JoURNALorVIROLOGY, Nov. 1974, p. 1245-1252
Copyright X 1974 American Society for Microbiology PrintedVol. 14, No. 5inU.SA.
Expression of the Major Internal Viral Polypeptide in Cells
Transformed
by Wild-Type and Temperature-Sensitive Murine
Sarcoma Virus
GAD SPIRA, GORDON R. DREESMAN,MATILDA BENYESH-MELNICK, SAUL KIT,ANDKENNETH
D. SOMERS 1
Department of Virology and Epidemiology and Division of Biochemical Virology, Baylor College of Medicine, Houston, Texas 77025
Received forpublication13 May 1974
Phenotypic expression of the murine intraspecies and interspecies antigenic
determinantsofthe major type C viral structural 30,000-dalton polypeptide,p30,
was measured by radioimmunoassay inhibition in cell lines from different
species. Uninfected normal rat kidney (NRK) cells did not contain detectable
levels of murine intraspecies and interspecies p30 antigen, whereas rat cells
transformed byand producing murine sarcoma virus (MSV)-Moloney leukemia virus (M-MSV-MuLV) contained high levels of both murine intraspecies and interspecies p30 antigen. Significant amounts of murine intraspecies and interspecies p30 antigen were detected in wild-type MSV-transformed
nonpro-ducer NRK cells. The control of p30 antigen expression was examined in temperature-sensitive MSV-transformed nonproducer cells
[NRK(MSV-lb)]
which are cold sensitive for maintenance of the transformed phenotype. Both murine intraspecies and interspecies p30 antigens were detected in
NRK(MSV-lb) cells when grown at the permissive (39 C) or nonpermissive
(33C)temperature,suggestingthatp30 antigen expression isnotcorrelatedwith maintenance of the transformed phenotype. The results demonstrate that
previously undetectablep30antigens are expressed inMSV-transformed
nonpro-ducerNRKcells, and suggestthat theexpression ofp30antigen maybe a useful
markerfor viral geneexpression in mammalian cells. Control of viral gene expression in murine
sarcoma virus (MSV)-transformed cells can
occur at various levels. Attempts to elucidate the level of viral gene regulation have been greatly facilitated bytheisolationofconditional lethalmutantsof MSV (30,32).The existence of
these mutants permitsan examination of viral gene products required for the initiation and maintenance ofthe transformedphenotype.We have previously described the isolation and characterization ofa cold-sensitive mutant of
MSV (32). Infection of normal rat kidney (NRK) cells by the mutant MSV results in
nonproducer transformed cells which are cold
sensitive for maintenance of the transformed
state.The cellsexpress the transformed
pheno-typeatthe permissivetemperature (39C),but appear phenotypically normal at the nonper-missive temperature (33 C).The MSV genome
canberescuedatboth temperaturesby
superin-fection of the cells with Moloney murine leu-IPresentaddress: Department ofMicrobiologyand Cell
Biology,EasternVirginiaMedicalSchool,Norfolk,Va. 23507.
kemia virus (M-MuLV), and the rescued MSV is cold sensitive for transformation of NRK cells. In anearlierstudyitwasshownthatcells grownatpermissiveornonpermissive tempera-tures containedquantitatively similaramounts ofviral-specificRNAasmeasuredbymolecular hybridization with a single-stranded DNA
probe synthesized bythe virionpolymerasewith
the endogenous viral RNA as template (33).
This indicated that the phenotypic expression of transformation was not being controlled at
the level of transcription. Similar conclusions
were reached by Scolnick et al. (30). In addi-tion, metabolic inhibitor studiesdemonstrated that the phenotypic expression of
transforma-tion was not affectedby inhibitors ofDNA or
RNAsynthesis,butwasblockedinthepresence ofinhibitors ofproteinandglycoprotein
synthe-sis, suggesting controlattheleveloftranslation orpost-translational modifications (33). In the present study, radioimmunoassay procedures (25, 29, 33a) were used to measure levelsofthe major murine internal virion polypeptide, the
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group-specific (gs) antigen (11, 13, 24) in trans-formed and normal cells. This antigenpossesses intraspecies (gs-1), interspecies (gs-3), and type-specific antigenic determinants (10, 28, 37, 39) which reside on a single polypeptide chain of approximately 30,000 mol wt (12). Since othertype C viral polypeptides have also been shown to possess group-specific antigenic determinants (38, 41, 42), the major murine
leukemia virus (MuLV) structural polypeptide
willbereferredtoasp30,basedonitsmolecular weight. Previous studies have reported unde-tectable levels ofthemurine intraspecies antigen innonproducerratcellstransformed byHarvey,
Kirsten, orMoloney strains of MSVevenusing
sensitive radioimmunoassays (2-4, 7, 19, 27,30,
41). This report describes the detection of the
MuLV intraspecies and interspecies antigenic reactivities in MSV-transformed nonproducer rat cells and demonstrates that the expression of the p30 antigen is not blocked in cells cold sensitive for maintenance of the transformed phenotype.
