Restriction endonuclease mapping of unintegrated viral DNA of B- and N-tropic BALB/c murine leukemia virus.

0022-538X/80/09-0812/12$02.00/0

Restriction

Endonuclease Mapping

of

Unintegrated

Viral

DNA of B- and N-Tropic BALB/c

Murine

Leukemia

Virus

ERIC RASSART"2AND PAULJOLICOEUR' ;

Institut deRecherchesCliniques de Montreal, Montreal H2W1R71;Departement deMedecine2 and Departement deMicrobiologie et d'Immunologie,3 Universite de Montreal, Montreal, Quebec, Canada

Unintegrated linear and closed circular DNAs of B- andN-tropic endogenous

BALB/c murine leukemia virus (MuLV) were extracted from newly infected

mouse cells and cleaved with EcoRI, XhoI, PvuI, HindIII,

SalI,

XbaI, KpnI,

SmaI, and PstIrestriction endonucleases. The DNAfragments wereseparated by electrophoresis and

analyzed

by the Southern blot

hybridization

procedure. EcoRI didnotcleavethe two genomes. Aphysicalmap of15

cleavage

siteson

B-andN-tropicgenomes wasconstructed with the other restrictionendonucleases.

Identicalcleavage sites ofB-andN-tropic MuLV DNAswerefound with all these

enzymes. However,the N-tropiclinear genome was foundtolack about 75 base

pairsateach end of the molecule.PstI,KpnI,andSmaIrecognizeacleavagesite atboth ends ofthe linear molecules. Andsequencesderived from the 5' end of

the RNAgenome werefound in the third left end of the linearDNAandatits

extremeright-endterminus, suggesting thepresence of redundantsequences. Two

speciesofclosed circular viral DNA were observed. The largerspecies has the

samesize as the linearmolecule and appearsto bea circularized form of linear

DNA. Thesmallerspecies containssequences commontoboththelinear andthe

larger circular viral DNA but seems to be deleted from sequences present at

eitherone orboth endsofthelinear DNA.

Therefore,

the

general

structureof the

linear and circularDNAspecies of these B- and

N-tropic endogenous BALB/c

MuLVappearsanalogoustothestructurefound with otherretroviruses.

Naturally occurring

ecotropic

murine leuke-mia viruses(MuLV)canbeclassifiedas

N-tropic

orB-tropic withrespecttotheir hostrange(13). N-tropic viruses infectNIH cells

(N-type)

more

efficiently

than

BALB/c

cells

(B-type),

whereas B-tropic virusesgrow more

efficiently

onBALB/

ccells thanonNIHcells. Thissusceptibility of

murine cells to the

naturally occurring

MuLV

has been shown to be determined

by

a

single

allelic

genetic locus,

the Fv-1gene

(28,

for review

see 20, 27). The restriction of N- or

B-tropic

MuLV inFv-1-resistantcellsoccurs

early

inthe

virus cycle, after

synthesis

of viral DNA but

before itsintegration into the cell genome (23,

37). Our recent results indicate that accumula-tionof circular viralDNAispreventedin

Fv-1-resistant cells (24). The viraltropism

determi-nant which is required for this restriction has

been shown to be aprotein because ofitstransfer

by phenotypic mixingtoother viralgenomes(3,

19, 25, 29). Evidence obtained with recombinants

of N- andB-tropicviruses(16, 32)andwith

NB-tropicviruses derived from

B-tropic

viruses(17)

indicated thatp30virionproteinmightcarry the

tropismdeterminant.

N-andB-tropicendogenousviruses obtained from BALB/c mice have been studied

exten-sively.Theirgenomesare

highly

homologous

as

shownbymolecular hybridization(5,22, 30) and

by Ti oligonucleotide fingerprintanalysis (7, 8).

N-tropic specific oligonucleotidesofN-tropic

re-combinantvirusesand NB-tropic specific

oligo-nucleotides ofB-- NB-tropic viruses were

iden-tified, and bothwereshown to be located at the

5'endof the genome (7, 8). Sequencingof these

specific oligonucleotides revealed only few

dif-ferences, asexpected for allelicsequences (31).

To determine the exact location of the gene

coding for theviraltropism determinantand to

study the integrationof B- and N-tropicMuLV,

wehaveconstructed a physical map of the

cleav-age sites oftheirDNAwith several restriction

endonucleases. We have purified unintegrated

linearand closed circular viral DNAs of B- and

N-tropic MuLV from acutely infected mouse

cells. Duringtheseexperiments, we found a

de-leted form ofclosed circular viral DNA. Since

then, reports have been published on the

ex-istence of asmallercircular form ofviral DNA

duringreplication of other retroviruses (11, 18, 33, 34, 43).

MATERIALS AND METHODS

Cellsand viruses. TheoriginsofJLS-V9,Sim-R,

and NIH/3T3 cells have been described (21). Cells weremaintained inDulbecco-modified Eaglemedium 812

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supplemented with 10% calfserum (GIBCO Labora-tories,Grand Island, N.Y.). The cloned N-tropic (N-C1-35) and B-tropic (B-Cl-11) endogenous BALB/c viruseshave been described (19). Both viruses were grownonFv-I-permissiveNIH/3T3(N-CI-35)or JLS-V9andSim-R (B-Cl-11) cells (21, 23).

PreparationofviralDNA.N-Cl-35 andB-Cl-11

unintegrated viral DNAs were obtained by infecting NIH/3T3 orSim-R and JLS-V9 cells, respectively, at

amultiplicity of infection ofapproximately2,inroller bottles in the presence of 8 ,ug ofPolybreneperml. At 16 to 24hafter infection, a Hirt extraction (15) was performed, and the Hirt supernatant DNA was sub-jected to propidium iodide-cesium chloride centrifu-gation as described (24). DNA from the lower band containing theclosed circular molecules (FormI)and DNAfrom the upper band containing linearmolecules

(Form III)wereextracted withNaCl-saturated

isopro-panoltoremovethepropidium iodide and precipitated with 2volumes of ethanol at-20°C. Form I DNA was suspended in water and used as the substrate for restrictionendonucleases. DNA from the upper band wassuspended in0.01M Tris(pH 7.8)-0.02 M EDTA

andlayeredon a 15 to30%(wt/vol) sucrosegradient

in0.01 M Tris-hydrochloride (pH 7.5), 0.1 M NaCl,

and5mMEDTA.Centrifugationwasperformed in an

SW41 Spinco rotor at 23,000 rpm for 16 h at 40C.

