Monomer and multimer covalently closed circular forms of Rous sarcoma virus DNA.

Vol.29, No. 2 JOURNALOFVIROLOGY, Feb.1979,p.799-804

0022-538X/79/02/0799/06-0000$02.00/0

Monomer and Multimer Covalently Closed

Circular Forms of

Rous

Sarcoma Virus DNA

GERARD GOUBIN AND MIROSLAV HILL*

Departmentof Cellular and Molecular Biology and Equipe de Recherche No. 148 du Centre National de la RechercheScientifique,Institute

of

Cancerology

and

Immunogenetics, Villejuif,

France

Received for publication 21 August 1978

Covalently closed circular moleculesof viral DNA synthesized in virus-infected

cells are composed mainly of monomers sedimenting at 22 to 27S in neutral

sucrosegradients.These monomers are detected by annealing with

complemen-taryDNAortransfectionassay.However, 11%oftheinfectious circlessediment

faster thanmonomers. There isa peakat 32S whichmaycorrespondto dimer

molecules. Traces of infectivity (about 3%) found between32Sand65S suggest

thepresenceof higher oligomers.Inalkalinesucrose gradients,covalently closed

monomers arefound at 64 to 71S.Infectivity ofthese monomers isreduced by

alkalitreatment toless than one-tenth, and, perhaps for thisreason, noinfectious

dimersorhigheroligomersareobserved. It has been shown thatupon

resedimen-tation the dimers of 95S can be separated from monomers and detected by

hybridization.

One of the DNA intermediates synthesized

duringthe lifecycleof thetypeC retrovirus has

a closed circular conformation (7, 26) and is

thoughttoarise fromalinear duplex of6 x 106

daltons (23).Giannietal. (6) firstmeasured the

size of the DNA circles and found that, in the

case of murine leukemia virus, the contour

length is characteristic of a molecule with a

molecular weight of about5.5 x106. In the case

of avian sarcoma virus, similar contour length

measurementrevealed thepresenceof different

molecular speciescorresponding in size to

non-defective, transformation-defective (td), and

de-fective genomes containing deletions of from

one-thirdtotwo-thirds of the viralinformation,

respectively (11). Sedimentation coefficients of

genome-sizeformIDNA in neutral and alkaline

sucrose gradients were 30S and 68 to 70S for

murineleukemia virus (7), 24 to 29S and62 to

65S foranaviansarcomavirus (10, 11),and 21S

and32S foranavian reticuloendotheliosisvirus

(5),respectively.

Supercoiled self-replicating circles of

mito-chondrial (22) or simian virus 40 (SV40) (20)

DNA,forexample, have been shownto contain

asmallpercentage of double- and multi-length

DNAforms.These forms are supposed to result

from errorsin replication, from recombination,

or from both (1, 20, 22). Retrovirus DNA was

never showntobeself-replicating. However, in

thispaper weshowthatlargerforms resembling

dimeric and oligomeric circular molecules are

found aswell asthemonomericforms.

Schmidt-Ruppin strain ofRous sarcoma virus

(RSV), subgroup D (SR-D), no. 304, and its td

derivativeno.300used in this workwereisolated

earlier(14). Viral DNAwaspurifiedfrom

virus-infected chicken cells 30 h after infection as

described elsewhere (13). Briefly, chicken

em-bryo fibroblasts (CEF; C/E phenotype,positive

for chickhelper factor)weregrown inmonolayer

cultures. About24hafterseeding, the cultures

were infected, in the presence of2 ,ug of

Poly-breneperml, withSR-Dor tdSR-Dat a

multi-plicity of 1 to 5 focus-forming units or 1 to 5

infectious units per cell, respectively. Thirty

hourslater the cellswerefractionatedaccording

toHirt (17), and the nucleic acidswerepurified

from the Hirtsupernatantand bandedto

equi-librium in a cesium chloride-ethidium bromide

densitygradient asshown inFig. 1.ViralDNA

was detected by

"2'I-labeled

RSV DNA probe

andfound in twobands atdensities

character-istic ofsupercoiled (form I)andnonsupercoiled

(forms II and III) DNAmolecules. The

differ-enceindensityof thesetwomolecularspeciesof

viral DNA and the corresponding density

markersseen in Fig. 1 wassupposed tobe due

to the different guanine plus cytosine contents

(N.Stedman,R.Mariage,G.Goubin,J.Hillova,

and M.Hill,J.Gen.Virol.,inpress).Thisfigure also showsthatform IRSVDNAcanbereadily

