Isolation of recombinant DNA clones carrying complete integrated proviruses of Moloney murine leukemia virus.

0022-538X/81/010181-10$02.00/0

Isolation

of

Recombinant

DNA

Clones Carrying Complete

Integrated Proviruses of Moloney Murine

Leukemia Virus

LEE BACHELERt* AND HUNG FAN

The Tumor Virology Laboratory, San Diego, California 92138

EcoRI DNA fragments from a Moloney murine leukemia virus

(M-MuLV)-infected mouse fibroblast line (M-MuLV clone A9) were cloned in lambda phage

Charon4A cloning vector toderiveclones containing integrated M-MuLVproviral DNA. A 10- to 16-megadaltonclass ofEcoRI fragments was chosen for cloning, based on (i) its ability to induce XC-positive virus upon transfection of NIH/3T3 cells, and (ii) its content of a 0.8-megadalton viral KpnI fragment diagnostic for M-MuLV. Six recombinant DNA clones were isolated which contain a complete M-MuLV provirus, as judged by (i) restriction endonuclease mapping and (ii) the

fact that all ofthe clonesgave rise toXC-positive, NB-tropic virus upon DNA infection in NIH/3T3 cells. The sizes of the inserts were 12.0 (for three clones) or 12.5megadaltons (for three clones). Restriction mapping indicated that these six

clonesrepresentfive different M-MuLV proviral integrations into different

cel-lularDNA sites.

Moloney murine leukemia virus (M-MuLV),

likeother murine and avian retroviruses,

estab-lishesastable associationwith the host cell that

it infects. During infection, one or more DNA

copies ofthe viralRNAgenome are covalently

integrated into the chromosomal DNA of the hostcelltoform proviruses (26). In both avian

and murine systems, multiplesites for integra-tion of retrovirus DNA sequences into cellular DNA have been demonstrated by restriction enzyme mapping (1, 8, 14, 16, 24). Although it seems clear that integrationis not limited to a

singleor avery small number ofchromosomal locations,it is as yetunclearwhetherintegration

into cellular DNA sequences occurs totally at

random or whether there are

regional

and/or

sequence

preferences

forM-MuLV

integration.

Although

multiple integrations

of M-MuLV andother retrovirusescanoccurina

single cell,

notallsuchintegrationsare

functionally

equiv-alent. Keshet and Temin(3, 18) studied

integra-tions of reticuloendotheliosis virus and

spleen

necrosisvirusDNAs.

They

demonstratedawide size rangeof

provirus-containing

restriction

en-zyme fragments in both acute and

chronically

infectedcells.Viral

infectivity,

asmeasuredina

transfection assay, however, resided in a

single

restrictionenzymefragmentsize(3,4).Jaenisch has derived a series of mouse strains which

genetically transmit M-MuLV DNA sequences

insertedatdifferentchromosomallocations

(17,

17a). The patterns ofviral gene

expression,

as

tPresent address: Fels Research Institute,Temple Univer-sity SchoolofMedicine,Philadelphia,PA 19140.

wellas theeffects on mouse development, vary among the different proviral integrations and

providestrongevidencefor "positioneffects," or

regulation

ofthe expression ofthe inserted M-MuLV DNA.

We (7)have indirectly studied the

transcrip-tionalactivity ofintegrated M-MuLV genomes in a numberof infected fibroblast linesby

mea-suring

the relative DNase I sensitivity of M-MuLV DNAsequences in isolated nuclei.

Wein-traub and Groudine (29) have shown that the DNA ofactively transcribed genes is preferen-tially digested when nuclei or chromatin is treated with

pancreatic

DNase I. In cellswith

several integrated copies of M-MuLV, only a

minority (as fewasonecopy)of the viralDNA sequences werefoundin apreferentiallyDNase

I-sensitive chromatin configuration. Most pro-viralcopies were resistant,suggestingthat many

oftheintegrated M-MuLV

copies

inthese cells

were notactively transcribed. Thus, fibroblasts, productivelyinfectedwithM-MuLV,apparently contain both transcribed and nontranscribed

copiesofproviralDNA.

Further studies of the fine structure and

expression of

integrated

M-MuLV genomes would be greatly facilitated by the availability

ofrecombinant DNA clones which contain

in-tegrated M-MuLV

proviral

DNA and the

sur-rounding cellular DNA sequences. We report heretheisolationand

preliminary

characteriza-tion of a series of lambda

phage

recombinant DNA clones whichcontain such

integrated

M-MuLV

proviral

DNAs from an infected mouse fibroblast line.

181

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All cells were maintained in Dulbecco-modified Ea-gle medium supplemented with 10% calf serum (GIBCO Laboratories, Grand Island, N.Y.). The deri-vation of M-MuLV clone no. 1 (10) and M-MuLV cloneA9(9) has beenpreviously described.