Fractions of0.4mlwerecollected from the bottom of the tube (9,40). A sample of each fractionwasscreened by agarose gelelectrophoresis, and DNAwas

trans-ferredto nitrocellulose. Viral DNA wasdetected by hybridization to 32P-labeled complementary DNA (cDNA). Fractions containing DNA sedimenting at about 20S werepooled and precipitated with2

vol-umes of ethanol at -20°C and kept as full-length

double-strandedlinearviral DNA.

Synthesis and purificationof viral

[32P]cDNA.

Representative3P-labeledB-Cl-il DNAwas

synthe-sizedbyincubating bandedB-Cl-li virions with calf

thymus oligonucleotide primers, as described

previ-ously(24). Itsspecificactivityvariedbetween2 x 108

and4x

10W

cpm/ug.

[32P]cDNA5'wassynthesized under the same con-ditions except that calf thymus oligonucleotide primerswereomitted (14). The reactionwasstopped after1hbyphenolextraction.[32P]cDNAwaspurified

on aSephadexG-50 column (1.5 x 35cm) and

incu-bated at 370C for 18 h in 0.3 N NaOH (24). After neutralization and ethanol precipitation,

['P]cDNA

chainswereseparatedbyelectrophoresison10% acryl-amidegelasdescribed (14).The strong stop cDNA 5' ofaround140baseslongwasdetectedby autoradiog-raphy. It constituted the major short cDNA species made. Itwaseluted from thegelasdescribed (14) and

usedas[32P]cDNA5'.

In vitro synthesis of double-stranded viral

DNA.Single-strandedviral DNAwasfirst synthesized

in vitroessentiallyasdescribed (42).Banded

B-Cl-il

virions wereincubated for 14 to 16hat

370C

in 50 mMTris-hydrochloride (pH 8.5); 2mMeachdATP,

dCTP, dGTP, dTTP; 50,uCi of [3H]dTTP (53 Ci;

mmol);6mMmagnesiumacetate;2mMdithiothreitol;

0.01%NonidetP-40;40

Mg

ofactinomycinDper ml in

afinalvolume of1ml. The mixturewas extractedwith phenol, and theaqueousphasewasloadedona

Seph-adexG-50 column, as above. Fractionscontaining viral DNA werepooled, adjustedto0.3NNaOH,incubated at370C for 12 h, neutralized and ethanol precipitated. The cDNA recovered by centrifugation waslayered on a 15to30%alkalinesucrosegradient (0.9 MNaCl, 0.3 N NaOH, 5 mM EDTA) and centrifuged in an SW41 rotorfor 16h at 35,000 rpm. Fractions of the gradient corresponding tothe largest cDNA species werepooled and ethanol precipitated in the presence of100Mg of calf thymus DNA primers (39).

This (-) strand cDNAwasmadedoublestranded (42) by incubation at 170Cfor 6 h in 20 mM Tris-hydrochloride (pH7.5); 4mM dithiothreitol; 10mM magnesium acetate;60mMNaCl; 1mMeachdATP,

dCTP, dGTP,anddTTP;50,uCiof[3H]dTTP; 200Mug

of calf thymus DNA primers (39); and Escherichia coli DNApolymerase Iinafinalvolume of0.05ml. Viral DNA was phenol extracted, treated with Si nuclease (24), layered on a 15 to30%neutralsucrose

gradient, and centrifuged as described above. Frac-tionsof the gradientwerecollected,andasampleof each fractionwasscreened for the presence of viral DNA by agarose gel-DNA transfer procedure and hybridizationto[32P]cDNAasdescribed below. Frac-tionscontainingviral DNAmigratingasless than full-length molecules (between1x106 and3x106daltons)

werepooledand ethanolprecipitated.

Restriction endonuclease cleavages.

Restric-tion endonucleases SmaI, XhoI, BgII, BgIII, PvuI, XbaI, andKpnI werepurchased from NewEngland Biolabs (Beverly, Mass.); HpaI and Sail were pur-chased from Miles Laboratories (Elkhart, Ind.). EcoRI,HindIll, BamHI,andPstIwerefrom Boehrin-gerMannheim Corp.,Montreal, Canada. Restriction endonucleasedigestion conditionswerethose

recom-mendedbythesupplier.Completionof each reaction

was monitored by including 0.5Mug of lambda DNA

(Boehringer) in the reactionmixtures.

Agarose slab electrophoresis and transfer.

Samplesof25

p1

(2to 5Mgof cellDNA)werelayered

into separatechannels ofaverticalslab gel (20 by20

by0.3 cm) castwith 1.4% agarose (SigmaChemical

Co.,St.Louis, Mo.).DNAwasseparatedby

electro-phoresis in 40 mM Tris (pH 8.3), 50 mM sodium acetate, and 1 mM EDTAfor about 18h at 35mA/ gel. Afterelectrophoresis, thegelswerestainedwith

0.5,ugof ethidium bromide per ml andphotographed with Polaroid filmby UV illumination. The DNAwas

then denatured, neutralized in situ, and transferred

onto a nitrocellulose membrane (0.45Mum; Millipore

Corp.,Bedford, Mass.)asdescribedbySouthern (36).

Hybridization procedure. After transfer of the

DNAtothenitrocellulose,the membraneswerebaked

at800Cfor2hand then soaked for10 to 12hatroom

temperaturein 25to 100mlof3x SSC solution

con-taining 50%formamide, 100Mugof calfthymus DNA,

and supplementedwith 0.02% each ofFicoll,

polyvi-nylpyrrolidone, and bovine serum albumin, as

de-scribed elsewhere(33). Thewetmembraneswerethen incubatedat390Cfor 30 to 48 h inplastic bagswith 0.5 to 1ml of thesamesolution containing3x 10W to

6 x 106 cpm of

[32P]cDNA.

