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Molecular properties of a gag- pol- env+ murine leukemia virus from cultured AKR lymphoma cells.

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Vol.41, No. 2 JOURNAL OF VIROLOGY,Feb. 1982,p.626-634

0022-538X/82/020626-09$02.00/0

Molecular

Properties of

a

gag- pol-

env'

Murine Leukemia

Virus from

Cultured

AKR

Lymphoma

Cells

ALANREIN,'* DOUGLAS R.LOWY,2BRENDAI. GERWIN,3SANDRA K.RUSCETTI,3AND

ROBERTH. BASSIN3

Biological Carcinogenesis Program,FrederickCancerResearch Center,Frederick, Maryland21701,1 and DermatologyBranch2and Laboratoryof Tumor VirusGenetics,3National Cancer Institute, Bethesda,

Maryland 20205

Received 6July 1981/Accepted21September1981

We have described the isolation of a replication-defective murine leukemia virus from a culture of AKR lymphoma cells [Rein et al., Nature (London) 282:753-754, 1979]. To facilitate the characterization of this murine leukemia virus, we transmitted it to mink cells and analyzed its genome by restriction mapping of theminkcellular DNA. This genome resembled the Akv genomequite closely,but it hadanadditional KpnI cleavage site at 1.3 kilobase pairs from the 5' end of the provirus and a small (-50-base-pair) deletion between 1.8 and 3.0 kilobase pairs from the 5' end. When we tested these mink cells by immune precipitation or by competition radioimmunoassay, we found that they synthe-sized gPr82env, but contained no detectable gag or polproteins. It seems likely that the KpnIcleavage site at 1.3kilobasepairs reflects an abnormal sequence at ornearthebeginningof the gag gene, which prevents gag or pol translation by introducing a frameshift or termination codon into this region.

Geneticvariants of virusesareof fundamental importance in analyzing the roles of individual gene products in viral life cycles. However, relatively fewvariants of the mammalian retro-viruses have been characterized in detail. We havedescribedtheisolation of a non-condition-ally defective murine leukemia virus (MuLV) from a cultureof AKR lymphoma cells (21). The lesionin this MuLV appeared to be quite severe; although the virus was detected by its ability to induceXC plaque formation in the presence of an appropriate helper virus (the complementa-tion plaque assay) (22), cells containing the defective MuLVcontained no virus particles or clear virus-specific structures when they were examinedby electronmicroscopy (21).

This defective MuLVis alsounique inthatit was obtained from cultured AKR lymphoma cells. AKR micepossessanendogenous ecotro-pic MuLV, designated Akv, which is expressed

athighlevelsthroughout the life of the mice (27) and appears to play some causative role in the developmentof spontaneousthymic lymphomas in these mice (5, 7, 10, 14, 15). Since Akv is produced at high levels by normalAKRtissues and cell lines (27), it was surprising that itwas

not produced by cultures of AKR lymphoma cells (16,17). These findings raised thequestion of theorigin of the defective MuLV. One possi-bility is that it is a previously undescribed

en-dogenousvirus which is inheritedindependently from Akv and isexpressed specifically in thymic

orleukemic cells; alternatively, itmightbe de-rived from Akv in the somatic tissues of the mouse by mutation or recombination.

Further-more,the defectiveness ofthis virusmight

rep-resent asubstitution of nonviral sequences,asin

the mammalian sarcoma viruses.

In this paper we present apartial molecular characterization of the defective MuLV. This virus apparently synthesizes an env protein but nogag or pol gene product. The restriction map of the defectiveprovirus is quite similar to that ofAkv, and our results arefully consistentwith the hypothesis that the defective virus arose from Akv while replicating in the somatic tissues ofthe mouse. One difference fromAkv, which

occursin theextreme5'portion ofthe gag gene,

may be responsible for the lack of detectable gagorpol protein synthesis.

MATERIALSANDMETHODS

Cellsand viruses.Thegeneraltissue culture

proce-duresused in thisstudyhavebeendescribed

previous-ly (24). The S+L- focus assay, which detects all

nondefective MuLV's thatreplicate on mousecells,

and thecomplementation plaqueassay,which detects

replication-defectiveaswellasnondefectiveecotropic

MuLV'sby usingtheXC testin the presenceofan

XC-negativehelper virus,wereperformedasreported

previously(2, 22), except that thehelpervirus used in

thecomplementation plaqueassaywasMoloneyclone

83 (21).

The AKR2B cell line (26), aline of AKR mouse

626

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VOL. 41,1982

embryo fibroblasts which contain the Akvgenomebut

do not normally produce virus, was obtained from

Sisir K. Chattopadhyay, National Cancer Institute (NCI). MinkcellcloneCCL64wasobtained from Paul

Peebles (NCI) and was grownin Dulbecco modified

Eagle medium containing 10ofetal calfserum.

Feline leukemia virus(subgroup C)was agift from

Charles D. Sherr (NCI); baboon endogenous viruswas

obtainedfrom chronically infected Cf2Th cells, which

were kindly supplied by Ellen Cusick and George

Todaro (NCI).

Acloneof minkcellsthatwereproductively

infect-ed withWN1802N MuLV (BALB/c-S2N MuLV) (6)

wasoriginally isolatedby P. 0.Weislogel and R. H. Bassin (unpublished data). The minkcells were infect-ed with a phenotypically mixed stock ofWN1802N

MuLVwhich also contained xenotropic MuLV, and then cloned 1 day after infection. One clone isolatedin this experiment produced -10' XC plaque-forming units ofN-tropic MuLVperml, withapurely

ecotro-pic hostrange.This clonewasusedas acontrol in all

of theexperiments described below. Since identical

mapsfor WN1802N and Akvwerederived inarecent

study in which 12 restriction endonucleaseswereused (20), for convenience werefertothis cloneas

mink-(Akv) below. Although the DNA of this clone

con-tainedallof therestriction fragments expected in the Akvgenome, wealso detected additional fragments.It

appearsthatatleastoneother MuLV-relatedgenome,

possiblyadefective derivative of xenotropic MuLV, wasalsopresentin this cell line.

