beeni trypsinzized anld reseeded as described in

Materials and Methods were harvested 48 hr after

mock infectiont andmixedpriorto Proniase treatmenzt

with 3H-SV40DNA1 (2jig). Tlhe,i the totalDNA was

isolatedasdescribedantd sedimented


a/lalkalinie sucrose gradienit (C). The DNA extract layered on

thegradienit represenits 7.5 X 105cells. Sluperimposed

on this clurve is the sedimentationi patterni of

3H-labeled3T3cellDNA isolatedas described (A). The

numberofcellsfrom whichthecellDNA wasextracted and layered




was the same as above.

Sedimentationi patter/i of DNA extracted from

in-fectedcells either with (0) or without ara-C (0) at

(b) 1, (c) 24, (d) 48 anid (e) 72 hr



nlumber of cellsfrom which the DNA was extracted

andlayeredoni each gracdienit was (b) 1.2 X J01, (c)

5 X 105, (d) 7.5 X 105, amid (e) 1.05 X 105. hI each

case, thesedimemitationi


ofmark-erSV40 DNA I

(arrows) was determinedby mixiiigina reconstitution

experimenit3H-SV40DNA1 (2Ag) withm9)ck-imifected

cellstrypsimiizedamidreseeded as described. Themiumber

of mock-ilifected cellsfrom whichthe DNAextractwas

prepared amid mixed with the marker DNA wasfor

each time period the same as imidicated above for



cultures. Alkumline suicrose gradients were

428 J. VIROL.

on November 10, 2019 by guest



menting component stillappeared at 48 hr post-infection but in a considerably reduced amount. This is in agreement with our earlier observation that the total viral radioactivity persisting in infected cells was considerably enhanced incells that were allowed to undergo division (12).

We also did control experiments to eliminate

the possibility that SV40 DNA I might, under

these experimental conditions, form aggregates

and contaminate the cellular DNA region of

the gradients. These control experiments (Fig. 3aandarrowmarkersinFig. 3b-e)weredoneas

follows. Mock-infected cultures which had been trypsinized and reseeded as indicated for the

infected cultures were harvested at 1, 24,48, and 72 hr postinfection. At each time after celllysis, 3H-SV40 DNA I was added to the lysate, and

Pronase treatment and phenol extraction were

performed as described. This control lysate that

was mixed with the marker SV40 DNA I was

prepared from the same number of cells as the infected cell lysate. For each time period


(Fig. 3b-e),

the same amount of DNA

extracted from controls and from infected cells was layered on the respective alkaline sucrose

gradients. Fig. 3a presents the typical radio-activitypattern of marker SV40 DNA Ias deter-mined insuchareconstitutionexperiment.It can be seenthatnoviralDNAradioactivity


in the cellular DNA




and thatmixingSV40 DNA I with cells afterlysisand

before further extraction did not convert this

viral DNA to slower-sedimenting forms.

Super-imposed on this curve is the sedimentation

pattern of 3T3 cellular DNA determined in a similar manner






Thisalkaline-stable bindingofradioactivityto fast-sedimenting cellular DNA in the infected cultureswasnotdueto





of viralDNAinto


synthe-sized DNA. Inhibition of DNA



ara-C treatment


% in these


did not prevent the appearance of the




radioactivity (Fig.


This conclusion will be

supported by experiments


bedescribedin the lastsection.

Cellular nature of


component. If the


component described above




indeed represents viral DNA



then itis necessary to


establish that this component does have cellular


and does not represent either aggregates of viral DNA or circular

polymers of SV40 which may cosediment with


DNAisolated from 3T3 cells exhibited a much

higher banding density in alkaline equilibrium

gradients than superhelical SV40 DNA I (Fig.