After annealing,

mem-braneswerewashedin 6xSSC-0.5% sodium dodecyl sulfate solution for12hat670Cand thenin the same

solutionat370Cfor6 to 8h(33). Membraneswereair

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driedand exposed at -20°C to Kodak-RP-Royal X

Omat filmwithaCronexLightningPlusintensifying

screen(DuPont Co., Wilmington, Del.) (33).

RESULTS

Mapping of in vitro B- and N-tropic

MuLV DNAs with EcoRI, HindIII, XhoI, Sall, PvuI,and XbaI. Toanalyzethe size and the structure of B- and N-tropic DNAs, we

partially purified linear and supercoiled viral

DNAmoleculesbycentrifugationonpropidium

iodide-cesium chloride gradients and further

purified theupperbandon sucrosedensity

gra-dients. The selected 20S linear viral DNA

mi-gratedas adiscrete band of 5.7x 106daltonson

agarose gel, as expected for a genome-length

molecule of double-stranded structure. DNA

from the lower band of the propidium

iodide-cesium chloridegradient contains closed circular

viral DNA which could be resolved into two

faster-migrating species byagarose gel

electro-phoresis,asreported for other retroviruses.

The linear and circular forms of B- and

N-tropic MuLV DNAweredigested byavarietyof

site-specific endonucleases.The DNAfragments

generatedbyenzymedigestionwereresolvedby

agarose gelelectrophoresisand detectedbythe

Southern procedure with a [32P]cDNA probe.

Molecularweightsweredeterminedby

compar-ison with Eco RI- or HindIII-digested lambda

DNArunonparallel lanes. Eco RI didnotcleave

the linear nor the circular forms of B- and

N-tropicMuLV DNA(Fig. lg, Fig.

2f).

XhoI,PvuI, HindIII,SalI,and XbaI all cleaved both linear

viral DNAsonce,thusgeneratingtwofragments,

the molecularweights of whichaddeduptothat

offull-length linear DNA. XhoI generatestwo

fragments of 2.9x 106 and 2.8x106 daltons(Fig.

la); HindIII generates two fragments of 3.8 x

106 and1.9x106 daltons(Fig. lc);SalIgenerates

two fragments of 3.05 x 106 and 2.65 x 10"

daltons(Fig. le). PvuIgeneratesonedetectable

fragment of 5.3 x 106 daltons (Fig. lb). The

other fragment of 0.4 x 106 daltons was not

detected in this experiment, but since the

en-zyme cleaves the circular DNAonlyonce (Fig.

2b), its existence is deduced. XbaIgenerates two

fragments of4.95 x 106and 0.75 x 106 daltons

(Fig.

lf),

but thesmallerfragment whichappears

as averyfaint bandherewasstudied further.

Amore precise comparison of the molecular

weights of the shorter fragments derived from

B- and N-tropic viral DNAs revealed that the

smaller PvuI fragment (0.4 x 10' daltons) and

thesmallerXbaIfragment (0.75x 106daltons)

generated from theN-tropic MuLV DNAwere,

in fact, both smaller in size than their

corre-sponding fragments derived from B-tropic

MuLV DNA(Fig. 3a-d).This size differencewas

estimated to be 0.05 x

10'

daltons (approxi-mately 75 base pairs) in each case. This small difference couldnotbe observed in theprevious experiment (Fig.

1)

because B- and N-tropic DNAfragments were not run on thesame gel. Since Pvu Iand Xba Icleave, respectively,atthe left andattheright ofthe linear viral DNA(Fig.

4),such adifferencecould either result froman

additional cleavage site in each of these regions for both restriction endonucleases or from the absence ofa75-basepairsequence atboth ends of the linear molecule (seebelow).

As shown in Fig. 1, the presence of other

discrete bandswasalsoobservedafter digestion with various restrictionendonucleases, andmore

especially with XbaI

(Fig.

1B, f). These addi-tional bands were not detected with all DNA preparations (Fig. 4c), and their presence was

markedly reduced indigested linear DNA

prep-arationsthat had been selected onsucrose

gra-dients. The sum of the molecular weights of these additional XbaI fragments does not add

up tofull-length linear DNA. It isunlikelythat

they

are the products of partial digestion, as

complete digestion of lambda DNA was

ob-served for each reaction. Theymostlikelyarose

eitherfrom the cleavage of contaminating cel-lular DNA, subgenomic linear viral DNA of

discrete size, or from partially single-stranded viral DNA.

The evidence that these enzymes cleave

B-and N-tropic MuLV DNAsat onlyone site was

confirmed by digesting the circular forms of

these DNAs. As shown in Fig. 2, all these

en-zymes transform the two circular species into

twolinear forms. Theupperband ofthe doublet

migratedas agenomic linearDNAmolecule of

5.7 x 106daltons.Thelower band has a

molec-ular weight of5.35x

106,

indicatingthat the two

circular species really differ in size and not in

theamountofsuperhelical twist.

Mapping

of in vivo B- and N-tropic MuLV DNAs

with

KpnI,

SmaI,

and

PstI.

SmaI endonuclease

cleavage

oflinear B-tropic MuLV DNA yields three detectable fragments of 2.95

X

106,

1.25 x

106,

and 1.15 x 106 daltons (Fig.

5B,b). Cleavageoflinear N-tropic MuLV DNA

with the same enzyme alsogenerates three

de-tectablefragmentsof 2.95x

106,

1.20x

106,

and

1.15 x 106 daltons (Fig. 5B, d). Two of these

fragments (2.95 x

10'

and 1.15 x

10'

daltons)

from B- and N-tropic MuLV DNA comigrate.

The otherfragments(1.20 x

10;

and 1.25 x.