MATERIALS AND METHODS
Cells. Cells were grown in Auto Pow minimal essential medium(Flow Laboratories) supplemented with 10%fetalbovineserum(GIBCO). The cell lines examined are listed in Table1. 78A1areWistarrat
embryo cells transformed andproductively infected withtheMoloney strain ofmurinesarcoma-leukemia virus(M-MSV-MuLV) (5). NRK cells and NRK cells productively infected with M-MSV-MuLV or
M-MuLV alonehave been described(31).
NRK(MSV-la) is a MSV-transformed nonproducer NRK cell
linederived by infection of NRK cellswithacloned MSV isolate [MSV-1(NRK)]that iscapableof
trans-forming newly infectedratcells,but has lost replica-tion funcreplica-tions (31). NRK(MSV-lb) cells, cold
sensi-tive for maintenance of the transformed phenotype, are MSV-transformed nonproducer cells derived
from a single focus of MSV-1 (NRK) transformed
NRK cells (32). Both NRK(MSV-1a) and
NRK(MSV-lb) cell lines are classified as
nonpro-ducer cells andproduce neither infectiousnor
nonin-fectious virusasmeasuredby focusassays,XCplaque
assays,electron microscopy, and['H]uridinelabeling
ofsupernatantfluids of the transformed cells(31, 32). NRK(RaLV) cells obtained from S. Aaronson are
NRK cells infected withrat leukemia virus (RaLV) (1). The XC cell line (40) was established from a
Wistarrat tumor induced with the Prague strain of Rous sarcoma virus (RSV). The NIW3T3 Swiss mousecellline(16)wasreceived fromG. Todaro and cloned in our laboratory. The HT-1 cell line was
derived fromanM-MSV-induced nonproducer ham-ster tumor(14).GLOH cells (17)arehamsterembryo
cells productively infected with hamster leukemia virus (HaLV). The cell line wasobtained from D.
Baltimore.
RadoimmunoassayformurinetypeCvirusp30
anligen. The radioimmunoassay-inhibition testhas been developed and describedelsewhere(33a),Briefly, thep30antigen ofdetergent-disrupted M-MSV-MuLV
wasiodinated and isolated by isoelectricfocusing.The
iodinated antigen wasshown to be a 29,000-mol wt
polypeptide by sodium dodecyl sulfate-polyacryla-midegelelectrophoresis. The specific activity of the
purified iodinated p30 antigen ranged from 5 to 10
pCi/4sg ofprotein. Antisera against thep30 antigen
was obtained by repeated immunization of rabbits
TABLz 1. Properties ofcelllinesused for detection ofp30 antigensa TypeC Rescuable
Species Cellline Morphology viruspro- MSV Derivation
duction genome
Rat 78A1 Transformed + NA M-MSV-MuLV-infectedWistar
ratembryocells
NRK(M-MSV-MuLV) Transformed + NA M-MSV-MuLV infected
NRK(MSV-la) Transformed - + Wild-type MSV-1(NRK)
infected NRK(MSV-lb)
Grown at 39C Transformed - + tsMSV-1(NRK)infected;
focus-derived
Grownat33C Normal,CI - +
XC Transformed - - RSV-induced Wistar rat tumor
NRK(M-MuLV) Normal,CI + - M-MuLV infected
NRK(RaLV) Normal,CI + - RaLV infected
NRKclone2 Normal, CI _ - Uninfected
Mouse NIH/3T3clone1 Normal, CI _ - Uninfected
Hamster HT-1 Transformed _ + M-MSV-induced hamster
tumor
GLOH Normal, CI + - HaLV-infected hamster embryo
fibroblasts
Abbreviations: CI, contactinhibited; NA, notapplicable; ts,temperature-sensitive. 1246
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VOL.14,1974 POLYPEPTIDE EXPRESSION INMSV-TRANSFORMED CELLS with purifiedM-MSV-MuLVp30. Goatantisera (OS
268) prepared against Tween-ether-disrupted feline leukemia virus (FeLV) was kindly supplied by R. Wilsnack, Huntingdon Research Laboratories, Balti-more. Precipitatingar.'tibodyagainst rabbit
immuno-globulinG(IgG)wasproducedingoatsby
immuniza-tion with rabbit (IgG) was produced in goats by immunization with rabbit IgG purified by DEAE
chromatography. Precipitating antibody againstgoat IgGwassimilarly producedinrabbits. Reactiontubes
for the radioimmunoassay-inhibition test contained 0.1 ml of 1%normalrabbit or goat serum correspond-ing toantiserumused, 0.1 ml of 10% normalhuman
serum,0.1 mlof the unknownantigen (cellextracts), and0.1 ml of rabbitanti-M-MSV-MuLV p30serum or goat anti-Tween-ether-disrupted FeLV serum at adilution known toprecipitate 40% ofthe iodinated
M-MSV-MuLV p30 antigen. The rea'ction mixture
wasincubated 2 hat 37C. A portion(0.1 ml)of125I
labeledp30 antigen(approximately 2,000count/min, 0.5 to 1.0ng) was thenadded, and thesampleswere
incubatedanadditional2h at 37 C and 18 h at 4C.