Isolationofhigh-molecular-weight DNA and unin-tegrated M-MuLV viralDNA, the synthesis of high-specific activity 32P-labeled M-MuLV representative complementary DNA (cDNA), and blot-transfer hy-bridization (23) have beenpreviously described (2, 11). Restriction enzyme digestions, gel electrophoresis inhorizontal agarose gels, and blot transferto nitro-cellulosewereperformedaspreviously described(2). For preparative isolation of DNA fragments, DNA samples wereelectrophoresed into thick (2-cm) 0.6% agarosegels, the DNAwasvisualized under UVlight after staining with ethidiumbromide, andappropriate regionsof thegelwereexcised. Thegelwasdissolved byheating in 10volumesof8MNaClO4at60°Cfor 1to 2 h and thenpassedover acolumnof hydroxy-apatite (BioRad), equilibratedin 0.05MPB (sodium phosphate buffer, pH 6.8) at60°C. The columnwas washed with 10 column volumes of1MNaCl04-0.05 M PB, and bound DNAwas eluted with 0.5MPB. The elutedDNA wasdialyzed against0.1 M ammo-niumacetateandconcentratedbylyophilization.

Ligation. A 1-pg amount ofgel-purified cellular DNAwasmixed with2jigofanequimolarmixtureof Charon4Alambdaphage left andrightendfragments

andlyophilized.TheDNAwasresuspendedin0.01M Tris(pH 7.4)-0.01MMgCl2-0.05MNaClat a

concen-tration of300,tg/ml and incubated at42°Cfor 1 h. Thesolutionwasadjustedto 0.4mMATPand1mM DTT, 2 U of T4 DNA ligase (Bethesda Research Laboratories,Bethesda,Md.)wasadded,and incuba-tionwascontinued for16to48hat12°C.

In vitropackaging. Products of theligation

re-actions were packaged into lambda phage particles

with purified lambdaproteinA, freeze-thaw lysates,

and sonicextractsof induced lambdalysogens bythe protocol of Blattner and co-workers (25; H. Faber, D. Kiefer and F.Blattner,personal communication, pro-tocolsdistributed with Charon vectors, 1978).Inone

600-pl

packagingreaction, 2.1,tg ofligatedDNA

re-sulted in 2.2 x 104recombinantphage. Ina second

600-,ul packagingreaction,7.2x105recombinantphage

resulted from thesame amountofinput DNA. The differences in efficiencies may have been dueto differ-entextentsofligation in thetwoDNAsamples.

Allpackaging experimentsandsubsequent biologi-calmanipulations wereperformed in acertified P-2 containmentlaboratory.

Screening ofrecombinant DNA clones. Prod-uctsof thein vitropackaging reactionswereadsorbed toEscherichia coliDP50supFandplatedonto 12 15-cmpetridishescontainingNZYDTagar (6). Nitrocel-lulose replicasofthepetri dishes were prepared by the method ofBenton and Davis(5)andhybridized with 32P-labeled cDNA. Dextransulfate (10%) wasadded tothehybridizationstoaccelerate theannealingrate (28).Afterautoradiographyof thenitrocellulose rep-licas,plaquesontheagarplateswhich showed hybrid-ization withthe M-MuLV cDNAprobe werepicked

tion andscreening until more than 90% of the plaques reacted positively withtheM-MuLV cDNA probe.A final plaque was then picked and used to preparea high-titerliquid lysate.

In oneexperiment, approximately 5x 103 recombi-nant phage werescreened, and 12 plaques hybridized M-MuLV cDNA. Of these 12, 8 hybridized strongly and1wassubsequently foundtocontain a M-MuLV proviral integration. In a second experiment, approxi-mately 1.5 x 104recombinant phage were screened, and30plaques hybridized M-MuLV cDNA, of which 11strongly hybridized. Six of the strongly hybridizing clones were further analyzed, and five contained M-MuLVprovirus whereas the other contained MuLV-related endogenous virus sequences.

Preparation of recombinant phage. Bulk prep-aration of recombinant phase was performed by the protocol of Blattner (Blattner et al., charon phage protocols, 1978). Briefly, high-titer lysates were used toinfect E. coli DP50 supF, and mass liquid cultures were grown.After lysis, the supernatant was clarified andphage wereprecipitated by addition of polyeth-ylene glycol. Precipitated phage were then banded throughastepCsCl gradient followed by banding to

equilibrium in CsCl. In cases where multiple phage bandsresulted (seeDiscussion), the individual bands wereharvestedseparately.

DNA infections.NIH/3T3cellswereinfected with 0.5to 2.0

iLg

ofpurifiedrecombinantphage DNA per 5-cmdish1day after seedingNIH/3T3cellsat 5x105

cells.Infectionswerecarriedoutbyamodification of thecalciumphosphateprecipitation technique of Gra-ham andvanderEb (15) byusing calf thymus DNA carrier anddimethylsulfoxidetreatment asdescribed by Lai and Verma (19). Cultures were assayed for virus-infected cells by UV-XC plaque assay (22) or passaged several timesto establishachronically in-fected culture.