106

daltons)did notcomigrate as verified by a

diges-tion of a mixture of B- and N-tropic MuLV

linear DNAs. SmaI endonuclease cleavage of

circular B- andN-tropicMuLV DNA generates

the same fragments as those obtained after

cleavageoflinear DNA (Fig. 5B,aand c),

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A

a b c

d

e

13.7-

II

i

4.4

of

4.7

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-3.0

-a

S

,,

law

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FIG. 1. Digestion of B- and N-tropic linear viral DNAs with XhoI, PvuI, HindIII, Sall, XbaI, and EcoRI.

In vivo linearunintegratedviral DNAsofB-andN-tropicMuLVwerepurifiedonapropidium iodide-cesium

chloridegradientanddigested with different restriction endonucleases tocompletion. After digestion, the

samples were run on 1.4% agarosegels, and the DNA fragments were transferred onto a nitrocellulose

membrane and annealed to[32P]cDNA. Fragment size was estimated by running EcoRI- and

HindIII-digestedlambda DNAsasmarkers. (A) B-tropicDNA. (B) N-tropicDNA.DNA samplesweredigested uwith

XhoI (a), PvuI (b), HindIlI (c), HindIII and SalI (d), Sall (e),XbaI (f), EcoRI (g), and nondigested (h). Nondigested B-tropicDNAwasfromadifferent preparation than theB-tropic viral DNAused for digestion.

cating that these fragmentsareinternal and that

theenzymealso cleaves closetooneendorboth

ends of thelinearmolecule. Evidence thatSmaaI

cleaves B- and N-tropic linear MuLV DNAs

close to the left end was provided by double

digestion of linear viral DNA with

SmaI-HindIII and SmaI-Sall endonucleases. The

smaller HindIII fragment (1.9 x 10" daltons)

wascleaved by Sma Itogenerateafragment of

1.65 x

10'

daltons (Fig. 6b; Table 1). Similarly,

SmaI cleaved the smallerSailI fragment (2.65

x

10'

daltons) to generate a shorter fragment

(2.4x 10" daltons) (Fig.6c; Table 1). Evidence that SmaI cleaves B- and N-tropic linear MuLV

DNAsclosetothe right end of the moleculewas

provided by double digestion with XbaI-SmaI

(Table 1). ThesmallerXbaI fragment(0.75x 10"

daltons)wascleaved bySma Itogeneratea0.65

x

10'-dalton

fragment, indicatingthatSmaI

rec-ognizes acleavage site close to this end of the

viral DNA.

Cleavageof linearB-tropic MuLVDNA with

KpnI yields three detectablefragments by

hy-bridization, which have molecular weights of

2.45x

106,

1.8 x 106,and 1.1x 10" (Fig. 5,panel

A(b)). Cleavageof linear N-tropic MuLVDNA

with thesameenzyme alsogeneratesthree

de-tectablefragmentsof 2.45 x

10W,

1.8 x

106,

and

1.05 x

10'

daltons (Fig. 5A, f).The twolargest

fragmentscomigratedwith those of theB-tropic

MuLV DNA, but the small one (1.05 x 10"

daltons)wasslightlysmaller than that of the

B-tropic (Fig. 5A, candd). However, these three

fragmentsdonotaccountcompletelyfor thesize

of the linear DNA and add up to 5.35 x 10"

daltons. Digestion of the circular DNA yields

the same three corresponding fragments (Fig.

5A,aande), indicatingthat thesefragmentsare

internal and that,in addition tocleaving twice

inside thelinearmolecule, KpnI alsocutsclose

B

f

q

h

f

'- _- _

-

13.7

-

4.7

-

3.5

3.0

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A

a b c d e f

g

B a b c d e f

g

5.735-a

5.35/-FIG. 2. Digestion ofB- andN-tropic circular viral DNAs with XhoI,PvuI, HindIII, SalI, XbaI, andEcoRI.

Circular viral DNAsofB- andN-tropicMuL Vwereincubated withdifferentrestrictionendonucleases.After

digestion, the samples were run on a 1.4%agarose gel, and the DNA fragments weretransferred onto a

nitrocellulose membrane and annealed with [32P]cDNA. Closed circular viral DNA (Form I) wasnicked

duringstorage,and therelaxedform (FormII) migratedslower than the linear viral DNA.(A)B-tropic DNA. (B) N-tropicDNA. DNA digested with XhoI (a), PvuI (b), HindIII (c), XbaI (d), SalI (e), EcoRI (f), and nondigested (g).

a b c d

0.75-35 0.4

-FIG. 3. Detectionof thesn

atedby cleavageoflinearanc tropicMuLVDNAswithPvu]

Circularandlinear viralDP

MuLVwerepreparedfrom

in)

inthe text.After digestion u

cleases, the samples were ru

DNAfragmentsuweretransfe)

membrane and annealedwith tropic (a)andN-tropic (b) DI LinearB-tropic (c) and N-tr with XbaI. Circular B-tropii DNAdigestedwithSmiaI.Ci

N-tropic (h)DNAdigestedwi

e f g h to oneendorboth ends of the linearmolecule.

Evidence thatKpnIcutsclosetothe left endof

linear DNA was provided by double digestion

with HindIII-KpnI (Table 1). The smaller

HindIII fragment (1.9x

10'

daltons)wascleaved

by KpnIto generate a new fragment of 1.65 x

106daltons. ThisKpnIcleavagesite must be at

0.35- A Z the left end of theHindIII fragment. A similar

conclusionwasreachedbydoubledigestionwith

SmaI-KpnI, which leaves the 1.8 x 106-dalton

KpnI fragment intact(Fig. 6d; Table 1). If this

nallerfragmentsgener- fragment were terminal, it should have been

d supercoiledB- and N- cleavedbySmaI, which cleaves close to theleft

I,XbaI,SmaI,andPstI. end of the molecule. Therefore,thisKpnI

frag-VAsof B- and N-tropic ment is internal, and a KpnI cleavage site is

fectedcellsasdescribed present atthe left end ofthe molecule. Evidence 'ith restriction endonu- thataKpnIcleavagesiteispresentattheright

in on agarosegels, the end of linear viral DNA was also provided by

rredontonitrocellulose double digestion withKpnI-SmaI (Fig. 6d; Ta-i

12P

PJcDNA.LinearB- ble1).Thisdoubledigestionleaves the 1.1 x106

*opic (d) DNA digested dalton KpnI fragment intact. If this fragment

c (e) and N-tropic (f) were terminal, it should have been cleavedby

ircular B-tropic(g)and SmaI (which recognizes a cleavage site at the

thPstI. right endoflinear DNA) to give rise toa

frag-(0

0

5.7,_'