Molecular hybridization. Cellular DNA was

pre-pared by phenol-chloroform extraction, spooledoutof ethanol, treated with RNase, and reextracted with phenol and chloroform. Restriction endonuclease di-gestions,agarosegel electrophoresis,transferto nitro-cellulose membranes, hybridization to [32P]DNA probes, and autoradiography were performed as de-scribed previously (20, 29). 32P-labeled AKVprobes

were synthesized by nick-translation, using

Esche-richia colipolymerase and DNase (13), from plasmids containing the 8.2-kilobase-pair (kbp)PstIfragmentof Akv clone623(12) (i.e.,afragmentextending from 0.1

to8.3kbponthe8.8-kbp Akv genome)orthe1.9-kbp BamHI fragment of clone 623 (i.e., the fragment between 1.85 and 3.7 kbpontheAkv map) inserted intopBR322 (20). The latter probewas agenerousgift from Malcolm A.Martin, National Institute of Allergy and Infectious Diseases. Under the conditionswhich

we used, fragments of high-molecular-weight DNA

whichwere smallerthan 0.7kbpwere notvisualized routinely.

Radioimmunoprecipitation. All radioisotopes were

purchased fromNewEnglandNuclearCorp., Boston, Mass.Cellswerelabeledin minimal essential medium lacking labeledamino acid butsupplementedwith 1% dialyzedfetal calfserum.Afterlabeling,thecellswere

rinsed with cold phosphate-buffered saline and scraped into lysis buffer (0.02 M sodium phosphate, pH 7.5,0.1MNaCl,0.001 MEDTA,1% TritonX-100, 0.5% sodiumdeoxycholate,0.1% sodiumdodecyl

sul-fate). Then they were passed through a 19-gauge

needle, and theextractwasclarifiedby centrifugation

at6,000rpmfor 20mininaSorvall HS-4rotor. The resulting supernatant was incubated for 3 h with

normal goat serumand Formalin-fixed

Staphylococ-gag pol env'MuLV 627

cus aureus: this mixture was centrifuged for 3 h at

40,000 rpm in a Beckman 50Ti rotor.

A total of 2 x 106 acid-precipitable counts was precipitated with goat antisera to Rauscher MuLV proteins (provided by the Division of Cancer Cause and Prevention, NCI), and the immune precipitates were collected and washed as described previously (31) by using protein A-Sepharose (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.). The precipitates were removed from the beads by boiling in sample buffer(1.6%sodium dodecylsulfate, 0.01 MTris, pH 6.8, 15% glycerol, 2% mercaptoethanol, 0.002% brom-phenol blue). They were then analyzedby electropho-resis on 5 to 15% gradient polyacrylamide gels (9) and visualized by fluorography (4).

Thespecificity of the anti-p15 serum was tested as follows. Cells that produced an Akv type of MuLV (isolate WN1802B) were labeled overnight with

[3H]leucine and [3H]lysine. Virus was partially

puri-fied from the supematant of this culture by pelleting through 10% sucrose. Thepellet was then dissolved in sample buffer. A sample of this preparation was

pre-cipitatedwith theanti-p15 serum and analyzed on a 7

to17% gradient gel; only one protein, whosemobility indicated an apparent molecular weight of 15,000, was presentin this immune precipitate.

Radioimmunoassays. Competition radioimmunoas-says were performed by double-antibody precipita-tion, as previously described (28). The presence of viralgp70 was determined by using iodinated Friend MuLVgp7O and goat anti-AKR MuLV serum obtained from the Division of Cancer Cause and Prevention, NCI. Viral p30 was detected by using iodinated Rauscher MuLVp30 and goat anti-AKR p30 obtained from the Division of Cancer Cause and Prevention, NCI. The presence of viral p12 was determined by

using iodinatedAKR MuLV p12 anda rabbit

antise-rumto AKRp12that wasa kind giftfromJamesIhle, Frederick Cancer ResearchCenter.The levelof viral

protein expressionwas calculated onthe basis of the

amountof total cellularprotein(11) and thesensitivity ofeachassay andis expressed as nanograms of viral protein per milligram of total cellular protein.

RESULTS

Transmission of thedefective MuLV genometo

mink cells. In a previous report, we described

the isolation ofaclone of 3T3FL mouse cells,

(designated AK24)which contained the genome

of the defective MuLV produced by AKRSL2

cells (21). Although this genome was readily detectable in this clone by an infectivity assay

for defective ecotropic MuLV's,we noted that

novirus-likestructures wereevident inelectron

micrographsof these cells. We wished to devel-op arestrictionmapoftheviralgenome and also to determine whetherthis genome directed the

synthesis of any known MuLV-specific pro-teins. However, thepresence ofother,

endoge-nous MuLV genomes in the 3T3FL host cells

greatly complicated these studies; therefore,we

attempted to transmit the defective MuLV to

nonmurine cells. Thedefective MuLV was

res-cuedfrom clone AK24 cells by superinfection

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628 REIN ET AL.

with amphotropic MuLV, as described

previ-ously (23). Mink cells were infected with the

resulting virusstockatamultiplicity of infection

of0.2complementation plaque-forming unitsper

cell,and the cellswereclonedonthefollowing

day.Individual cloneswerepicked and screened

for the presence ofdefective ecotropic MuLV

bycocultivation with cells that produceda help-er virus, as described previously (21); two

clones, designated MAK26 and MAK71, were

positive in these preliminarytests.These clones

werethensuperinfected withaseriesof

nonde-fective,XC-negative viruses. Infectivity assays

ofthe viruses produced by these superinfected

cells (Table 1) demonstrated that these two

clones were indeed nonproducer clones since,

like clone AK24, they contained a rescuable,

defectiveecotropic virus which registered in the complementation plaqueassay.