RESULTS

The source of infected cell DNA for these

molecular cloning experimentswasthe cellline M-MuLVcloneA9. M-MuLVcloneA9cellsare

aclone ofNIH/3T3 fibroblasts productively

in-fected with M-MuLV, and we have previously

characterizedthemascontainingapproximately

4copies of M-MuLVDNA per haploid genome,

or 10 to 12 copies per cell (7, 9). EcoRI DNA fragments containing some ofthese integrated M-MuLV proviral DNAs (approximately 13.0, 12.0, 8.7, 5.4, and 4.6 megadaltons [Md]) have been identified in thesecells (1,2). In addition,

even though this cell line is productively in-fected, only a minority of the viral DNA se-quences exist in chromatin in a configuration which is preferentially sensitive to DNase I

digestion and therefore potentially

transcrip-tionallyactive (7).

We wishedtocloneM-MuLVproviruses from

an infectedmouse cell, the natural host for M-MuLV, and from a characterized cell clone to

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VOL. 37, 1981

facilitate further studies of the structure and function of individual M-MuLV integrations. A serious problem in attempting to screen for M-MuLV proviral DNAsequences in mouse cells

is the presence of an endogenous family of MuLV-related sequences which share partial se-quence homology with M-MuLV. Figure 1A

showsthe complex pattern of EcoRI fragments withnucleic acid sequence homology to a

rep-resentative M-MuLV cDNA probe (11) from uninfected mouse cells and M-MuLV clone A9 cells. Two EcoRIfragments present only in the

infected cell lineandabsent fromtheuninfected

cells canbeseenatapproximately 12 x106 to 13

x

10W

daltons, and they may contain integrated

M-MuLV proviralDNA. That these new EcoRI

fragments actuallycontain integrated M-MuLV DNA sequences is shown by their enhanced hybridization to an M-MuLV cDNA specific for

M-MuLV DNA (and whichdoesnot recognize endogenous MuLV-related sequences in

unin-fectedcells) asshowninFig. 1B (2).

Our experimental design involved cloning of

size-selected EcoRI fragments from total

M-MuLV clone A9 DNA in the lambda phage

charon4Acloningvector (6). This strategy was

employed for several technical reasons. First,

EcoRI doesnotcleave M-MuLV DNA(12), so

EcoRI fragments frominfectedcellDNAwould

containcomplete copiesof MuLVproviralDNA

with adjacent5' and 3'cellular sequences.

Sec-ond, the Charon4Acloningvectorwas designed

forcloning of EcoRI fragments,and the sizes of

insertedDNAfragments thatcanbe

accommo-datedby thisvector(7.6to20.6kilobasepairs or 4.85 to 13.2 Md) include most of the EcoRI

fragments containing M-MuLV proviralDNA in M-MuLVcloneA9cells. Third, size selectionof

theEcoRI

fragments

wasdeemednecessarydue

to the large number of EcoRI fragments

con-taining endogenous M-MuLV-relatedsequences present in both uninfected and infected cells. Generating lambdaphage clones from total

in-fected cellDNAwouldresult inalarge number of clonescontainingendogenous MuLV-related

sequences in additiontothesmaller number of desired M-MuLV

proviral

clones. Clones of theseendogenous virus-relatedsequenceswould also be

recognized during

the

screening

proce-dures. Thus, many clones which hybridize M-MuLVcDNAmighthave to bescreenedbefore

one containing a genuine M-MuLV provirus mightbeobtained.Asshown in

Fig.

1A,certain sizes ofEcoRI fragmentsfrom M-MuLV clone A9cells contain

relatively high

concentrations of M-MuLV

proviral

DNA in

comparison

to

endogenous M-MuLV-relatedsequences, for in-stance, the 12 x 106_ to 13 x 106-dalton range.

M-MuLV PROVIRAL CLONES

183

Sc

Ii

12-13MdEC

5.4- I 12

2-l3

Md

d 5.4

Representative Specific

c DNA c DNA

FIG. 1. Identification of M-MuLV proviral

frag-ments. UninfectedNIH/3T3 andinfected M-MuLV clone A9cellDNAs (10pg per channel) were digested with EcoRI and analyzedby agarose gel electropho-resis and blot transfer to nitrocellulose filters. As a mobility marker, linearunintegrated M-MuLVDNA

(5.4Md) isolatedfrom freshly infectedcellswasalso included

("viral'".

Hybridization to two replicate blots wasperformed with either representative M-MuLV cDNA('representativecDNA"' orwith cDNA

specificfor M-MuL V whichwasgeneratedby

prean-nealing representativeM-MuL V cDNA withalarge excessof AKR MuLV 70S RNA ('specific cDNA'9. Autoradiograms ofthehybridized blotsareshown.

Therefore, appropriatesize selection of the

M-MuLV cloneA9EcoRI

fragments

before

cloning

could

provide

an ixnportant enrichment of

M-MuLV

proviral

sequences over

endogenous

MuLV-relatedsequences. For thesereasons. we

chose toclone M-MuLV clone A9EcoRI

frag-mentsof12x 106to13x 106 daltons.