5.35""

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a b c

de f

4.95-

1.1

-

0.75-FIG. 4. Orientationof B-tropicMuL V DNA frag-mentsproducedby differentrestriction enzymes with respect tothe3' and 5' endsof the RNA molecule. Double-stranded viral DNAwassynthesizedin vitro

asdescribed in thetext. Less than full-length viral DNA wasselectedon sucrosedensitygradientand

usedfordigestion withdifferentrestriction

endonu-cleases.After digestion, sampleswere run on a1.4% agarosegel,and the DNAfragmentsweretransferred

onto nitrocellulose membrane and annealed with

[32P]cDNA.

In vitro-synthesized viral DNA

frag-mentsnondigested (aand

/)

anddigestedwith XbaI

(b)orKpnI (e).Asacontrol,invivo-extracted linear

B-tropic viral DNA was cleaved with XbaI (c) or KpnI(d).

ment shorter

by

0.1 x 106 daltons as shown abovewith XbaI and

SmaI.

Therefore,

this 1.1 x 106-dalton KpnI fragment is

internal,

and a

KpnI

cleavage

site ispresentatthe

right

end of themolecule.

PstI restriction endonuclease

produces

only

one detectable

fragment

of 5.35 x 106 daltons

afterdigestionof linear B- and

N-tropic

MuLV

DNA (Fig. 5C) and thus seems to cleave

only

once. However, cleavage of the two circular

DNAspeciesgenerates

only

onedetectable

frag-mentof5.35x

10W

daltons instead of

producing

theexpecteddoublet of5.7 x

106

and5.35x

10W

daltons (which is whatoneshouldexpect from

circular DNA withan enzymethat cleaves once).

Thisfragment comigrates withthesmaller frag-mentobtained after digestionof the two circular

species with HindIII (Fig. 5C,a),indicating that

PstI cleaves the smaller circular species once

and the larger circular viralDNA species at least

twice. Evidence that PstI cuts the linear viral DNA at both ends was provided by double

diges-tion with HindIII-PstI.Asshown(Fig. 7; Table 1), PstI cleaves the smaller HindIII fragment

(1.9 x

10'

daltons)to generate a fragment of 1.85

x

10'

daltons and the largerHindIIIfragment

(3.8x

106

daltons) togive riseto afragment of

3.5x

10'

daltons. Thesmallfragments generated

by digestion of linear DNA with PstI are too

smalltobedetected in this experiment.

A more detailed analysiswas alsoperformed

todetect thesmaller DNA fragments generated fromsupercoiled viralDNAby SmaI,Kpn I, and PstI. As shown, these three restriction

endonu-cleases cleave linear viralDNA atbothends and

generateidentical largeinternalfragments from

linear and circular viral DNA. Therefore, the

smaller DNA fragment generated by these

en-zymes in circular viral DNA should represent

the fused ends of the linear molecule. After

digestion ofB-andN-tropic circular viralDNAs

with SmaI (Fig. 3e and f) and PstI (Fig. 3g and h), we could observe a fragment of0.35 x

106

daltons undetected in previous experiments. We found that thisfragment derived from N-tropic viral DNA was approximately 75 base pairs

smaller than the B-tropic fragment. A similar resultwasobtained with Kpn I (data not shown).

Digestion of in vivo B- and N-tropic

MuLV DNA with otherrestriction endonu-cleases. We have useda number of other

re-striction endonucleases, including BglI, BglII,

HindII,

HpaI, and HpaIItocleave B- and N-tropic MuLV DNA. All these enzymes yield multiple fragments which have not been

or-dered. Cleavage of linear MuLV DNA with BamHIgives three detectable fragments of2.0

x

106,

1.25 x

106,

and 1.20 x

106

daltons. The

smallestfragment (1.20 x 106 daltons) appears

in someexperiments as adoublet and, in fact,

represents two fragments of almost identical

size.Usingdigestion of circular MuLV DNA and doubledigestion with

HindIll,

these fragments have been ordered. This gives the orientation

for the

BamHI

DNA fragments of

5'-1.2-1.25-2.041.2-3'.

Orientation of B- and N-tropic linear

MuLVDNAs. We haveperformeddouble

diges-tions on both B- and N-tropic viral DNAs to

orient thecleavagesites of the restriction

endo-nucleases with respect to each other. Double

digestionswere performed on B- and N-tropic

linearviral DNAs with HindIIIand each of the

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[image:6.510.85.226.72.369.2]

A

a b c d e f

B

a b c d

C

a b c d e f

a

IN as

5.357-2.95- t-_

1.25

1.15- -1.20

1.1- -,

FIG. 5. Digestion ofcircular andlinearforms ofB- andN-tropic MuLVDNAs with Kpn I,SmaI,andPst I.