recovered if gradient fractions containing the

denser (but notthe lighter) part ofthe form I

SV40DNA bandarepooled.

Inallexperiments describedhere,suchapool

ofgradient fractionswassubjected to asecond

equilibrium cesium chloride-ethidium bromide 799

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800 NOTES

_'1X~~~~~~~~~~~

-1.60 A4i

20-U

o aV ~~~~~~~~~~1.56ca1~

[image:2.505.69.260.61.320.2]

15- rm

1.54Bo-tto

FIG.1.~~~~Iqiiruceiu~Frchoieehiimbo

RSV RSVDNA wU

(p 7.)(.11S)toehrih05 5lo2ehdu

4'

15201~~6

U

-10idY2 mgm) inenldniy akr t1C labee'-r)IS4anliermueDA)ad

solidCsCltoa

finaldenstyof18

gm.Ci50

5

5 10

150

Top Fraction number Bottom

FIG. 1. Equilibriumicesiumpchioride-ethidium

bro-midedensity gradient centrifugation of supercoiled

RSV DNA. About 1.5x 10' CEFwereinfected with SR-D for 30 h and thenlysed, and thenucleic acids

werepurified fromthe Hirtsupernatantasdescribed

(13). One-quarter of the nucleic acidswasdissolved in 3.5 mlof0.015MNaCs-0.0015 M sodium citrate (pH7.0) (0.1xSSC) togetherwith0.5mlofethidium bromide (2 mg/ml), internal density markers ("C-labeledform ISV40and linearmouse DNAs), and solidCsClto afinaldensityof1.582g/cm'.

Centrif-ugation wascarried outin a Spinco Ti 50 rotor at

40,000rpm at150C for 70 h.Fractionsof0.3mlwere

collected from the top, and 0.2-ml samples were

weighedinacalibratedmicropipettetodetermine the

density. Samples of25

ILI

were deposited on glass

fiber filters and dried, and the radioactivity was

measured in a Nuclear Chicago liquidscintillation spectrometer.Inhybridizationexperiments, 50.2g of

carrier rat thymus DNA in 1 ml of CsCl solution

(density1.5g/cm3) wasaddedtoeachfraction.Then

ethidium bromide was removed by treatment with

isopropanol-water (9:1), andthefractions were

di-alyzedagainst0.1xSSC. NaOHwas addedtoafinal

molarityof0.3Mi thefractionswereheatedat80°C for 2h, cooled, andneutralizedwith 2N HColMf Tris solution, and the DNA wasprecipitated with ethanolandspundownbycentrifugation. The DNA

pelletwasdissolved in 50

jil

of0.6MNaCl-0.02M

Tris-hydrochloride (pH 7.5)-0.01 M EDTA solution

containing 2,000 cpm of "'L1labeled genomic RSV RNA (specific activity20x 106 to80X 106CPM/tg) prepared accordingto Commerford (3) with

modifi-cations(27).Hybridizationswerecarriedout at680C for72h. Then thesamplesweremixed with0.5mlof