To prepare size-selectedfragments, 10mg of

M-MuLV cloneA9cellDNAwas

digested

with

EcoRI and fractionated

by

electrophoresis

in

0.6% agarose

gels.

DNA was extracted from

slices of the agarosegel bydissolvingthe

gel

in

concentrated

NaClO4

and

binding

the DNAto

hydroxyapatite. Figure 2 shows the size distri-bution of each of these isolated

fractions,

which

arealso listed in Table 1. Each size fraction of infected cell DNAwastested for its contentof

integrated M-MuLV sequences by two addi-tional criteria. First, a portion of each DNA fraction was tested for thepresence ofan

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[image:3.496.252.451.64.313.2]

though not necessarily sequencecomposition,is

diagnostic of M-MuLV as opposedtoanyofthe

murineendogenous virus-related sequences (2). Figure 3 shows that this0.8 x 106-dalton

frag-ment present in unintegrated Moloney viral

DNA isalso detectedinthe totalcellular DNA of infected cloneA9.

Importantly,

no

virus-re-lated fragment of similar size is liberated

by

KpnI

digestion

of uninfected

NIH/3T3

cell

DNA. Bydigesting a smallaliquot of each

iso-lated DNA fraction with KpnI, the M-MuLV

diagnosticfragmentwasidentified in fractions 4, to 7

(Fig.

3). Fraction 4 seemed the mosthighly

enriched for M-MuLV proviral DNA, since MuLV-relatedKpnIfragmentspresent in unin-fected cellsbut not in M-MuLVviralDNAwere

largelyabsent.

In a second testfor thecontentof M-MuLV genetic informationof eachisolated DNA frac-tion,aportion ofeachfractionwasusedtoinfect NIH/3T3 cells in a DNA transfection assay.

EcoRI-cut DNA from uninfected NIH/3T3 cells, totalM-MuLV cloneA9DNA, and unin-tegrated viralDNAisolated fromthecytoplasm offreshly infected cells were also tested. After

one or more passages, eachtransfectedculture was assayed for M-MuLV infection by an XC

11 10 9 8 7 6 5

and total M-MuLV clone A9 DNA both

pro-duced XC-positive virus in the transfected

cul-tures, whereas uninfected NIH/3T3 cell DNA did not. Fractions 4 and 5 also induced

XC-positivevirus,confirmingtheir contentof

genet-TABLE 1. Characterization of size-selected EcoRI-cutDNAof M-MuLVinfectedcell line A9

Size range

M-MuLV-DNA fraction (X16dal- diagnostic Infectiv-tons) KpnI frag- ity

ment

Unintegrated viral 5.8 + +

DNA

EcoRI-cut3T3 DNA <1->20 - -EcoRI-cutA9DNA <1->20 + + A9fraction

1 9-20 -

-2 9-20 -

-3 13.5-18 -

-4 10.5-16 + +

5 9.4-12 + +

6 7-10 +

-7 6.5-9 +

-8 5.8-6.7 -

-9 5.0-6.0 -

-10 4.3-5.5 -

-11 4.1-4.7 -

-4 3 2 1 A9 3T3

- 14.7 - 10.7

- 6.7

- 5.

- 4.6

FIG. 2. Characterizationofsize-selectedDNAfractions. Aliquots of each size fraction(Ito11)ofpurified EcoRI-cut M-MuLVclone A9cellDNA wereelectrophoresedthrough a 0.6% agarose gel and visualized by staining with ethidium bromide. Alsoshown are total EcoRI digests of M-MuLV clone A9 cell DNA and uninfected3T3cellDNA. The positions of several restriction enzyme fragments of A DNA analyzed on the samegelareindicated,along with their sizes in Md. A DNA cut with EcoRI was mixed with the digested M-MuLVcloneA9DNAbeforepreparativefractionationtoprovide guides for slicing thegel.TheseA restriction enzymefragmentsarevisableinseveralisolated DNA fractions.

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[image:4.496.115.453.177.592.2]

icallycompetent M-MuLV (Table 1). It is

inter-esting that fraction 7 may have contained M-MuLVproviral DNA by the KpnI cleavage test, but did not yieldinfectious virus upon

transfec-tion.

DNA fraction 4, with a sizerange of 10.5 x 106

to 16.0 x

106

daltons, was cloned into the charon 4Acloningvector. The right and leftEcoRIend

fragmentsof charon 4A,purifiedbypreparative agarose gel electrophoresis, were ligated with

fraction4DNA, and thehigh-molecular-weight

DNA was packaged into lambda particles in vitro by using the in vitro packaging system

described by Blattner(seeMaterials and

Meth-ods). In vitro-assembled phage particles were directly plated as primary plaques on E. coli DP50

supF

without initial amplification. Re-combinant phageplaqueswere screened for se-quencehomologytoM-MuLV byhybridization of an M-MuLV cDNA probe to nitrocellulose

E NC 1 2 3 4

filter replicas of the initial plates (5). Plaques

with strong sequence homology to M-MuLV werepicked, andplaqueswerepurified.