4a). In these gradients, SV40 DNA oligomers, aswouldbe expected for superhelical molecules,

bandedclose to, butzfightlyless dense, than form

I (unpublished data). Addition of ethidium

bromideto suchgradients enhanced the difference

in banding positions between viral and cellular

DNA. This isprobably due to theability of the

superhelical viral DNA to bind relatively more intercalating dye than the denatured single-stranded



The fractions containing the radioactive com-ponent cosedimenting with cellular DNA in an


velocity gradient (similar to Fig. 3d,

open circles) were pooled, thereby eliminating degradation products of supercoiled viral DNA

containing at least one nick, which sedimented asform II or slower inthese velocity gradients. After neutralizationanddialysis,thiscomponent

x 2



°2 12




0 0


1.70 0 1.65 t

1.60 -c

1.55 1.50 3


10 20 30 40



FIG. 4. Alkaline CsClequilibrium centrifugation of

DNA isolatedfrom infected andnoninfected cells (a); eitherS,ugof3H-cell DNA (0)extractedasdescribed or purified SV40 DNA I (0) was mixed with the

CsCI-EtBr solutioni and centrifuged to equilibrium as

describedinMaterialsandMethods(b). The fractions

6 to 11 without ara-C treatment from



velocity gradient similar to that described in Fig. 3d were pooled, neutralized, dialyzed, and then

centri-fuged to equilibrium in alkalinie CsCI-EtBr as de-scribed above.

performed as described in Materials and Methods.

Thegradientswerecollectedon WhatmanGF/Cfilters whichweredriedpriortodeterminationofradioactivity.


on November 10, 2019 by guest




was centrifuged to equilibrium in alkaline CsCl withethidium bromide.Ascan be seen inFig. 4b, the bulkofradioactivityfrom this

fast-sediment-ing component assumed a banding position coinciding with the buoyant density of cellular DNA (Fig. 4a). The nature of the radioactivity appearing at evenhigherdensities atthebottom

ofthegradientis not knownatthepresent. Evidence for viral specific sequences in cellular DNA. As previously mentioned, inhibition of DNA synthesis with ara-C did not prevent the association of viral DNA-derived radioactivity with cellularDNA (Fig. 3). Although thisresult argues against re-utilization of radioactive pre-cursors from degraded viral DNA into newly synthesized DNA, a more rigid control

experi-ment was performed to further eliminate this possibility.

Cells wereinfected with unlabeledSV40DNA I, and48 hrpostinfectionthe DNA wasisolated and sedimented through an alkaline sucrose gradient. Various fractions of the gradient were

pooled and immobilized after denaturation on filters. In the control


the same procedure was followed with DNA prepared

under identical conditions from mock-infected

cultures. Then SV40 cRNA transcribed from

SV40 DNA I was


with the im-mobilized DNA. The


which was

specifically boundtotheimmobilized SV40DNA sequences


subtraction of


nonspecifically bound to DNA from

mock-infectedcells)isindicatedinFig.5by the vertical bars. As revealed from a


with the




ofa marker SV40DNA, SV40-specificsequenceswereindeed associated with the faster



DNA. The resultof this


experiment and the result obtained from the alkaline equi-librium


show that the fast-sedi-mentingcomponent seenin Fig. 3d and e repre-sents


DNA sequences covalently linked inan alkaline-stablemannerwith cellular

DNA. It is notclear,however,whether theentire viralgenome oronlypart of it is



The experiments described here report a

chronological study of the conformational

changes undergone by free supercoiled SV40 DNA I upon infection of nonpermissive 3T3 cells. The donor SV40 DNA I is first converted to aslowersedimentingcomponent and is then,

between 24 to 48 hrpostinfection, associated by an alkali stable linkage to fast-sedimenting cellular DNA.

From the actual radioactivity found in the fractions corresponding to the fast-sedimenting


10 0




<) .!

30 I CD


3X) i



20 X w

10 >

5 10 15 20 25


FIG. 5. Detectioni ofSV40 specific DNA sequences

from infected cells by hybridization with SV40 cRNA.