Aportion (0.1ml)ofa 1:8 dilution ofgoatanti-rabbit IgGorrabbit anti-goat IgG(correspondingtothe
anti-serum used) wasadded, andthesamples were
incu-bated a further 18 h at 4 C. The precipitate was collectedby low-speed centrifugation andwashed in coldsaline, and radioactivitywascounted for4min inanautomaticgammacounter.Preliminary
experi-ments demonstrated that the
radioimmunoassay-inhibition testcouldtolerate2to 4mgofnonspecific proteinwithoutlossofspecificity.
Preparation ofcellextracts forradioimmunoas -say. Confluent monolayer cell cultures were washed
twotimes withphosphatebuffered saline (PBS), pH 7.2,removed with arubber policeman, washed again
inPBS,andprepared asa20%(vol/vol)suspensionin PBS. The 20% cell pack wassubjected totwocyclesof
freezingandrapid thawing and sonictreatment for 45 s,followedby centrifugationat25,000 xgfor90min
at 4C. The supernatant fluid was tested for the
presence of thep30antigen. The protein
concentra-tion wasdeterminedbythe method ofLowry (21).
RESULTS
Properties ofcell linesusedfor detectionof p30antigen.Table 1 lists someof theproperties
ofthe cell lines examined for MuLV
intraspe-cies and interspecies antigen. The cell lines tested exhibit varying degrees of viral gene expressionand include representatives from rat, mouse, and hamsterspecies.Thephenotypesof
the cell lines examined can be categorized as follows: (i) transformed and productively
in-fected with M-MSV-MuLV, (ii) transformed and nonproductively infected with MSV or RSV, (iii) nontransformed and productively
infected with leukemiavirus,and(iv)
nontrans-formed and uninfected. The MSV-transformed
nonproducercell linesatthepassage levels used
in this study were examined for noninfectious
virus production by electron microscopy and
[8H Juridine labeling of culture supernatant fluids. No intercellular or budding viral
parti-cles were observed in thin sections of
nonpro-ducer MSV-transformed cells, whereas both intercellular and budding virions were readily
observed in sections of productively infected cells. Likewise, labeling of actively growing
nonproducer MSV-transformed cells for 24 h with 10
gCi
of [3H]uridine per ml and subse-quentconcentrationand purificationbysucrose gradient centrifugation (31) resulted in back-ground levels of radioactivity (100to200counts/ min) in thatregion of the gradientcorrespond-ing to the density of oncornaviruses (1.15 through 1.18). In contrast, supernatant fluids from virus-producing cell lines contained viral particles that bandedat1.15through1.18, and radioactivity in the peak fractions ranged from 2,000 to 10,000 counts/min. Thus, under our culture conditions, theMSV-transformed non-producer cells did not release noninfectious viralparticles. Furthercharacterization of these celllinesby reverse transcriptase assay and p30
radioimmunoassay of culture supernatant fluids is in progress.