Restriction mapping of the integrated MuLV

genome inclonesMAK26 and MAK71.To

com-pare the defective MuLV genome with the genome of Akv, we isolated

high-molecular-weight cellular DNAs from clones MAK26 and

MAK71, from mink(Akv) cells, from AKR2B mouse cells, which also contain the Akv

genome, and from uninfected mink cells. Hirt

supernatant DNAfrom cells that wereacutely

infected with Akvwasalsoprepared (20).These

DNAswerethendigested with restriction

endo-nucleases, subjected toagarose gel

electropho-resis, transferred to nitrocellulose filters, and hybridized with a 32P-labeled probe that was

representative of the full Akvgenome(PstI

8.2-kbp clone 623DNA). Figure 1A, lanesathrough

fshow the resultsobtained byPstI digestion of

these DNAs. PstI cleaves endogenousecotropic MuLVgenomesonly inthelongterminal repeat

(LTR), sothatdigestion ofafull-lengthproviral

DNA molecule generated an 8.2-kbp fragment

(Fig. 2). As Fig. 1A,lanes aand b show, PstI

digests of MAK26 andMAK71 DNAs did

con-tainan8.2-kbp moleculewhichhybridizedwith

the MuLV probe. As expected, this fragment

wasalso presentintheDNApreparations

con-taining the Akvgenome(Fig. 1A,lanesc, e,and

f) butnot inthe preparationfrom normal mink

cells (lane d). These results suggest that the

defective MuLV genome is approximately the

samesizeasthe standard Akvgenomeandthat it alsoresemblesAkv incontaining PstI sites inits

LTR.

TheAkvgenomeiscleavedoncebyHindIIIat

3.0kbp(measuredfrom the 5' end of the provi-rus) and once byXbaI at 7.7 kbp. To test the

defective MuLV genome for the presence of

these cleavage sites, we digested MAK26 and

MAK71 DNAswithHindIII plusPstI and with

XbaIplus PstI.AsFig. 1A,lanesgand hshow, HindIII-PstI digestion of these DNAs yielded fragments 3.0 and 5.2 kbp long, just as was

observedwithAkv(lanesi andk). Similarly,the

XbaI-PstI digests ofthese DNAs contained a

large fragment (Fig. 1A, lanes m and n) which

comigratedwith the 7.7-kbp fragment obtained

from Akv(lanesoandq).Theseresults indicate

that the defective MuLVgenomealsoresembles

the Akv genome in theplacement ofaHindIII

site andanXbaI site(Fig. 2).

Inaddition,digestionof MAK26 andMAK71

DNAswith EcoRI and PstIgaverisetoan

[image:3.490.59.448.452.607.2]

8.2-kbp fragment,justas wasfoundwithAkv; thus,

TABLE 1. Rescueofcomplementationplaque-forming units from clones MAK26 and MAK71

Cellline Superinfectiona CPFU/mlb FIU/mlc

MAK26 None <1 x 100 <1 x 100

AmphotropicMuLV 4x 104 5 x 105

Moloney clone 83 4x 103 3 x 10'

MAK71 None <1 x 100 <1 x 100

AmphotropicMuLV 8 x 104 1 X 106

Moloney clone 83 4 x 104 6x 105

Felineleukemia virus 3 x l0 NDd

Baboonendogenousvirus 1.6 x 103 ND

Mink None <1 x 100 <1 x 100

AmphotropicMuLV <1 x 100 8 x 105

Moloneyclone 83 <1 x 100 2 x 105

Feline leukemia virus <2 x 100 ND

Baboonendogenous virus <2 x100 ND

aCultures of MAK26

cells,

MAK71 cells, anduninfected mink

cells

weresuperinfected with amphotropic

MuLV, Moloney clone 83, feline leukemia virus, or baboon endogenous virus. After5days the culture fluids

from these preparations were assayed for replication-defective ecotropic MuLV by the complementation plaque assayand fornondefectiveMuLVbythe S+L- focus assay.

bCPFU, Complementationplaque-formingunits. cFIU,S+L-focus-inducing units.

dND, Notdetermined.

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gag- pol- env'MuLV 629

a b c d e f g h i i kI m n o p q r

23.7- * 9.5

-6.6

4.3

- 2.2-1.9

-0.6

-a

A

a b c d e f g h k rTl t} 0 0q

9.5 -4

66---- @ 4.3 - 10

Ii

2.2..,..

1t 9

06-C

FIG. 1. Southerngel analysisofproviralDNA. DNAswereextracted, digested,fractionatedbyagarosegel electrophoresis,blottedontonitrocellulosefilters,andhybridizedasdescribed in thetext.TheDNAs usedwere

cellular DNA of MAK26 cells(lanesa,g,andm),cellular DNA of MAK71 cells(lanes b, h,andn),cellularDNA ofmink(Akv)cells(lanesc,i,ando),cellular DNA of uninfected mink cells(lanes d, j,andp),cellular DNA of AKR2B cells(lanes f, 1,andr),and Hirt supernatant DNA from NIH/3T3 cellsacutelyinfectedwith Akv(20) (lanese,k,andq). (A)DNAsdigestedwith PstI(lanesathrough f),PstIplus HindIII (lanesgthrough 1),and PstI

plusXbaI(lanesmthrough r). (B)DNAsdigestedwith BamHI(lanesathroughf)andKpnI (lanesgthrough 1). (C) DNAs digested with BamHI (lanesathroughf), BamHIplus KpnI (lanesgthrough 1), andBamHI plus HindlIl (lanesmthrough r).The DNAswerehybridizedwith 32P-labeledPstI8.2-kbpclone623(AandB)or32p_ labeled BamHI 1.9-kbp clone623 (C). The molecularweightmarkers shownatthe left ofeachpanel were a

HindIII digest of 32P-labeled A DNA,and thenumbers represent thelengths (inkilobasepairs)ofthese markers. Thefragnents foundinuninfectedmink cells representmaterial whichhybridizeswith MuLVprobesandwere

notcharacterized further. Although the hostrangeof the infectious MuLVproduced bythemink(Akv)clone used is purely ecotropic, the restriction analysisshown here(panelA, lanescandi; panel B,lanec;panel C, lanescandi)indicates thatmorethanonetypeof MuLVgenomeisactuallypresentin thesecells,asnoted in the text.