2x SSC solution containing 100 pg of RNase A (Worthington; heated at 80°C for 10min) per ml, incubated at 37°C for 60 min, and trichloroacetic

densitygradient centrifugation, and formIRSV

DNA was recovered again from fractions

con-taining the heavier part of form I SV40 DNA

density marker. It was estimated (profiles not

shown) that after this purification procedure

formIRSVDNA wasvirtually devoid (lessthan

1.5%) of form II and III RSV DNA

contami-nants. Contamination by chromosomal DNA

molecules carrying an infectious provirus was

estimated to be even lower, not exceeding

0.02%. Thisdegree of contamination was

calcu-lated from the fraction of chromosomal DNA

foundafter Hirt fractionation in the Hirt

super-natant and from the specific infectivity of the

DNA in the Hirt pellet. We conclude that if

linear DNAmolecules sedimenting fasterthan

supercoiled circles of6 x 106 daltons

contami-nateformIRSVDNApreparations, theycannot

be detected by hybridization and transfection

techniques.

Figure2showsthe sedimentationprofile ofa

purifiedform IRSV DNA ina neutralsucrose

gradient.RSV DNAwasdetectedby annealing

with 3H-labeled complementary DNA (cDNA)

and foundtosedimentmainly ina peak of 25S

correspondingtosupercoiled circles of about 6

x

10'

daltons (2). A shoulder seen in Fig. 2 at

20Smaycontainopencircles of thesamesize.A

detectable amountof the RSVDNA, however,

sedimented much faster than 25S witha small

(andreproducible in two experiments) peakat

47 to58S. The evidence derived from transfec-tionassays was more compelling. It wasfound

that only about 89% of the infectious material

consisted of22 to 27S monomers. Asmallpeak

ofinfectivitywas observed in fraction 8 (32S),

consistent with the presence of dimeric circles

(2). The material in this peak and its leading

edge(fractions9and10),thoughhardlyresolved

byhybridization, representedabout8% of

infec-tivity. The rest of the infectivity (about 3%)

sedimented faster than32S,as seeninfractions

(except fractions14and20) between theleading

edge of thepeakand thebottomof thegradient.

In one experiment (not shown), gradient

frac-tions were collectedfrom the bottom and

ana-lyzed as in Fig. 2.Theinfectivity profileinthis

gradient resembledthat in Fig. 2,providing

di-rect evidence that the infectivityseeninlower

partsofthegradientinFig.2didnotarisefrom

contaminationbymonomerssituated in the top

acidprecipitated, andthe amountof acid-insoluble

[125IIRNA

-DNAhybrids was measured in a Packard gamma scintillation spectrometer. Background counts(65 cpm) determinedin control samples con-taining["26I]RNA andcarrier DNA only were sub-tracted.

J.

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NOTES 801

9 11325

I12

°°16 o

,,IR~

L-

cSm

n

..

N~~~~~~~ 0

5 1

DN in a-netaursgaiet uecie N

~~~~~~~~

2

~~~~~~~~~~.0

D

wa puiidfo bu . 0 iu-netdCE

j6 {20

q 1522

andtwice banded in cesium chloride-ethidium

bro-mide densitygradients as shown in Fig. 1. Pooled

gradientfractionscontainingthis DNAtogetherwith

GC-labeledform I SV4o DNAdensitymarkerand10 pg ofyeast tRNA were extracted with

isopropa-nol-water,dialyzed against O.lx SSC, and ethanol

precipitated. Theprecipitate wasdissolvedin 0.2 ml

of 0.lx SSCanddepositedon the top of a linear

10-mlgradient(5 to 20% sucrose in 0.1 MNaCI-0.01M

This-hydrochloride [pH7.41-0.001 MEDTA [NTE] solution) and a 0.5-ml cushion of 60% sucrose in NTE.