Circular and linear viral DNAsofB-andN-tropicMuL Vwerepreparedfrom infectedcellsasdescribed in

the text. After digestion with restriction endonucleases, the samples were run on agarosegels; the DNA

fragmentsweretransferredontonitrocellulosemembraneand annealedwith[32PJcDNA. (A)KpnI digestion ofB-tropiccircular DNA(a)andlinear DNA(b, c);andofN-tropiccircular DNA (e)andlinear DNA (d, f). (B)SmaIdigestion of B-tropiccircularDNA(a)andlinear DNA (b);andofN-tropiccircular DNA(c) and linear DNA (d). (C) PstIdigestion. B-tropic circular DNA digestedwithHindIlI (a) and PstI(b), B-tropic

linear DNAnondigested (c),anddigestedwith PstI(d).N-tropiccircular DNAdigestedwithHindIII (e)and PstI(f).

other restriction enzymes. Double digestions

with SmaI-KpnI,SmaI-SalI, SmaI-PvuI, and

SmaI-XbaI were also performed. The results

are summarized in Table 1. Double digestions

with SmaI-HindIII, SmaI-SalI, and

SmaI-KpnIareillustrated inFig. 6, and with Hin

dIII-SailIinFig. ld. For example, the double

diges-tionswithHindIII-SailI endonucleasesresulted

inanaltered mobility of the smaller SailI

frag-ment (Fig. ld). A new fragment of 0.75 x 106

daltons was generated which must originate

fromthecentralpartof thegenome. Thus, the

smallerHindIllfragment (1.9 x 106 daltons) is

located at the same end as the smaller SailI

fragment (2.65 x 106daltons). (See mapin Fig.

8.)

Orientation ofthe DNAfragments produced

by different restriction endonucleases with

re-specttothe 5'and 3'endsof the RNA molecule

was accomplished by restriction endonuclease

digestion of less thanfull-length in

vitro-synthe-sized double-strandedDNAasdescribed (42).It

has been shown that in vitro (-) strand DNA

synthesis initiates at the 5' end of the RNA

molecule and extends toits 3' end (for review,

see2). In themurinesystem,less thanfull-length

in vitro DNA represents the first 135 to 140

bases derivedfrom the 5' end of the RNA

mol-ecule, followed bysequencesrepresentingthe3'

end of the RNA molecule (2, 14). We used this

ordered synthesis to map the DNA fragments

relativetothe3' end of the RNA. Less than

full-length cDNAwasmade double stranded in vitro

with E. coli DNA polymerase I and used as

substrate forrestriction endonucleases.

Restric-tion endonuclease cleavage of less than

full-length, double-stranded in vitro-synthesized

DNA ofheterogeneous size should generate a

discretefragmentderivedfrom the 3' end ofthe

RNA, plus fragmentsofheterogeneoussize.

As showninFig. 4,cleavageof less than

full-lengthinvitro-synthesizedDNAwithXba I

pro-(x

x

2.45-

-1.8- N

J. VIROL.

I

I I I

I I i

ll

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[image:7.510.86.446.70.353.2]

A

a

b c d e

S.

1

3.7

-

:...iq

4.7

3.5-3.0-

S

B

a

b c

d

e

13.7

...

I

4.7

-3.5

-3.0

-e A

1.6-1.4

-.ff!!.,

> .4'

FIG. 6. Double digestion oflinearB- andN-tropicMuLV DNAs with restriction endonucleases. Double digestion oflinear B- andN-tropicMuLV DNAs with various restriction endonucleases was carriedout sequentially byaddingthe secondenzymewith its recommendedbufferto the reactionmix.After digestion, sampleswere run on a1.4%agarosegel;the DNAfragmentsweretransferredonto nitrocellulosemembrane and annealed with[32P]cDNA. (A) B-tropicDNA. (B) N-tropicDNA. (a)SmaI, (b)SmaI and HindIII (c)

SmaI andSall, (d)SmaI andKpnI, and (e) KpnI. The twoHindIII fragments (3.8 x 10 and 1.9 x 10'

daltons)and thetwoSalIfragments (3.05x106and 2.65x10 daltons)arenotshown,buttheywerealsorun

onthesamegeltolocalizeappropriatefragments.

duced thesmallerfragment (0.75x

10'

daltons)

whichcomigrated with the fragment generated

by thesameenzymefrom invivo-extracted viral

DNA.KpnIcleavage of this in vitro-synthesized

DNA generatedonly the smallerfragment (1.1

x

10'

daltons)whichagain comigratedwith the

fragment derived from in vivo-extracted viral

DNA.Similar digestion with SmaI only

gener-atedthetwosmallerfragments (1.15 x

106

and

1.25 x

106

daltons) (data not shown). These

results indicated that the smaller XbaI and

KpnI fragments and thetwosmallerSmaI

frag-mentsofB-tropic MuLVrepresentthe 3' end of

MuLV genomic RNA (Fig. 8). The ordering of

thefragmentsontheN-tropic MuLV DNA

rel-ativetothe 5'and 3'end of the N-tropic MuLV

wasnotdone but is presumedtobethesame as

the B-tropic MuLV because of their high

ho-mology.

Studies ofterminal sequences in B- and

N-tropic MuLV DNA. Unintegrated linear

DNA of aviansarcomaviruseshas been shown

to possess a direct terminal sequence

redun-dancy of about 300 nucleotides (34, 38) derived

from both the 3' and 5' endsofviral RNA (34).

The largerclosed circular avian sarcoma virus

DNAwasshowntocontaintwocopiesintandem

of this repeat sequence whereas the smaller

closed circular form contains only one copy of

thissequence (18, 34). Linear (4, 10-12, 18, 26,

33, 35, 43) and closed circular (12, 33, 43) viral

DNAs withasimilarrepeatstructurehave also

beenobserved in cells infected with other

retro-viruses.