EcoRI does not cleave the defective MuLV

genome(datanotshown).

We also investigated the defective MuLV

genome by using digestion with BamHI. This

enzyme cuts Akv four times, yielding internal

fragments that are 3.0, 1.9, and 0.4 kbp long.

Digestion of MAK26 and MAK71 DNAs also produced these fragments (the 0.4-kbp fragment

wasinferred, since it is located between the

1.9-and 3.0-kbp fragments), exceptthat the 1.9-kbp

bandmigrated slightly faster(1.85 kbp) than the

1.9-kbp band from Akv (Fig. 1B, lanesathrough

a b c d e f g h k

S

r-;

X0.

- 0.6

--U,

B

6 a

eav

U, _

0

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630 REIN ET AL.

KsLb

0 1 2 3 4 5 6 7 8 8.8

B HKBB

,,Il

KDB HKB B

50bp Deletion

BKX P K

I Akv MuLV

BKX P K

I I I IFh (J Defective MuLV 50bp

Addition

FIG. 2. Restrictionmapof thedefectiveMuLV.The restrictionmapofthedefectiveMuLVinMAK26 and MAK71 cellsis compared with that of Akv (20).P,PstI; K, KpnI; B, BamHI; H,HindIII;X,XbaI. Therewere

noEcoRIcleavage sites.

c). (These1.85-kbp fragments in Fig. 1B, lanesa

and b, which ran ahead ofthe 1.9-kbp marker

and the 1.9-kbp fragment in lanes c, e, and f,

werepoorly visualized because theywere

super-imposed on a 1.8-kbp endogenous mink

frag-ment. Theywereclearlyvisualized withaprobe

that was specific for this region of thegenome

[Fig. 1C, lanes a and b].) This difference is

discussedbelow.

We alsodigested the defective MuLVgenome

with KpnI. KpnI cleavage of Akv yields three

internal fragments, which are 3.9, 2.8, and 1.4

kbp long. As Fig. 1B, lanesgand h show, KpnI

digestion of MAK26 and MAK71 DNAs gave

risetofourfragments, whichwere

approximate-ly 3.9, 2.0, 1.4, and 0.8 kbp long. The fact that

the 2.8-kbp fragment of Akv was replaced by

fragments that were 2.0 and 0.8 kbp long

sug-gests that the defective MuLVgenomecontains

allof theKpnI sites of Akv, but also hasaKpnI

site within the 2.8-kbp fragment. Since this

additionalKpnI site is apparently 0.8 kbp from

one end of the 2.8-kbp fragment, it is located

either 1.3 or 2.5 kbp from the 5' end of the

provirus (see Fig. 2).

This additional KpnI site was localized in

further studies by using the 1.9-kbp BamHI

fragment of Akvas aprobe. This fragmentspans

theregion between 1.85 and 3.7 kbp in Akv. As

Fig. 1C,lanes aand bshow, this probe reacted

only with the -1.9-kbp fragment of

BamHI-digested MAK26 and MAK71DNAs,as

expect-ed.Ifthe added KpnI site in these DNAswere

located in the 3' portion of the 2.8-kbp KpnI

fragment (i.e., at 2.5 kbp), then KpnI would

cleave the1.9-kbpBamHIfragment twice,

yield-ingBamHI-KpnI fragments thatare0.7, 0.8, and

0.4kbp long. On the otherhand, if the additional

KpnI site in the defective MuLVgenome werein

the5' halfofthe 2.8-kbp KpnIfragment (i.e.,at

1.3 kbp), then KpnI cleavage of the 1.9-kbp

BamHI fragment wouldgenerate asingle large

fragment that is -1.4 kbp long plus a 0.4-kbp

fragment, justasoccurswithcleavage of the Akv

genome.The results of theBamHI-KpnI double

digestion (Fig. 1C,lanesgandh) supported this

latter prediction and thus indicate that the

addi-tionalKpnIcleavage site of the defective MuLV

genome is at 1.3 kbp (Fig. 2). This conclusion

wasalsoconfirmed by digestion with KpnI plus

HindIII. We found (datanotshown) that HindIII

cleaved the 2.0-kbp KpnI fragment in MAK26

and MAK71 DNAs; since this KpnI fragment

therefore includes theHindIII siteat3.0kbp, it

mustextend from 1.3to3.3kbp rather than from

0.5to2.5kbp.

We also detected two other differences

be-tween the defective MuLV and Akv in these

studies. First, the -1.4-kbp KpnIfragmentwas

slightly larger in MAK26 and MAK71 DNAs

than in theAkv-containing control DNAs used;

weestimate that itwas 1.45kbplong (Fig. 1B,

lanes gand h). Double digestion with PstI and

KpnI localized this -50-base-pair (bp) size

dif-ferencetothe 3' side of the 3' PstI site(i.e., in

the LTR) (datanotshown).