Recombinant DNA phage with strong se-quence homology to M-MuLV could represent clones of intact integrated M-MuLV proviruses, rearranged or otherwise aberrant integrations of

allorpartof theM-MuLVgenome, orclones of

endogenousMuLVrelatedsequences which

re-sideonEcoRIfragments of the size selected for cloning.To distinguishbetweenthese possibili-ties,DNA was purified from each recombinant

clone and further characterized with respect to its restriction enzyme cleavage pattern. Each DNAsamplewasdigested withKpnI to testfor the presence ofaM-MuLV-diagnostic0.8x 106-daltonfragment which is liberated from the 3'

region oftheviral genome. Figure 4 shows the

patterns of restriction enzyme fragments ob-tained from eight recombinant clones. Blot

5 6 7 8 9 10

0

j;' .

I

3.5

Md-2.5

-1.8

-0.8

-Eadogemeos

Fro o*nfs

- M-M.LV

[image:5.496.105.399.289.591.2]

Specific

FIG. 3. KpnI cleavageofsize-selectedDNAs.Aliquotsofthesize-selected EcoRIfragments fromM-MuL V clone A9 cells were secondarily cleaved with KpnI and analyzed by electrophoresis and blot transfer

hybridizationbyusingarepresentativeM-MuL V cDNAprobe.Forcomparison,total cellularDNAfrom M-MuLVclone A9 anduninfected NIH/3T3cells, aswell asunintegratedM-MuLV linearDNA, was also digestedwithKpnIandanalyzed.Themobility ofthe M-MuLV-diagnostic0.8-MdKpnIfragment,aswellas anendogenous 2.5-MdKpnIMuL V-relatedfragmentpresent inuninfectedcells butnotcontainedingenuine

M-MuLVprovirus,isindicated.

37,

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cDNA is shown. Six of the eight clones tested contained theM-MuLVdiagnostic fragment of

0.8Md. Thissuggeststhatthey containatleast the3'portion ofanM-MuLVgenome,sincethis fragment is actually a doublet oftwointernal

viral DNA fragmentsliberated from the 3' end of the M-MuLV DNA sequences. These six clones also liberate a doublet of

M-MuLV-hy-bridizing KpnI fragments of1.8Md which arise from the 5' endofM-MuLV. This fragment is

not diagnostic ofM-MuLV, however, since the

vast majorityof theMuLV-related endogenous

sequencesalsocontain a1.8-Md KpnIfragment (Dolberg, Bacheler andFan, submitted for pub-lication).Indeed,clone27contains such a

virus-related 1.8-Mdfragment, butlacks the diagnos-tic 0.8-Md fragment. It is possible that clones which fail to release the M-MuLV-diagnostic 0.8-Md KpnI fragment carry M-MuLV

se-quences, either as

partial

integrations

or

rear-rangedsequences,butsuchclones are not

can-didates for complete, potentially functional

M-MuLVintegrations. We conclude that clones48, 61, 63, 73, 75, and 76 are good candidates for

containing complete integrated M-MuLV

ge-M-MuLV-diagnostic

fragment

liberated

by

Bam digestion(1) whichcomigrated with the 1.9-Md

Bamfragmentfromunintegrated M-MuLV viral

DNAand whichhybridizedtoM-MuLVcDNA

(datanotshown).

Figure5 showsadetermination of the sizes of

theinserted cellularDNA sequences.DNAfrom

each clonewas cutwithEcoRI whichseparates

the vector sequences from the inserted DNA and analyzed by gel electrophoresis. The six clones identified as containing M-MuLV-diag-nostic fragmentsfallinto twosizeclasses, having

inserts ofapproximately 12.0and 12.5Md. The sizesareverysimilartothe 12 and 13Md of the EcoRI fragments previously identified in total M-MuLV clone A9 DNA as containing

inte-gratedM-MuLV DNA sequences. The sizes of

the insertssuggestedthat atleasttwodifferent integrations of M-MuLV DNAwerecloned.

Thebiological activity of each ofthe sixclones

of integrated M-MuLV DNA sequences was

tested by using a portion of the phage DNA (after digestion withEcoRI) toinfectNIH/3T3 cells in atransfection assay. Each ofthese six

clones induced the fornation of XC-positive

2 so

1 46 *1 43 ,n Vs #

do

d ~~~*-J,

I

Al* %^ f

I

p

1.8 Md -

la

q_

0.8 - _

*ipe

i

-_

_

-- M-MuLV

specific

FIG. 4. Test of recombinantclonesforM-MuL Vprovirus. DNAfromisolated recombinant clones or from linearunintegrated M-MuLV viral DNApurifiedfrom freshlyinfected cells('viral")was digested with KpnI andelectrophoresed througha 1% agarose gel. Hybridization to a blot transfer of this gel with M-MuLV-representative cDNA is shown. Themobility of the M-MuLV-diagnostic 0.8-Md KpnIfragmentis indicated. The fulldesignations of the clones are Ch4A-A9-4- followed by the clone number, butfor simplicity are referred to by the clone number.Inthefigure, Ch4A-A9-4-48 is indicatedas clone 48.