Centrifugation was carried out in a SpincoSW41

rotor at39,000rpm for 3 h at4°C.Fractions of 0.6 ml

were collected from the top, and the density was

measuredasinFig. 1. Forhybridization, 0.1-ml

sam-plestogetherwith 20 pg ofcarriercalfthymus DNA

weretreatedwithNa2H, ethanolprecipitated, then

dissolvedin the hybridization buffer containing7(W)

cpmof3H-labeled tdPR -RSV-CcDNAprepared

ac-cordingto Taylor et al.(25) andhybridizedat68wC

for 17 has inFig. 1.Afterhybridizationthesamples

weremixedwith2mlof 0.03 M sodiumacetate (pH

4.5)-0.2 MNaCI-0.0i3MZnSO4solutioncontaining

10 pg ofsonically disrupted and alkali-denatured

calf thymusDNAand 41.l ofSlsingle-strand-specific nuclease(24)andincubatedat50°C for2h. The Sl-resistant material([3H]cDNA-DNA hybridsand self-annealed "4C-labeled SV40 DNA)wastrichloroacetic

acid precipitated and assayed for radioactivity. Background tritium counts (8 cpm), determined in

controlsamples containing[3H]cDNA and carrier DNAonly, weresubtracted. Theposition ofthe 14C-labeledformISV40 DNA,usedas aninternal sedi-mentation markerof 21S,isgiven byan arrow.

Sed-imentationcoefficients ofRSVDNAwerecalculated 12

:io

0

'-8

x

a-6

(NI ("4

2

12

oC

-o

4) N

E

L 0

C

0

Z

L

'-2 FormI SV40 DNA

53S

643

1.20

I~

~~~~~~~~~~~

,r1'

/1.124

.

11.:

/6ois,

1

1.06

Top Fractionnumber Bottom

FIG. 3. Sedimentation ofthe supercoiled tdSR-D DNA in an alkaline sucrosegradient. Supercoiled DNAwaspurified fromabout 7.5x108virus-infected CEFas inFig. 2, and deposited togetherwith 3H-labeledformISV40DNA(53S)onthetopofalinear 10-mlgradient (5to20%sucrose)in0.3 MNaOH-0.6 MNaCl-0.01 MEDTA)anda0.5-mlcushionof60% sucroseinthesamesolution.Centrifugationwas car-riedoutinaSpincoSW41rotorat30,000rpmfor1.5 hat4°C.Gradientfractionswerecollectedand an-alyzedasdescribedinFig. 2,except that32P-labeled PR-RSV-CcDNA (700 cpm)wasused,and hybridi-zationswerecarriedoutat68'C for48 h. For

trans-fectionassay, a 0.5-mlportion ofeachfraction was neutralized with 1.1 M HCl-0.2MTris. Then carrier DNAwasadded,and the DNAwasprecipitatedwith ethanol. Thisprecipitatewasdissolved and titrated forinfectivitytoend-pointdilutionasinFig.2.Other

conditionsasinFig.2.

accordingtoMartinand Ames(21). Infectivityassays wereperformed using the calciumphosphate tech-nique (9) asdescribed(15).A 0.5-mlsample of each fractionwasprecipitatedwithethanol in thepresence of10pgofcarriercalf thymusDNAand 0.3 MNaCl. Then the DNAprecipitatewasdissolved in0.1xSSC, seriallyfourfold (or threefold)diluted in N-2-hydrox-yethylpiperazine-N'-2-ethanesulfonicacid (HEPES)-buffered saline,supplementedwithcarrierDNAtoa

finalconcentrationof10pg/ml,andassayedateach dilution inthree(or two) CEF cultures.After2or3 weeks, the cultures were examinedfor the reverse transcriptaseactivityinthe culturemedium(16).The infectivityintermsof infectiousunitswasdetermined by end-point dilution from the fraction of positive cultures (12) and normalized for 109 virus-infected CEF.

VOL. 29,1979

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[image:3.505.47.247.62.316.2]

5 10 15 Top Fraction number Bottom

6

i

5

4

0

"- 4

x

E

0-U

a-CV)

31

2

1

Form I SV 40 DNA B

53$

71$

V

1I

1.12

lI

Ii I I

b 110E

6~~~~~~~10

** I C

I s I I

\0w,8I 0

Q __

5

10

15

Top Fraction number Bottom

Form I SV40 DNA 53S

95$ 74S

I

E.o8

E

U

1.06

-1.04 :

0

.I II

-II

- ;,I.

I'

.I 0

II

II 0 0

'o.

Il Dill

Ii 'I , I.I

., . I. II

Ii

I I.

\ p

5 10 15

Top Fraction nunber Bottom

FIG. 4. Sedimentation analysis of the supercoiled SR-D DNA in alkalinesucrosegradients. Supercoiled

DNAwaspurified fromabout 4x108virus-infected CEF and, in (A), sedimented together with the3H-labeled formISV40DNAthroughanalkalinesucrosegradientasinFig. 3exceptthat the time of centrifugationwas

3h. Virus-specificDNA wasdetectedin 0.2-mlportions of gradient fractions by annealing with32P-labeled

PR-RSV-C cDNA(800 cpm)at68°C for40 h. Therest(0.4 ml) of the gradientfractions, in regions shown by

bars b andc,werepooled, neutralized, supplemented with 10pgofyeasttRNA, and ethanol precipitated. The precipitates b andc werecollectedby centrifugation,dissolved in 0.2 ml of 0.1x SSC, addedto2,000cpmof

3H-labeled formISV40DNA,andsedimentedthrough alkalinesucrosegradients in (B) and (C),respectively, asabove, exceptthat this time the60%osucrosecushionswereomitted and thegradientswerecentrifuged for

3.5 h. Gradientfractionswereanalyzed forthepresenceof virus-specificDNAbynucleicacidhybridization

asin(A), exceptthat theradioactivity oftheinput 32P-labeledPR-RSV-C cDNAprobeamountedto500cpm.

Other conditionswere asinFig.2.

6

0

x E

Q4

a-CV)

Form I SV 40 DNA A

53 $

l

1 118

71 S

l

1

< 116

I'

~

E

112

'l

/

c

I