Second, as notedabove, the 1.9-kbp BamHI

fragment migrated slightly faster in MAK26 and

MAK71 DNAs than in Akv (Fig. 1C, lanes a

through c). Thus, this region of the defective

MuLVgenomeappearedtobeslightly (-50 bp)

shorter than the corresponding region ofAkv.

Further information on this difference was

ob-tainedby double digestion ofthe cellular DNAs

and hybridization with the 1.9-kbp BamHI

probe. As Fig. 1C shows, this difference was

presentin the -1.4-kbpBamHI-KpnI fragment

(Fig. 1C, lanesgthrough i)andwasalsopresent

in the 1.2-kb BamHI-HindIII fragment (lanesm

through o). These findings show that the

differ-enceis in theregionbetween theBamHI siteat

1.85 kbp and the HindIII siteat 3.0kbp inthe

defective MuLV genome (Fig. 2). The results

shown in Fig. 1C also suggestthat this region

from MAK71 DNA may have been slightly

shorter than the corresponding region from P K

PK

(

50bp Addition

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Vgag

pol env'MuLV 631

MAK26 DNA,but this differencewasnot repro-ducibleinothergels.

MuLV-specificproteins in MAK26 and MAK71 cells. Theanalysisof the defective virus genome by restriction mapping revealed three major differences between this genome and Akv. As discussed above, the defective genome has a slightly larger LTR, an additional KpnI site at 1.3 kbp, and a small deletionbetween 1.8 and 3.0kbp. We decidedtodeterminewhether any ofthese abnormalities in the genomewas reflect-ed in an aberrant pattern of virus-specific pro-teinsynthesisin MAK26 and MAK71 cells and whether defects in the viral proteins could ac-countfor the inability of the defectivevirus to replicate itselfin the absence ofahelpervirus (21).

Initially, MAK26and MAK71 cells were

ex-amined for the presence ofMuLV-specific pro-teins as follows. Cultures of these cells were

pulse-labeled with

[35S]methionine;

as controls, uninfected mink cellsand mink(Akv)cellswere

labeledinparallel.Cytoplasmicextractsofthese cultureswere thenprecipitated with antisera to allofthemajorMuLVproteins,and the labeled proteins that bound to the antisera were

ana-lyzedbysodiumdodecylsulfate-polyacrylamide gel electrophoresis.Figure 3 shows the results of onesuchexperiment, inwhichthefourextracts

were exposed to normal goat serum (Fig. 3, lanes 1 through 4), anti-plO (lanes 5 through 7 and 9), anti-p12 (lanes 10 through 13), anti-reversetranscriptase(lanes14through 17),

anti-1 2 3 4 5 6 7 8 9101112131415

68

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p30 (lanes18through21), andanti-p1S(lanes 22 through 25). This figure showsthat with eachof these sera, the MAK26 and MAK71 extracts (Fig. 3, lanes 2, 3, 6, 7, 11, 12, 15, 16, 19, 20, 23, and24) were indistinguishable from the extract ofuninfected mink cells (lanes 1, 5, 10, 14,

18,

and 22). In contrast, M5gag and Prl80gagPoI were both readily detected with all of the im-mune sera in the productively infected cells (lanes 9, 13, 17, 21, and 25); gPr80gag and a

40,000-dalton gag cleavage intermediate were also visible in several of these preparations. When the MAK26 and MAK71 extracts were precipitated with anti-gp7l, there was a band at 82,000 daltons (data not shown); this env precur-sor isdiscussed below.

The results shown in Fig. 3 suggest that the

gag

proteinsand the reverse transcriptase areall completely absent from MAK26 and MAK71 cells. However, these results did not eliminate the possibility that p15 was present in these cells, since the p15 encoded by Akv contains no methionine and hence would not be detected in these methionine-labeled extracts (18). This seemed particularly important since p15 is the N-terminal protein in PrO5gag and many defec-tive viruses contain an N-terminal fragment of the gag gene product (1, 8, 19, 25, 32). Accord-ingly, we pulse-labeled MAK26 cells, MAK71 cells, and controls with [3H]leucineand [3H]ly-sine. Extracts of these cells were precipitated withmonospecificanti-p15 serum; Fig. 4 shows gels displaying the precipitated proteins. This

617 18192021 22232425

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28-9-68

-28 -12.3

FIG. 3. Analysis of[35S]methionine-labeledminkcells for MuLV-specific proteinsby radioimmunoprecipita-tion. Cellswerelabeledfor 30 min withL-[35S]methionine (250,uCi/ml)andprocessedasdescribed in thetext. Extracts of minkcells(lanes 1,5,10, 14,18, and22), MAK26 cells (lanes 2, 6, 11, 15, 19,and23),MAK71cells (lanes 3, 7, 12, 16, 20, and 24), andmink(Akv) cells (lanes 4, 9, 13, 17, 21, and 25)wereprecipitated with normal goat serum (lanes 1 through 4), anti-plO (lanes 5, 6, 7, and 9), anti-p12 (lanes 10 through 13), anti-reverse transcriptase (lanes 14through 17), anti-p30 (lanes 18 through 21), and anti-p15 (lanes 22 through 25). The

followingmolecularweight markerswereused:bovineserumalbumin(68,000), carbonicanhydrase(28,000), and

cytochromec(12,300).

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632 REIN ET AL.

