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 minkcells
weresuperinfected with amphotropicMuLV, 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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[image:4.490.46.447.58.446.2]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
(
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[image:5.490.110.391.65.181.2]Vgag
pol env'MuLV 631MAK26 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)cellswerelabeledinparallel.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. This617 18192021 22232425
-..nf ..
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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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[image:7.490.256.451.558.642.2]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
VOL.41, 1982
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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.
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