~~~~~~110-X C

b- c~~0I

I

1080A

A)t0bs ~~~~1!06

z / ~~b

\ / t~~~~~~~~~~'

b

c0'-b- b

c°os~~~~~~

2 9

0

x

E

a-U

a-C',

cV)

o

2

11

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[image:4.505.67.460.36.587.2]

NOTES 803

fractions. We conclude thatatneutral pH form

IRSV DNA is composed of 25Smonomersand

also oflarger forms resembling dimers. These

forms sediment at 32S. Traces of infectious

ma-terial found between 32S and 65S suggest the

presenceofhigheroligomers.

Large covalently closed circles of RSVDNA

werefurther studied under alkaline conditions

to seewhetherthey denatureintosingle strands

or, alternatively, in the absence of alkali-labile

bonds, intoacompactrandom coil.

Sedimenta-tion inan alkalinesucrosegradientis shownin

Fig.

3. The monomer form of tdSR-D DNA

sedimentedat64S. This correspondstothe

sed-imentation coefficient ofa form I DNA of6 x

106 daltons under denaturing conditions (2).

Transfection assays show that the monomers

are infectious, though their infectivity was

re-duced to less than one-tenth ofthat found at

neutral pH in Fig. 2. Infectious material

sedi-menting faster than denatured monomers was

found onlyat125S (fraction8ofFig. 3), andno

infectious material wasfound in lowerparts of

thegradient. This could be due toreduction of

thespecific infectivity of covalently closed viral

DNA inalkalinegradients.TheSi-resistant

ra-dioactivity found in fraction 11was not

consid-ered to be relevant to the virus-specific DNA

sincenosuchradioactivitywasfound in further

experiments (Fig. 4A).

To show the alkali resistance of large DNA

formsmoreconvincingly, different size classesof

the denaturedform I SR-D DNAcomposed of

71S monomers and putative 95 to 160S

oligo-mers (Fig. 4A), respectively, wereresedimented

under thesameconditionsinalkalinesucrose as

shown in Fig. 4B and C. In Fig. 4B monomers

resedimentedatabout thesameposition of 74S.