I*A

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[image:6.496.137.371.355.601.2]

plaques in the infected cell cultures, whereas

other clones isolated in the same experiment,

which did notcontain M-MuLV diagnostic frag-ments, did not (clones 27 and 28) (Table 2). Supernatant virus from each

XC-positive-in-fectedculture was further characterized by

titra-tionon NIH/3T3 and BALB/c 3T3 cells. The XC-positive virus in each case titered with equal

efficiencyon bothcelltypes,indicatingthat the virusesliberated after cloned DNA infection had

theNB tropic host range ofM-MuLV itself. This NBtropic host range is also not characteristic of anyknownendogenous murine virus. This fur-ther supports theidentificationof these

recom-binant DNAclones ascontainingintact integra-tionsofM-MuLV DNA.

Thetwo sizes of cellular DNA inserts contain-ing integrated M-MuLV genomes suggested that at least twodifferent integrations of M-MuLV

DNA intocellularDNAsequences were cloned. Tofurther characterize the inserted sequences, restriction enzyme maps of the six clones

con-tainingintegrated M-MuLV proviral DNA were

constructed by sequential restriction enzyme

digestionsandby blot transfer-filter

hybridiza-tionswith M-MuLV cDNA.Figure 6shows re-striction enzyme maps for the EcoRI inserts of

A 1 2 3 4 5 6 7 8 9

13.22-

4.54-3379

3.22-these sixclones. The mapshave been drawn to align the 5' end of the integrated viral DNA sequences and indicate that theintegratedviral DNAsequences are located in different positions in several of the clones. One 12.0-Md cellular DNAfragment was cloned twice, on clones 61

[image:7.496.96.444.206.585.2]

and76, inoppositeorientations with respect to the Charon 4A cloning vector. Restriction en-zyme sites in the flanking cellular DNA se-quencesindicatethat clone 73contains a differ-entM-MuLVintegrationthan thatcontained in

TABLE 2. Characterization of isolated M-MuLV-relatedrecombinant DNA clones

Size of in- M-MuLV-

Infectiv-

NB tro-Clone no. sert(x106 diagnostic ity pism

daltons) fragments

48 12.5 + + NB

61 12.0 + + NB

63 12.5 + + NB

73 12.0 + + NB

75 12.5 + + NB

76 12.0 + + NB

70 7.7 - NDa

27 9.2 - -

-28 8.9 - -

-0ND, Not determined.

B

*

2 3 4 5 6 7 8 9

2.34-FIG. 5. Sizeofcloned inserts. DNAfromseveral recombinant DNA clones withstrongsequencehomology

toM-MuLVwas cutwithEcoRIandelectrophoresedthrougha0.6%agarosegel.The mobilitiesofmarker AEcoRI DNAfragmentsco-electrophoresedin thesamegelareindicated, alongwith their sizes (Md). (A) Ethidium bromidestainingofthegel;(B) hybridizationwith M-MuL V cDNAtoa blottransfer of thesame

gel.(Lane 1) MixtureofADNAcutwithHindIII,detectablebyethidium bromidestaining (A), and uncut M-MuLVunintegratedlinearDNA, detectedbyhybridizationwith M-MuLV cDNA(B);(lane2) Ch4A-A9-4-48; (lane 3)Ch4A-A9-4-61; (lane 4)Ch4A-A9-4-63; (lane 5) Ch4A-A9-4-73; (lane 6)Ch4A-A9-4-75; (lane 7) Ch4A-A9-4-76;(lane 8)Ch4A-A9-4-70; (lane 9)Ch4A-A9-4-27.

.'*.

0 .M 14 I

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M-MuLV RNA 8 Xh

I,

RXL

)L".I H

ID

PXKPsPs

Xh

Xh 8a HD 8 8 B R1

R

P K P P XXK KK PX K RI

Xll 8a HD BaS

I I" X

Ps K\ ,, P,,,\X Ps L

Ps PXK PsPs P K P P XXK KK PXK PS 'XL

R1

XL

---HD ND B Xh

PX K Ps Ps P

88 ND

K P .,

K P P XX K

8 8 HD RI

KK PXK

48

61

76

63

ND BHD Xh

Ps

X,iR

-5

v "IPPP

PS P X KPSPS P

0

88 NHD B ND B

''I I KKPXK

K P P XXKH KK H PXK Ps H

Xh 88 HD H B H Xh

5

Kb

10

..XR

a

...~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

73

XL 75

s5

8-BamHI, HD-HindM, Ps.PstI, P-Pvu, H-HpaI, X.XboI, Xh.XhoI, K-KpnI, RI-EcoRI

FIG. 6. RestrictionmapsofM-MuLVproviralclones. Restrictionmapsforthe sixM-MuLVproviralclones areshown. For convenienceoforientation,coordinatesareshown with the M-MuLVproviralportions ofeach clonespanning from0to8.9 kilobasesonthemap, andtheyareshown in theconventional orientationwith respect to M-MuLV RNA. Adjacent cell sequences to the 5' side ofthe viral DNAs have negative map

coordinates, andadjacentcellsequencestothe 3' side havepositivecoordinatesgreaterthan 8.9 kilobases.