Radjoimmunoassayof murineintraspecies
and interspecies antigen. To quantitate the degree of murineintraspeciesp30 and interspe-ciesp30 antigen expression in various celllines, a radioimmunoassay-inhibition test was used. Rabbit antiserum to the purified
M-MSV-MuLV p30 was usedto detect theintraspecies antigenic determinant, and goat antiserum raisedagainstTween-ether-disruptedFeLV was used to detect theinterspecies antigenic deter-minant. The two antisera were titrated using
125I-labeled M-MSV-MULV p30 antigen puri-fied bIsoelectric focusing. Over 80% of the 12II-labeled p30 antigen was precipitated at a 1:3,200 dilution with the rabbit anti-M-MSV-MuLV p30 serumand at a 1:400 dilution with the goat anti-FeLV serum (Fig. 1). For the
radioimmunoassay-inhibition test, antisera
wereusedat adilution whichbound40 to50%of the 12II-labeled p30antigen. At this dilutionof rabbit anti-M-MSV-MuLV p30, theantiserum detected mainly murine intraspecies and no interspecies determinants based on titrations
using labeled p30 antigen derived from Rauscher MuLV and simian sarcoma virus (SSV-1) (datanot shown).The specificityand
sensitivity of theradioimmunoassay-inhibition
test is the subject of another report (33a). Briefly, the test is capable of detecting 1 to 3 ng ofp30antigen. For the purposeofthisstudy, it was necessary to demonstrate that extracts
ofcells producing M-MSV-MuLV gave
inhibi-tion curves parallel to those obtained with the crudep30antigen derived from purifiedvirions. 1247
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z Anti-M-MSV-MuLVp30
I0
--:
Anti-FeLV
B. 040
20-SM
[image:4.498.61.250.51.231.2]200 800 3200 12,800 51,200 204.800 RECIPROCAL OF SERUM DILUTION
FIG. 1. Titration of rabbit anti-M-MSV-MuLV p30serum (A) and goatanti-Tween-ether-disrupted
FeLVserum(A) bytheradioimmunoassay
precipita-tion test.
Ingeneral, compoundscontainingagroup
iden-ticaltothatofthe antigenicdeterminant inthe
specific antigen will give parallel inhibition of
binding(affinitysimilartothespecific antigen),
whereas others givenonparallel inhibition (affin-ity dissimilar) (6). Figure 2 demonstrates that extracts of M-MSV-MuLV producing cells,
78A1, (Fig. 2A) gave inhibition curves parallel tothoseobtainedwithp30antigen frompurified M-MSV-MuLV (Fig. 2B), thus indicatingthat identical antigenic determinants were being
measured. Competition experiments with the
purified 30,000-dalton viral polypeptide gave
inhibition curves with slopes similar to those
inFig. 2A and B, thus demonstratingthe
speci-ficity of theradioimmunoassay-inhibition test. For the standard radioimmunoassay-inhibition
test, results are expressed as the amount of proteinrequiredtoinhibit by40%thebindingof
purified 125I-labeled p30 antigen to limiting dilutionsofantibody.
Detection ofp30 antigensinuninfectedand
oncornavirus-infected cell lines.Thecell lines
listed in Table 1 wereexamined for thepresence
ofMuLV intraspecies and interspecies antigen by the radioimmunoassay-inhibition test. Cell
extracts (20%
vol/vol)
were prepared for each cellline,and theresultsoftheinhibition assays arepresentedinTable2.Rat cellsproductivelyinfected with M-MSV-MuLV [78A1 and
NRK(M-MSV-MuLV)]orM-MuLV
[NRK(M-MuLV)
]
contained high levels of intraspecies antigen.Only2 to 9jig
oftotalcell protein was required to give 40% inhibition. NonproducerMSV-transformed
NRK cell lines[NRK-(MSV-la) and NRK(MSV-lb)] also contained
detectable amounts of murine intraspecies p30
antigen. However, the levels of murine intra-species antigen in the nonproducer NRK cells were considerably lower than amounts detec-table in virus-producing NRK or 78A1 cells. A range of 10 to 500 times more protein was
required to give significant inhibition.
Ad-ditionally, thelevelsofmurineintraspecies p30
antigenexpression in thetwo nonproducercell lines,NRK(MSV-la) andNRK(MSV-lb),were different, indicatingthatexpressionofp30 anti-gen can vary considerablyin cells with
compa-rablephenotypes.Murineintraspecies p30 anti-gen was undetectable inuninfected NRK cells
using up to 1.5 mg of cell protein in the
inhibition test. Extracts from a number of control cell lines had undetectable levels of murine intraspecies p30. The amount of com-peting protein tested from the various control
cell lines ranged from 1.1 to 2.0 mg of cell protein, and neither rat cells [XC and
NRK(RaLV)] nor hamster cells (HT-1 and
GLOH) contained detectable levels ofmurine intraspecies p30 antigen (Table 2). NIH/3T3 cells of mouse origin had low levels of murine intraspecies and interspecies p30 (Table 2), confirming the findings of others who demon-strated a low level ofp30antigen expression in uninfectedmousecells (27).