1 2 4 5 6

*.*,

68

46 -28 12.3 FIG. 4. Analysis of 3H-labeled mink cells for MuLV-specific proteins by radioimmunoprecipitation. Cellswerelabeled for 30 min with L-[3H]leucine and

L-[3H]lysine (50 ,Ci/ml each) and processed as

de-scribed in thetext. Extractsof mink cells (lanes1and 5), MAK26 cells (lanes 2 and 6), MAK71cells(lanes 3 and 7), and mink(Akv) cells (lanes 4 and 8) were

precipitated with anti-p15 (lanes 1 through4)and anti-gp71 (lanes 5 through 8). The following molecular weight markerswereused:phosphorylase b (92,500), bovine serum albumin (68,000), ovalbumin (46,000), carbonic anhydrase (28,000), and cytochrome c

(12,300).

figure shows thatanti-p15 precipitated Pr6g5ra, gPr80a9, andPrI8099arPlfrom theproductively infected cells (Fig. 4, lane 4), but that nothing

wasprecipitated fromMAK26 and MAK71 cells

(lanes 2 and 3). In addition, the precipitates

obtainedfrom3H-labeled cell extractsby using

this antiserumwere run on a7to17%

polyacryl-amidegradient gel. Undertheseconditions,

pro-teins considerably smaller than p15 were

re-tained on the gel. As in Fig. 4, no proteins

precipitated from MAK26 and MAK71 cells

were detected on this gel (data not shown). Based on these results, we conclude that

MAK26 and MAK71 cellscontainnogag orpol

geneproducts. (The possibility thatvery small fragments of p15 or other gag proteins were

presentcouldnotbeexcluded.)

Figure4also shows the results ofprecipitation

of these 3H-labeledcellextractswithanti-gp7l.

As shown inFig. 4, lanes 6 and 7, aprecursor was readily detected at a molecular weight of

approximately 82,000.Theamountandmobility

of this protein were similar to the amount and

mobility of the wild-type protein (Fig. 4, lane8). Thus, MAK26 and MAK71 cells do contain the env gene product gPr82env.

As anindependent approach to the question of which MuLV proteins are present in MAK26 and MAK71 cells, we also tested extracts of thesecellsfor p30, AKR-type p12, and gp7l by competition radioimmunoassays. As Table 2 shows, these cells were negative for the gag proteins but did contain substantial levels of proteinswith gp71 determinants, as determined by these assaysaswellasby radioimmunopreci-pitation.

DISCUSSION

Previously, we reported the isolation of a

replication-defective MuLV from the AKRSL2 line of cultured AKR leukemia cells(21). In this work we analyzed thedefectivenessof thisvirus in molecular terms andcompardit withAkv,the endogenous ecotropic virus ofAKR mice. We studied the genomes of these two MuLV's by mapping with a variety of restriction endonucle-ases. Three relatively small differences were found(Fig.2).First,thedefective virus contains aKpnI cleavagesite at 1.3kbpfrom the 5' endof the provirus, whereas this site is not found in Akv; second, the defective genome is approxi-mately 50 bp shorter than Akv in the region between 1.8 and 3.0 kbp from the 5' end; and third, the LTRof the defective genome is -50 bp longerthan the LTRof Akv.

Itis not known whether the defective genome differsfrom the genomeofAkvbyonlyasingle base pair at 1.3 kbp or whether the sequences around the additionalKpnI sitearealsodifferent fromthe sequences in the corresponding region ofAkv. Since relatively small restriction

frag-mentsfromthisregionweredetectedby

hybrid-ization with MuLV probes, the majority ofthe sequences in this part of the defectivegenome

are clearly MuLV related. We estimate that a

[image:7.490.54.242.58.293.2]

substitution of nonviralsequencesin thisregion could span 400bpatmost.

TABLE 2. Analysis of MAK26 and MAK71 cells for MuLVproteins by competition

radioimmunoassay

Amtof:a

Celiline

p12 p30 gp7l

Mink <1 <1 <10

MAK26 <1 <1 531

MAK71 2 <1 288

Mink(Akv) 750 ND" 1,380

a Results are expressed as nanograms of MuLV

proteinper milligram ofcellularprotein.

"ND, Not determined.

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gag pol env' MuLV 633

As noted above, the defective MuLV could have originated from Akv in the somatic tissues of the mouse which gave rise to the AKRSL2 leukemia cell line, or it could be a distinct endogenous virus. The fact that the region be-tween 1.8 and 3.0 kbp is -50 bp shorter in the defective genome than in Akv shows that, if the defectivegenome is derived fromAkv, then its originmusthave involved deletionsor recombi-national events or both, not just single-base changes.

Finally, the defective genome differs from the Akv isolates used as controls in these experi-ments with respect to the length of the LTR. However, recently the endogenous ecotropic MuLV'shave beenfound to be polymorphic in this regard. The LTR of the defective virus is the same size as the LTRs in other nondefective MuLV's (20); thus, its size is fully consistent with normal LTR functions, such asreplication, integration, and transcription of the viral DNA. Thefact that the defective MuLVreplicates to a hightiter in the presence of a helper virus (Table 1)(21) suggests thatallthree ofthese functions are performed efficiently by the defective genome. In particular, the efficient rescue ob-served implies that full-length,genomicRNAis present at normal levels in the cytoplasm of infected cells, whereas the production of env

protein in nonproducercells(Fig.4and Table2) suggests that the env message is synthesized, processed, and translatednormally.

We conclude that the defective MuLV pro-duced by AKRSL2 cells has arestriction map which issimilarbut notidentical to that of Akv. These results areconsistent with the hypothesis that the defective MuLV was derivedfromAkv, but do not completely exclude the possibility that this virus is a distinct endogenous virus. Furtherstudies will berequiredbeforetheorigin and significance of the defective virus can be established. (We also cannot exclude the possi-bility that the defective viral genome was changed by mutation or recombination in the tworeplicative cycles which occurred between its releasefromAKRSL2cellsand itsanalysisin MAK26and MAK71 cells.)

Wealso tested cells that contained the defec-tive MuLV forvirus-specific proteins.No prod-ucts of the gag or pol genes were detected in

thesecells (Fig. 3and 4and Table2). This defect

obviously accounts for the inability of the defec-tiveMuLVtoreplicate in the absence ofahelper virusand forthe absence of any particles visible by electron microscopy (21).