The peak at about 20S shows that they were

partly converted into single-stranded circular

and linear forms of about 3 x 106 daltons. On

the other hand, resedimentation of the 95 to

160S material (Fig. 4C) separatedDNAforms of

about95S frommonomers of74S. Further evi-dence that the peak at 95S represents a

dis-cretely sedimenting specieswas obtainedwhen

the experiment shown in Fig. 4 was repeated

under thesameconditions.Againapeak ofDNA

formssedimenting faster (92S) than the

mono-mers wasresolved.The differencebetween the sedimentation coefficients of monomers of 6 x 106daltons and theirdimers,predicted from the

data obtainedinearlier studieswithotherDNAs

(2), wouldresemble that observed inthesetwo

experiments.

The demonstration in this paper oflarge

cir-cular

fonrs

of RSV DNA raises further

ques-tions. Aretheseforms composedofgenomic or

both genomic and subgenomic submolecules?

Are these molecules unicircular or circular

catenated? Finally, are they generated from

monomersbyadouble recombination(4, 18)or, although this isunlikely, byaberrantreplication

(8, 19)ofviral DNAmoleculesorrecombination

of viral and cellular DNAmolecules?It is

con-ceivable that recombinationeventsinviralDNA

circles would explain the generation of

recom-binant virusesindouble-infected cells.

We thank DominiqueStehelin for kindly providing cDNA probesandMonique Kalekine for excellent technical assist-ance.

Thisinvestigationwassupported by Institut Nationalde la Santeetde la RechercheMedicale,contractsA.S.R.no.1and A.T.P. 28.76.60, and Delegation Generale a la Recherche ScientifiqueetTechniquecontract76.7.1656.01.

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citro.Biochemistry 10:1993-1999.

4. Flory,P.J., Jr.,and J.Vinograd. 1973. 5-bromodeox-yuridinelabelingofmonomeric and catenated circular mitochondrial DNA in HeLa cells. .J. Mol. Biol. 74: 81-94.

5. Fritsch, E., and H. M. Temin. 1977. Formation and structure ofinfectious DNA ofspleennecrosis virus. J. Virol. 21:119-130.

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9. Graham, F. L., and A. J. van der Eb. 1973. A new techniqueforthe assay ofinfectivityofhuman adeno-virus 5 DNA.Virology52:456-467.

10. Guntaka,R.V.,B. W. J.Mahy,J. M.Bishop, andH. E. Varmus. 1975. Ethidium bromide inhibits appear-anceofclosed circularviral DNA and integration of virus-specificDNA in duck cells infected by avian sar-comavirus. Nature(London)253:507-511.

11. Guntaka,R. V., 0. C.Richards, P. R.Shank, H.-J. Kung,N. Davidson,E.Fritsch,J. M.Bishop,and H. E. Varmus. 1976.Covalently closedcircular DNA ofavian sarcoma virus:purification fromnuclei of in-fectedquailtumorcells andmeasurement byelectron microscopy andgelelectrophoresis.J. Mol. Biol. 106: 337-357.

12. Hill, M.,and J. Hillova. 1976. Genetic transformation of animal cells with viral DNA of RNA tumor viruses. Adv. CancerRes. 23:237-297.

13. Hill, M.,N.Stedman,R.Mariage, G.Goubin,J. Hil-lova, andM. Roussel. 1978. Unintegratedand inte-grated viral DNA in Rous sarcoma virus-infected chickencells,p.155-177.In S. Barlati and C. de Giuli-Morghen (ed.), Avian RNA tumor viruses. Piccin, Padua.

14. Hillova, J.,D.Dantchev,R.Mariage,M.-P.Plichon,

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andM. Hill. 1974.Sarcoma and transformation-defec-tive virusesproducedwith infectious DNA(s) from Rous

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17. Hirt,B. 1967.Selective extraction ofpolyoma DNA from infectedmouse cellcultures.J. Mol.Biol. 26:365-369. 18. Hudson, B., andJ.Vinograd.1967.Catenated circular DNA molecules in HeLa cell mitochondria. Nature

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