Themaps werederivedby cleavage ofrecombinantphageDNAs with various restrictionenzymes(singlyor incombinations) followed byagarosegel electrophoresis. The totalfragment patterns weredetermined by ethidium bromide staining, and those which contained viral sequences were identified by blot-transfer hybridizationwith M-MuLV cDNA. Theabbreviationsforthedifferentenzymesaregivenin thefigure;sites shown above the line havebeenmapped throughouttheinsert,whereas additional sitesmayexistforenzymes

shown below the line. The locationsoftheleft andrightarns ofthe Charon 4A cloning vectorarealso

indicated.

clones61and76,eventhough both integrations arecontained inEcoRI fragments ofverysimilar

size.Similarly, clones 48, 63, and 75 each

repre-sent different integrations of M-MuLV DNA

sequencesinto cellularDNA,eventhough each

provirus is contained in anEcoRI fragment of

12.5Md. In contrasttothediversity of restric-tionenzymesites in the cellular DNAsequences

flanking each integrated M-MuLVgenome,the viral DNAsequencesin eachcaseshow identical

restrictionmapswhich alsoagreewell with the

restrictionenzyme mapfor M-MuLVderived by

Gilboaetal. (13).

DISCUSSION

We have isolated six recombinant lambda phage DNA clones which contain both proviral M-MuLV DNAsequencesand the surrounding

cellular DNA sequences into which the viral

DNAwasinserted. Five different integrations of

M-MuLVarerepresentedontheseclones, even

though several of the EcoRI DNA fragments containing these integrationsareofverysimilar

size.We consider theseintegrations distinct

be-causeeach showsaunique distribution of

restric-tionenzymesites in the cellularDNAsequences

surrounding the proviral DNA sequences. The

number of different integrations of M-MuLV present in EcoRI fragments of 12.0 x 106and 12.5 x 106daltons wasinitiallysomewhat

sur-prising, since we had previously assumed that

the new EcoRI fragments of this size seen in

EcoRI digests of M-MuLV clone A9 cellular DNAwereeach the result of theinsertion ofa

single M-MuLV proviral DNA sequence. The

isolation of five different integrations is, how-ever, compatible with our previous estimates

that this cell line contains 10 to 12 integrated copies of M-MuLV (7). Furthermore, the fact thatthesameintegration sitewasindependently

isolated twice among six clones suggests that

most of the M-MuLV integrations in EcoRI fragments of this size have been cloned.

Although the cloning efforts with the M-MuLV clone A9 cell DNA successfully yielded the M-MuLV proviral clones described here,

effortstoperform similar experiments withtwo

other DNA fractions from other productively infected cells were unsuccessful. EcoRI

frag-mentsof6to10Md from thecellline M-MuLV

clone E7 (9) which carries integrated M-MuLV DNAsequences onEcoRIfragments of9.5, 8.6,

6.6, and 3.75 Md (2) were cloned, and 22,500

P,,PSPIP K KK, P

P X KPIPSP K P P XXK KK PX K Ps

-1 I I

RI

I

RI

X

L- -

k

RI

B1

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http://jvi.asm.org/

[image:8.496.57.452.62.276.2]

recombinant phage were screened. Ten phage

cloneswerefoundtohybridize M-MuLV cDNA and four strongly hybridized. However, none of the clones contained a M-MuLV provirus, al-though some contained endogenous

M-MuLV-related sequences. Similarly, EcoRI fragments

of6 to 10 Md fromM-MuLV clone 4Acells (9) were cloned, and 2.3 x

105

recombinant phage were screened; 34 phage clones hybridized with

M-MuLV cDNA, and 15 strongly hybridized.

Again,none of theseclones contained a genuine

M-MuLV provirus. In the second experiment,

the 6- to 10-Md 4A EcoRI fragments were

di-gested with BamHIand found to yield a 1.9-Md

Bam fragmentdiagnostic of M-MuLV. In

addi-tion, this DNA gave rise to XC-positive virus upon DNAinfectionof NIH/3T3cells.Bythese

criteria,atleasttheM-MuLV clone 4AcellDNA

fractionshould havecontained intact M-MuLV

proviruses. Several possibilities might explain

the failureto obtainM-MuLV proviral clones in two cases. (i) Not enough recombinant phage may have been screened. This may have been the case forthe experimentswith theM-MuLV

cloneE7DNA. However, sufficient numbers of phage were screened for the M-MuLV clone 4A

trialsthat itcouldbecalculatedthat anysingle copy DNA sequence present should have been

cloned. (ii)Theparticular DNAfractionschosen may have containedrelatively higher concentra-tions of endogenous MuLV-related sequences. This wasprobably the case,sincethe 6- to