3T3cellswereinfectedwithunlabeledSV40 DNAIas described in Materials and Methods (6 ,ug of SV40 DNA Iper milk dilution bottle). The DNA was ex-tractedfrom theinfectedcells 48 hr postinfection and centrifuged through an alkaline sucrose gradient as described in Fig. 3d. The samples were thenpooled, neutralized, and dialyzed. After immobilization on

filterpads, the sampleswerehybridized with 3H-SV40 cRNA. Theresults indicatedbythebars are corrected by subtraction of the radioactivity nonspecifically bound (170 to 200 countsper minperfraction) to a control gradient ofDNA from uninfected cells (in both gradients identical numbers of cells were em-ployed). Superimposed is the profile of a marker 14C-SV40 DNApreparedas describedinFig. 3a.

cellular DNA

(Fig. 3d,



one can calculate that a substantial number of viral

genome equivalents must be integrated. The



oftheviralDNAis39, 100 counts per min per ,ug,and the


ofanSV40 DNA

moleculeis 5 X 10-18 g


X 106 g permole per 6 x 1023 molecules per mole). Therefore, it follows that the 200 counts/min in the


cellular DNA fraction in



(open circles, fractions 6 to 11) correspond to

1 x 109 SV40 genome equivalents. As there are

7.5 X




itmustbeconcluded that approximately 1,200 SV40 genome

equiva-lents areintegratedper cellat48hr


This is a surprisingly large number in view of recent findings where as little as four or less SV40 DNA


per cell have been re-ported

(5, 8)

in established virus-fiee

SV40-transformed 3T3celllines.

Anothercalculation, based onthe DNA-RNA hybridization assay for SV40 DNA, also revealed that a large number of SV40 genomes must be

integrated per cell 48 hr after infection. The

430 J. VIROL.

on November 10, 2019 by guest



amount of radioactive cRNA that binds to the

fast-sedimenting DNA in Fig. 5 (fractions5to8)

can be calculated from the 1,300 counts/min bound to this DNA. From the results of the

calibration experiments described in Materials and Methods, wecalculate that there are 3.5 x

10-3 Ag of SV40 DNA in these fractions. This

correspondsto7 x 108 SV40genomeequivalents,

and there are 6 X 105 cells per gradient in this case. Hence, we conclude again, by using the

DNA-RNA hybridization data, that there are

about 1,100to 1,200genomesintegratedper cell.

There isan apparent discrepancy between the small number of SV40 genome equivalents in

established transformed cell lines (5, 8) and the large number of viral DNA equivalents linkedto

cellular DNAat 2days after infection. It is pos-sible that presence of only a few viral genome equivalents may offer selective advantages to a

cell during the course of in vitro propagation. Thus, cells harboring initially many-fold the number of viral DNA equivalents mayeither be eliminated from the cultures and beoutgrown by cells with fewer viral DNA equivalents, or viral genomes may get lost owing to excision and degradation processes. Which of these explana-tions may turn out to apply remains to be


Our results, along with the results of others (6, 11), give strongevidence that SV40 DNA is

integratedas alinearpiece ofDNAinto host cell DNA. Oneimplicationof thisfindingis that the

donor superhelical form IDNA must be nicked

in one of its backbone phosphodiester bonds before its integration into the linear host cell molecule. Ourresultsclearly show thatthesecond

part of this overall reaction, the conversion of

nicked DNA into anintegration state must, on theaverage,takeatleast several hours. This can be deduced from the fact thatno integration of

the SV40DNA into the ceHular DNA can be

detectedat24 hrpostinfection,eventhoughat 1

hr postinfection approximately 60% of the

superhelical input DNA has been converted toa nickedform and after at 24 hr all of the donor

DNA hasbeen nicked.

To establish rigorously that viral DNA se-quenceswerecovalentlylinked tocellularDNA, it was shown by equilibrium analysis that the

radioactively labeled fast-sedimenting component from the alkaline sucrose gradients repiesented indeed linear single-stranded molecules. As

pointed out previously, single-stranded SV40 molecules were eliminated in thisexperiment by the prefractionation procedure. It has been

shown, therefore, that viral radioactivity was

associated with molecules that cosedimented

with cellular DNA in alkaline sucrose gradients

and displayed thecharacteristics of linear single-stranded cellular moleculesin alkaline equilibrium

gradients. This finding, as well as the controls indicated in thetext, eliminatedthe possibility of

viral DNAaggregatesorpolymers accounting for

theobserved fast-sedimenting peak.

Furthermore, the presence of viral DNA

se-quences in this cellular fraction was demon-strated by DNA-RNA hybridization analysis.