As discussed above, the defective MuLV genome containsaKpnIcleavage site at 1.3 kbp, whichis notpresent in nondefective Akv. Since

thegag coding regionisbelieved to begin at 1.1

kbp (C. van Beveren and I. Verma, personal

communication), this cleavage site is probably in the middle of the p15 gene. One possible expla-nation forth'e absence of any protein that was precipitable with ouranti-p15 serum is that the abnormal nucleotide sequence at 1.3 kbp intro-duces aframeshift or termination codon into the gene. In this case a protein containing an N-terminal portion of p15 would be synthesized. Our failure to detect thisprotein might be due to its small size orinstability; it is also possible that this p15 fragment lacks antigenic determinants recognized by ourantiserum. Alternatively, the defectivegenome could also be altered upstream from 1.3 kbp, so that gag translation is com-pletely prevented by a frameshift or termination codon or by some other alteration.

Althoughcellscontaining the defective MuLV appear to lack gag and polproteins,theyclearly containanenv geneproduct. The presence ofan

env gene product was not unexpected, since cellscontaining both the defective MuLV andan

XC-negative helper virus fuse XC cells (21). Several lines of evidence (23, 30, 33) suggest that XC cell fusion is a property of ecotropic env molecules; our present finding that the defective virus that exhibits this phenotype apparently codes onlyfor the envproteinconstitutes strong additional support for this conclusion. (The re-quirement for coinfection with a nondefective helper virus is not yet understood). We also observed that when the defective MuLV was rescuedbyahelper virus which normally is not able toinfect mouse cells, such asfeline leuke-mia virusorbaboon endogenous virus, the re-sulting pseudotype particles registered in the standard complementation plaque assay on mouse cells (Table 1). It seems likely that the defective genome encodes afullyfunctional env protein, which can be incorporated into virions and gives virions the ability to infect mouse cells. Further analysis of this protein is now underway.

Besmeretal. (3) have described amutant of Moloney MuLVwhich synthesizesan env pro-tein and a 45,000-dalton fragment of the gag geneproduct.To ourknowledge, however,this is the first description of a nontransforming mammalian retrovirus genome which synthe-sizes a complete envprotein but no detectable gag orpol geneproduct. Itshould benotedthat a defective virus of this type would not be detectedbymostvirus assays, sinceitdoesnot

produce virus particles, has no DNA

polymer-ase activity, and is negative in sensitive

immu-nologicaltests forthe viral coreproteins(Table 2). Cellscontaining this type of defective virus shouldbeusefulreagents foravarietyofgenetic experiments. Inaddition, theyshouldprovidea

unique opportunity to analyze the processing

andsubcellularlocalization ofthe envproteinin

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634 REIN ETAL.

the absence ofgag proteins and thus to deter-mine the role, if any, of gag proteins in env

maturation.

ACKNOWLEDGMENTS

We thank Anne Soria, Melody McClure, Elaine Rands, Wen-PoTsai, AliceBrown,andBrendaWallaceforexcellent technical assistance, Richard Mural and Alan Schultz for helpful discussions, Sisir Chattopadhyay forcritical contribu-tions,and JeannieClarke forpreparationof themanuscript.

LITERATURECITED

1. Barbacdd,M.,J. R.Stephenson,and S. A.Aaronson.1976. gag gene mammaliantype-CRNA tumorviruses. Nature (London)262:554-559.

2. Bassin, R. H., N. Tuttle, and P. J.Flschinger. 1971.Rapid cell culture assay for murine leukaemia virus. Nature (London)229:564-566.

3. Besmer,P.,H.Fan, M.Paskind, and D. Baltimore. 1979. Isolationandcharacterization ofamousecell line contain-ingadefectiveMoloney murine leukemia virusgenome. J. Virol.29:1023-1034.

4. Bonner, W. M., and R. A. Laskey. 1974. Afilmdetection methodfortritium-labeliedproteinsandnucleic acidsin polyacrylamide gels.Eur.J. Biochem. 46:83-88. 5. Furth, J., H. R. Seibold, and R. R. Rathbone. 1933.

Experimental studiesonlymphomatosisofmice.Am.J. Cancer19:521-604.

6. Hartley, J.W., W. P. Rowe, and R. J. Huebner. 1970. Host-range restrictions of murine leukemia viruses in mouseembryo cellcultures.J.Virol. 5:221-225. 7. Huebner, R. J., R. V.Gilden,R.Toni, R. W.Hill,R. W.

TrImmer, D. C.Fish, and B.Sass. 1976. Prevention of spontaneous leukemia in AKR mice by type-specific immunosuppression of endogenous ecotropicvirogenes. Proc. Natl.Acad.Sci. U.S.A.73:4633-4635.

8. Khan, A. S., and J. R.Stephenson.1977. Feline leukemia virus: biochemical and immunologicalcharacterizationof gaggene-codedstructuralproteins.J.Virol.23:599-607. 9. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature(London) 227:680-685.

10. Lilly,F., M. L.Duran-Reynals, andW. P. Rowe. 1975. Correlationofearly murineleukemia virus titer and H-2 typewithspontaneousleukemiainmice oftheBALB/cx

AKR cross:ageneticanalysis.J.Exp.Med.141:882-889. 11. Lowry,0.H., N. J.Rosebrough, A.L.Farr,andR.J. Randall.1951.Proteinmeasurementwiththe Folinphenol

reagent.J. Biol. Chem.193:265-275.

12. Lowy, D. R., E. Rands, S. K.Chattopadhyay,C. F.Garon, andG. L. Hager. 1980. Molecularcloningof infectious murine leukemiavirus DNAfrom infected mousecells. Proc.Natl. Acad.Sci. U.S.A.77:614-618.