The restriction analysis presented above

showsthat linear viral DNAs of B- andN-tropic

MuLV containonePstI,KpnI,andSmaIsiteat

bothends of the molecule. Inaneffortto

estab-lish whetherthegeneralstructure of B- and

N-tropic BALB/c MuLV might be similar tothe

(0 0

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[image:8.510.130.374.76.381.2]

structure of other retroviruses, we used strong stop [32P]cDNA 5' as a probe to determine whether 5'-derived sequences were present at

both ends of the linear molecule. As shown in

Fig.7B, a,thetwoHin dIII fragments of B-tropic

MuLV linear DNA hybridized tocDNA 5', in-dicating that this sequence is present on both halves of the DNA molecule. Thesame conclu-sion could be reached after annealing ofboth SaII fragmentswithcDNA 5'(datanotshown). However, when double digestionwith HindIII-PstI (whichcuts atbothends of the linear DNA)

wasperformed, only thesmallerfragment (1.85

x106daltons) couldstillbedetected with cDNA

5' (Fig. 7B, b). This indicates that the larger HindIII fragment, which represents the 3'

se-quence of genomic RNA, contains at its right

terminus asequence homologoustothe 5' end of the RNAgenomeand which iscleaved off by PstI. Since the smaller HindIII fragment can

still be detected with cDNA 5' after cleavage with PstI, it appears that the 5'-derived

se-quence present on thisfragment isnotlocated

at theextremeterminus. Thesameexperiment

has also been done with the N-tropic linear MuLV DNA, and the results (not shown) are

TABLE 1. Sizeof fragments fromlinearB-tropic DNA ab

Restrictionendonu- Size

(X1Ob

daltons) clease

5'-a

3'end of RNA

HindlIl 1.9 3.8

SmaI 2.95 1.15 1.25

HindIII+SmaI 1.65 1.3 1.15 1.25

SaiI 2.65 3.05

HindIII+Sai 1.9 0.75 3.05

SmaI+

Sail

2.4 0.65 1.15 1.25

KpnI 1.8 2.45 1.1

HindIII+KpnI 1.65 2.45 1.1

SmaI+KpnI 1.8 1.23 1.15 1.1

XbaI 4.95 0.75

HindIII+XbaI 1.9 3.05 0.75

SmaI+XbaI 2.95 1.15 0.5 0.65

XhoI 2.9 2.8

HindIII+XhoI 1.9 1.0 2.8

PvuI 0.4 5.3

HindIII+PvuI 0.4 1.5 3.8

SmaI+PvuI 2.8 1.15 1.25

PstI 5.35

HindIII+PstI 1.85 3.5

BamHI 1.2 1.25 2.0 1.2

aThe size of some fragments is given with two decimals.Although the second decimalisnotaccurate,

ithas been included to distinguish some fragments fromoneanother.

bThe underlinedfragmentsare shorterby0.05 x 106 daltons for theN-tropic linearDNA.The size of all other fragments of B- and N-tropic linear DNA wasidentical.

A

B

a

b

a

b

3.8

-x

1.9-FIG. 7. Analysis of 5' derived sequences. B-tropic linear viral DNAwascleaved with HindIII (a) and

withHindIII-PstI (b).After digestion, samples were

run on a1.4% agarose gel; the DNA fragments were transferred onto nitrocellulose membrane and annealed with representative [32P]cDNA (A) or

[32P]cDNA

5'(B) prepared as described in the text.

5' END OF RNA 3' END OF RNA

B-TROPIC DNA

Hindm Sal I XhoI XbaI PvuI PstI KpnI SmaI

P.1 1 .

.1 I I L

I I I

N-TROPIC DNA

o-,

_-FIG. 8. Restrictionendonuclease mapsoflinear

B-andN-tropic MuLV DNAs.Cleavagesitesonlinear B- andN-tropic DNAsproduced byHindIII, SalI,

XhoI, XbaI, PvuI, PstI, KpnI, and SmaI. Linear DNA isoriented with respecttothe5'and3'endsof

the RNA. Molecular weights ofthe fragments are giveninTable1.

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identicaltothoseobtainedwithB-tropic MuLV

DNA. Inaddition,ourstudyofB- and N-tropic

MuLV DNA showed that the N-tropic linear

molecule,ascompared to the B-tropic molecule,

is shorter at both ends byapproximately75 base

pairs.Taken together, these results suggest the presence of terminal repeated sequences on lin-ear B- and N-tropic MuLV DNAs as found in

otherretrovirus DNA.

DISCUSSION

In this

study,

we have

mapped

the

cleavage

sites of

eight

restriction endonucleases on

BALB/c

endogenous

B- and

N-tropic

MuLV DNAs, using the Southern agarose

gel-DNA

transfer

procedure.

We have concentrated on

restriction

endonucleases thatrecognizea

hex-anucleotide

sequenceand henceare

expected

to

cleaveaDNA

molecule

of about6x 106daltons onlyafew times. The DNA

fragments

have been ordered relativetothe 5' and 3' ends of the viral RNA molecule. The

general

structure of both linear andcircular B- and

N-tropic

MuLV DNA has been studied with sucharestriction endo-nuclease

analysis.

Wehave found three enzymes

(PstI,

KpnI,

andSmaI) ableto

recognize

a

cleavage

siteat

both

ends

of the linear viral DNA within a

fragment of0.35 x

10'

daltons

(approximately

550base

pairs).

We havealso shown that the 5'-derivedsequences presentatthe

right

terminus oflinear DNA are

repeated

somewhere within the left third end of the linear DNA. The pres-ence of a

5'-derived

sequence at the extreme

right

end of linear viral DNA has been foundon

other

retroviral

DNA

(11,

34,

35).

The

precise

organization

of the distal ends of B- and

N-tropic

MuLV DNAs has not been

fully

elucidated. However, our restriction enzyme

analysis

and cDNA 5'

annealing

dataareconsistentwith the

presence of terminal redundant sequences on

the linear viral DNA of B- and

N-tropic

MuLV

as

found

with other retrovirus DNA

(4, 10-12,

18, 26, 33-35, 38, 43). Definitive evidence will

require

sequencing

data.

We havealsodetectedtwocircular DNA

spe-cies in B- and

N-tropic

MuLV-infected cells. After

cleavage

of these circular DNAs with

re-strictionenzymeswhichcleave thegenome

only

once

(Sall,

HindIII, XbaI,

XhoI, PvuI),

oneof

the linearmoleculesgeneratedhadthesamesize

(5.7 x

10W

daltons) as the linear molecule

ex-tracted fromcells(Fig. 2).Thissuggests that the

largerof the twocircularDNAspeciesof B-and

N-tropic MuLV is a circularized form of the

linear viral DNA. The smaller circularspecies

appearsto berelated tothe

larger

species.