This result, as well as the results with ara-C, eliminated the possibility thatthe degradation of

labeled donor viral DNA to mononucleotides and thereutilization of the released 3H-thymidine monophosphate in cellular DNAsynthesis could

account for the observed phenomenon.

Ourresults indicate that integrationof theviral DNA into host cell DNA occurs when thecells

areundergoing mitosis but doesnotappeartobe dependent upon cell DNA synthesis. Doerfler (3) and Hiraietal. (6) have also found that viral DNA integration appears to be independent of cellDNA replication. In light of these observa-tions and our own previously reported observa-tions (12), itseemslikelythat although cell DNA

synthesis may enhance integration it is not an

essential prerequisite. This enhancement maybe

the result of replicating DNA rendering more

sitesavailable for the attachment and integration

of theincomingviral DNA.


We thankUrsula Leis for able technical assistance and Allan Fried for kindly providing the labeled complementary SV40-cRNA and for valuable discussion.

Thisinvestigationwassupported by the Deutsche Forschungs-gemeinschaft.


1.Aaronson,S. A., andM. A.Martin.1970. Transformationof

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baby hamster kidneycells. J.Virol. 6:652-666.

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proteinsincellsproductivelyinfected withsimianvirus 40.


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mouseand virus-transformed cell genomes.J. Mol. Biol.


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simianvirus 40deoxyribonucleicacid intothe deoxyribo-nucleic acid of primary infected Chinese hamster cells.

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infectedmousecell cultures. J.Mol.Biol. 26:365-369.

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Diamandopoulus, and J. F. Enders. 1970. Virus-specific deoxyribonucleicacid in simian virus 40-exposedhamster

cells:correlation withSandTantigens.J.Virol.6:199-207.


on November 10, 2019 by guest




9. Levine, A. J., and A. K.Teresky. 1970. Deoxyribonucleic acid replication in simianvirus 40-infected cells. J. Virol. 5:451-457.

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Dul-becco. 1968.Theintegrated state of viral DNA in SV40-transformed cells. Proc. Nat. Acad. Sci. U.S.A.


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SV40-transforimed and non-transformed monkey kidney cells. Z.Krebsforsch. 74:40-47.

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SV40 genome in productively infected and transformed

cells.Proc. Nat.Acad.Sci.U.S.A.61:1256-1263. 15. Todaro, G. J., and H. Green. 1966. Cell growth and the

initiation of transformation by SV40. Proc. Nat. Acad.


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host DNAby simianvirus40:asimianvirus 40


17. Trkula, D., S.Kit, T. Kurimura, and K. Nakajima. 1971. Infectivity of molecularforms of simian virus40 DNA.

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and SV40-transformed cell lines. Proc. Nat. Acad. Sci. U.S.A.59:1158-1165.

19. Williams,A.E.,andJ.Vinograd.1971. The buoyant behavior of RNAand DNAin cesium sulfate solutions containing dimethylsulfoxide.Biochim.Biophys. Acta 228:423-439. 20. Winocour, E. 1971. The investigation of oncogenic viral

genomesintransformed cellsbynucleic acidhybridization. Advan. Cancer Res. 14:37-70.

432 J. VIROL.

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FIG.1.padsplasticbyactivitykeptresuspen2dedtrichloroacetictrichloroaceticdextrantofculturescatedcountssalinievested3T3werewas pelletinig deoxyribontulclease Uptake of '4C-labeled SV40 DNAI into cells
FIG. 2.freshDNAcells0.2asturesofanidcover described. phosphate-buffered Relationiship betweeni uptake ofSV40 DNA I production of T-anitigeni
FIG. 4.fugedDNAscribeddescribedeitheror6wereCsCI-EtBrvelocity to purified Alkaline CsCl equilibrium centrifugation of isolated from infected and noninfected cells (a); S ,ug of 3H-cell DNA (0) extracted as described SV40 DNA I (0) was mixed with the soluti
FIG. 5.filterfromployed).bothcontrolbound14C-SV40byDNAdescribedneutralized,cRNA.centrifuged3T3describedtracted Detectioni of SV40 specific DNA sequences infected cells by hybridization with SV40 cRNA


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