10-Mdrangeof EcoRI fragments from infected or

uninfectedmousecells containsmany more

frag-ments with sequence homology to M-MuLV

than the12- to 13-Mdrange. However,the fail-ure to isolate asingle M-MuLV proviral clone

contrastssharply with the relativeeaseof clon-ing from M-MuLV clone A9 cells. (iii) Some proviral integrations may not be clonable in bacterial systems. Evidence that certain

se-quences in the murine mammary tumor virus genomecannotbe cloned has been obtained

by

several research groups

(G. Hager, personal

communication; H. E.

Varmus, personal

com-munication), and it is

possible

that some

M-MuLV

proviral integrations

may have similar properties.Inthecaseof

M-MuLV,

the

putative

unclonable sequences would not be located in the viralsequencesthemselves

(since

other M-MuLVproviruseswere

successfully cloned),

but

rather inparticularadjacentcellular sequences. (iv) M-MuLV proviral

integrations

may have beencloned and

subsequently

lost.

Instability

of recombinant phage clonescarrying

proviral

se-quences has been observed for other murine retroviruses(20, 27).Retroviruses alsocarry

se-quencesresemblingclassical

procaryotic

and

eu-caryotic transposable elements, which may

re-sult in sequence arrangement during growth

(22a). Indeed, we have observed that many of therecombinant phageclones isolated(carrying both genuine M-MuLV proviruses as well as endogenous MuLV-related sequences) display

instability during propagation. Growthof virus preparations isfrequentlyaccompanied by gen-eration ofdeleted forms of the originalphage, which can bedetected by the presence of phage

particles with lowerbuoyant density. We have found it necessary to routinely band all phage preparations inequilibrium CsCl gradientsand useonlythe phage of greatestdensity to mini-mize these problems.

Wehavetestedthebiologicalactivity of each M-MuLV-containing recombinant DNA clone by using the isolatedphage DNA toinfect

NIH/

3T3 cells. Eachofthe clones identifiedas

con-taining M-MuLV based on the presence of

di-agnostic restrictionenzymefragments alsogave rise to infectious NBtropic virus,confirmingthe

identification of these clones. The observation that DNAfromeachclonewasinfectious differs somewhat from several other studies in which

proviruses resultingfrom recent viral infection

(as opposedtogenetically transmitted

endoge-nousproviruses) were isolated as recombinant DNAclones.Lowyetal.(20) have isolatedthree

clonesofintegratedAKR viral DNA sequences

froma masscultureofinfectedfibroblasts.One

ofthese clones was noninfectious, whereas the

other twocloneswereinfectious, althoughwith

approximately 30-fold difference in specific

in-fectivity. O'Rear et al. (21) have cloned

inte-grated spleen necrosis virus DNA sequences from chronically and acutely infected chicken

cells. Both infectious and noninfectiousclones

were obtained. Although infectious clones

con-tainedinserts ofsmaller size thanthe

noninfec-tiousintegrationscloned,sizedidnotappearto

accountfor thedifferencein

biological activity.

Mullins et al. (J. I.

Mullins,

M.

Nicholson,

J.

Casey,K.Burck and N.Davidson,

personal

com-munication) have cloned seven

integrations

of

feline leukemia virus from an infected human

cell line and obtainedbothinfectiousand

non-infectious isolates. Nomajordifferences between the viral sequences ofinfectious and

noninfec-tious clones could be detected. It is unclear whether theinfectious natureof all ofourclones is merely chance or whether M-MuLV

provi-ruses are more often infectious than AKR, spleen necrosisvirus, or feline leukemia

provi-ruses.

The infectivity of all five of the M-MuLV proviruses cloned stands in apparentcontrastto

theDNase Iresistanceand,

therefore,

potential

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http://jvi.asm.org/

grated copies of M-MuLV found in the A9 cell line. Weare currently investigatingthe specific infectivity ofindividualcloned M-MuLV

provi-ruses and thetranscription activity of these

in-dividual integrationsof M-MuLV in the A9cell line.

ACKNOWLEDGMENTS

Thisworkwassupported byPublic HealthServicegrants

R01CA-22829(L.T.B.),R01CA-15747 (H.F.),contract

N01-CP-71008(H.F.),andcoregrantCA 14195,all fromthe

Na-tional Cancer Institute.

Wethank FrederickBlattnerand his associatesfor

provid-inguswith thecharon phagecloning kit,and Jeffrey Browne for adviceonin vitropackaging. Jennifer Price and Patricia

Kreikemeierprovided excellenttechnical assistance, and Mau-reenBrennantyped themanuscript. Wethankmenmbers of

theTumorVirologyLaboratoryforvaluable adviceand dis-cussions.

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