13. Maniatis, T., A.Jeffrey,and D.G. Kleid.1975. Nucleotide sequence oftherightwardoperator ofphageX.Proc. Natl. Acad.Sci. U.S.A.72:1184-1188.

14. Mayer, A., M. L.Duran-Reynals,and F.Lilly.1978. Fv-1 regulation of lymphoma development and of thymic, ecotropic,andxenotropicMuLVexpressionin mice of theAKR/J x RF/J cross.Cell15:429-435.

15. Nowinski, R.C.,M.Brown, T. Doyle, andR. L.Prentice. 1979. Genetic and viral factorsinfluencingthe develop-mentof spontaneous leukemia in AKR mice. Virology 96:186-204.

16. Nowinski, R. C., and E. F. Hays. 1978.Oncogenicityof AKRendogenousleukemia viruses. J.Virol. 27:13-18. 17. Nowinski, R.C., E. F.Hays, T.Doyle, S.Linkhart,E.

Medeiros, andR.Pickering. 1977. Oncornaviruses

pro-duced by murine leukemia cells in culture. Virology 81:363-370.

18. Oroszlan, S., and R. V. Gilden. 1980. Primary structure analysis of retrovirus proteins, p. 299-344. In J. R. Stephenson (ed.),MolecularbiologyofRNA tumor virus-es.Academic Press, Inc., New York.

19. Oskarsson, M. K., J. H. Elder, J. W. Gautsch, R. A. Lerner, and G. F. Vande Woude. 1978. Chemical determi-nation ofthe MI Moloney sarcomavirus pP60"0' gene order:evidence forunique peptidesin thecarboxy termi-nus of the polyprotein. Proc. Natl. Acad. Sci. U.S.A. 75:4694-4698.

20. Rands, E., D.R.Lowy, M. R. Lander, and S. K. Chatto-padhyay. 1981.Restrictionendonuclease mappingof

eco-tropicmurine leukemiaviral DNAs: size and sequence heterogeneity of the long terminal repeat. Virology 108:445-452.

21. Rein, A., E. Athan, B. M.Benjers,R. H. Bassin, B. I. Gerwin,andD. R.Slocum. 1979.Isolation ofa replication-defective murine leukaemia virus from cultured AKR leukaemia cells.Nature(London)282:753-754. 22. Rein, A.,andR. H.Bassin. 1978. Replication-defective

ecotropicmurineleukemia viruses: detectionand quanti-tation ofinfectivity using helper-dependent XC plaque formation. J. Virol.28:656-660.

23. Rein, A., B. M. Benjers, B. I. Gerwin, R. H.Bassin,and D. R. Slocum. 1979.Rescue and transmission of a replication-defective variant of Moloney murine leukemia virus. J. Virol. 29:494-500.

24. Rein, A., B. I. Gerwin, R. H.Bassin, L. Schwarm, and G. S. Schidlovsky. 1978. A replication-defective variant of Moloney murine leukemia virus. I. Biological character-ization. J. Virol. 25:146-156.

25. Reynolds, F. J., Jr., T. L. Sacks, D. N.Deobagkar,and J. R. Stephenson. 1978. Cellsnonproductivelytransformed byAbelsonmurineleukemia virus express a higher molec-ular weight polyprotein containing structural and non-structural components. Proc. Natl. Acad. Sci. U.S.A. 75:3974-3978.

26. Rowe, W. P.,J. W. Hartley, M. R. Lander, W. E. Pugh, andN.Teich. 1971. NoninfectiousAKR mouse embryo cell lines in which each cell has the capacity to be activated to produce infectious murine leukemia virus. Virology 46:866-876.

27. Rowe, W. P.,and T. Pincus. 1972. Quantitative studies of naturally occurring murine leukemia virus infection of AKR mice. J.Exp. Med. 135:429-436.

28. Scolnick, E. M., W. P. Parks, and D. M. LivIngston. 1972. Radioimmunoassayofmammalian typeC viralproteins. I. Speciesspecific reactions of murine and feline viruses. J. Immunol. 109:570-577.

29. Southern, E. M. 1975. Detection of specific sequences among DNAfragmentsseparatedbygelelectrophoresis. J. Mol. Biol. 98:503-517.

30. Troxler,D.H., D.Low,R.Howk, H. Young, and E. M. Scolnick. 1977. Friend strain of spleenfocus-formingvirus is arecombinant betweenecotropic murine type C virus and theenvgeneregion of xenotropic type C virus. Proc. Natl. Acad. Sci.U.S.A. 74:4671-4675.

31. Versteegen, R.J.,andS. Oroszlan. 1980. Effect of chemi-calmodificationandfragmentationonantigenic determi-nants ofinternal protein p30 and surface glycoprotein gp7OoftypeCretroviruses. J.Virol. 33:983-992. 32. Witte, 0. N., N.Rosenberg, M. Paskind, A.Shields,and D.

Baltimore. 1978. Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblasts and lymphoid cells. Proc. Natl. Acad. Sci. U.S.A. 75:2488-2492.

33. Zarling, D. A., and I. Keshet. 1979. Fusionactivity of virions of murineleukemia virus. Virology 95:185-1%.

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Figure

TABLE 1. Rescue of complementation plaque-forming units from clones MAK26 and MAK71
FIG. 1.electrophoresis,cellularHindlIlAKR2Bof(C)(lanespluslanesusednotlabeledHindIIIThe mink(Akv) Southern gel analysis of proviral DNA
FIG. 2.MAK71no Restriction map of the defective MuLV. The restriction map of the defective MuLV in MAK26 and cells is compared with that of Akv (20)
FIG. 4.weightbovinecarbonicgp71MuLV-specificCellsprecipitatedand5),[3H]lysinescribed Analysis of 3H-labeled mink cells for proteins by radioimmunoprecipitation

References

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