In-deed, cleavage of thetwocircular species with

enzymes which recognize more than one site

(SmaI,

KpnI, and PstI) generates fragments identicaltothose obtainedafter digestion of the

linear molecule (Fig. 5). This result indicates

that thesefragments areinternalandthat they

arecommon to both circular species as

well

as

to the linear form. Therefore, if the

smaller

circular speciesis derived from the linear viral

DNAorfrom the larger circular species, it must

be deleted from sequences outside the

internal

SmaI, KpnI, and PstI fragments.

PstI

restric-tionendonuclease analysissuggests, in fact, that

the smaller circular species is deleted from

se-quences present at one or bothends of the

linear

viral DNA because PstI cleaves linear viral

DNAclosetoboth ends of themolecule. From

thetwocircular DNA species, PstIgenerates a

single 5.35 x

106-dalton

fragment. Most likely,

PstI

cleaves the

larger

circular DNA species twice in regions correspondingto both ends of

linear DNA and the

smaller

circularDNA

spe-cies once to generate a 5.35 x

106-dalton

frag-ment.Therefore, the

smaller

circular DNA

spe-ciesseems tobedeleted fromasequence present

at one (orboth) end(s) of the

linear

viralDNA.

Thesedata

confirm

and extend previousresults

onthestructureofcircular DNA ofretroviruses

(12,18, 33, 34, 43).In eachcase,the

smaller

viral

DNA species appears to lack one copy of the redundantsequence.

Out of14enzymestestedon B-and

N-tropic

MuLV

DNAs,

only

one

(Eco

RI)

failedtocleave thetwoMuLVDNAmolecules.

Eight

restriction endonucleases

(XhoI, PvuI, HindIII,

SalI,

XbaI,

KpnI, SmaI,

andPstI) cleaveB- and

N-tropic MuLV DNAsatidentical sites. This

sim-ilarityinthe restriction endonuclease

pattern

of the two genomes was

expected

since several

groupshave

reported

that

BALB/c

B- and

N-tropic MuLV genomes are

highly homologous

(5, 7,8, 22,30). However, ourdata

provide

evi-dence thatN-tropic viral DNA used inourstudy isnotidenticaltothe

B-tropic

viral DNA. With four restriction endonucleases

(PvuI, XbaI,

KpnI, andSmaI), wefound that the

right

and leftterminal sequences of

N-tropic

linear viral DNA were shorter than the

B-tropic

terminal

sequences

(Fig.

3and

5).

Wealso found that the

smaller fragment generated by digestion

of

N-tropic,

supercoiled

viral DNA with

SmaI,

KpnI,

andPstI wasshorter than thecorresponding

B-tropic fragment (Fig. 3). This

small

fragment

derived from

supercoiled

DNAis

likely

to

rep-resent the fused fragments from both ends of

the linear molecule and, therefore, to contain

onecopy of the redundantsequence. This

differ-encebetween theterminalfragmentsof B- and

N-tropic viral DNA is unlikely to arise from

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additional cleavage sites, since itappears at both

ends of the molecule and is observed with five different restriction endonucleases.Mostlikely, this difference could be accounted forby dele-tion ofashortsegment (approximately 75base pairs) in the terminal redundantsequence. Ob-viously, suchasmall differenceinsizecouldnot

be detectedwith XhoI, HindIII, and SalI

be-causetheyproduceonly large fragments.

Physical mapping of theDNAof three

differ-entexogenousmurine retroviruses

(Mo-MuLV,

Mo-murinesarcoma virus, Ha-murine sarcoma

virus)has now beenreported (1, 4, 6, 10, 12,

41-43).Theavailability of thephysicalmapsof the

DNA of two MuLV endogenous to BALB/c

mouse should provide the basic information

needed tostudy the numerous endogenous

se-quences present in the mouse genome. Wehave

already found that restriction endonucleases able to cleave in the redundant sequences of BALB/c endogenous MuLV DNA give a very

simplified band pattern of virus-specific DNA aftercleavage ofmouseliver DNA from various strains (E.R. and P.J., unpublished data). This work should also allow comparisons of B- and N-tropic MuLV variants and give the informa-tion to map the tropism determinant onviral DNA. Thesize difference observed between the terminal sequences of N-Cl-35 and

B-Cl-il

MuLVDNAsisunlikelytoberelatedassuchto

thetropism determinant.Indeed, thispartof the

genome is notthoughttobecoding foraprotein,

and thetropism determinantseems tobea virus-coded protein (3, 17, 19, 25, 29). Moreover, an

endogenous B-tropicMuLV fromC57BL/6 mice hasrecently beenshown to havealso ashorter terminal sequence than

B-Cl-li

MuLV DNA

(E.R. and P.J., unpublished data). Therefore,

thesequence

coding

for the

tropism

determinant

hasnotbeenidentified by thepresentrestriction analysis, since all the restriction sites studied

wereidenticalonB-andN-tropic viralDNA. A

finer

analysis with cloned DNAandconstruction of

specific

recombinantswillbeneededtosolve thisproblem. Alongthis line and using the in-formation provided bythe present map, we have recently cloned both B- and N-tropic MuLV DNAmoleculesinCharon21A.

Aftercompletion of this work, we learned that

aphysicalmap ofN-tropic MuLV DNA of AKR

mice had beenderived inWeinberg's laboratory

(R.A.Weinberg,personal communication).Five

restrictionendonucleasesusedin common

gen-erated identicalfragmentswith the same 3' to 5'

orientation, suggestingthat AKR N-tropic and

BALB/c B-tropic and N-tropic MuLV DNAs

share ahighly homologousstructure.Thisclose

homology of ecotropic MuLV from different strains is interesting in view of the fact that

AKR and BALB/c strains were derived from

differentancestors.

ACKNOWLEDGMENTS

Wethank Marcelle Bedard forpreparing this manuscript. This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada to P.J., and agrant from the Cancer Research Society Inc. to E.R.

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