The amount ofinterspecies p30antigen was alsomeasuredinvariouscell extracts (Table 2).
C type virus-producing GLOH, NRK(RaLV),
78A1, NRK(M-MSV-MuLV), and
NRK(M-MuLV) cells had high levels of interspecies antigen. Nonproducer NRK(MSV-la) and
NRK(MSV-lb) cells were also found to express the interspecies antigenic determinant;
how-100-
o
~40
[image:4.498.262.453.458.582.2]160 40 10 2.5 640 160 40 10 2.5 COMPETING PROTEIN g1P9 COMPETING PROTEIN Ing)
FIG. 2. Radioimmunoassay inhibition of (A)78A1
cell extract (0) and (B) p30 antigen frompurified M-MSV-MuLV(0).Rabbitanti-M-MSV-MuLVp30
serum was used at adilution capable of binding 40 to
50% of the 1251-labeledM-MSV-MuLV p30 antigen,
purifiedbyisoelectricfocusing.
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VOL.14,1974 POLYPEPTIDE EXPRESSION IN MSV-TRANSFORMED CELLS
TAmz 2. Amountof p3O antigenin differentcell lines"
Antigenic
reactivity (sgofprotein)"
Species Cellline MuLV MuLV intra-
inter-species species
p30 p30
Rat NRKclone2 C
NRK(MSV-la) 1,000 165
NRK(MSV-1b)
Grownat39C 155 133
Grownat 33C 50 44
NRK(M-MSV-MuLV) 9 74
78A1 2 8
NRK(M-MuLV) 3 12
NRK(RaLV) - 280
XC - 115
Mouse NIH/3T3clone1 1,180 625
Hamster HT-1 -
-GLOH 140d
aAllcell lines, with theexceptionof
NRK(MSV-lb), weregrownat36C. Cellextracts wereprepared and assayed for p30 antigens by radioimmunoassay inhibitionasdescribedinMaterialsandMethods.
bTheamountofproteinfromcellextractsrequired
toreduceby40%thebindingof"2q-labeled M-MSV-MuLVp30antigentolimiting dilutionsofantibody. The values are based on at least three separate preparations.
£_,Nocompetingantigen detected usingbetween 0.75and 2.0 mg ofproteininthe radioimmunoassay-inhibition test.
dThisamount of protein gave 75% inhibition.
ever, unlike the large differences in levels of murine intraspecies antigen in these two cell lines, the amounts ofinterspeciesantigen were
quite similar. The XC and HT-1 cell lines, although both negative for murineintraspecies p30 antigen, were found to differ in their ex-pression ofinterspecies antigen. Extracts from
XC cells contained levels of interspecies anti-gen comparable to those found in the
virus-producingNRK(M-MSV-MuLV) cells,whereas HT-1cells contained nodetectable interspecies antigen with up to 1 mg of cell protein. Like-wise, NRK-clone2cells containedundetectable interspecies antigen with up to 750
Ag
of cell protein. It is conceivable that with greateramounts ofcompetingprotein, low levels of
in-terspecies p30 antigen expression
might
be detected.Expression of p30 antigen in NRK cells transformedby temperature-sensitive MSV. The ability to detect murine intraspecies and
interspecies antigen in MSV-transformed
non-producer NRK cells, coupled with the observa-tion that NRK(MSV-lb) cells grown at the nonpermissive temperature contained approxi-mately threefold more p30 antigen relative to
the levels detected at the permissive tempera-ture (Table 2), prompted us to ask whether the p30 antigens were differentially expressed at
permissive or restrictive temperatures in cells temperature sensitive for maintenance of the
transformed phenotype. Extracts of
NRK(MSV-lb) cells grown at the permissive (39 C) or nonpermissive (33 C) temperature were tested for murine intraspecies and in-terspecies antigensby radioimmunoassay inhi-bition (Fig. 3). Analysis of the inhibition curves indicated that cells grown at the nonpermissive temperature had quantitatively more intraspe-cies(Fig.3A) and interspecies (Fig. 3B) antigen than cells grown at the permissive temperature. Using the anti-M-MSV-MuLV serum to mea-sureintraspecies reactivity,maximal saturation wasobserved at 56% inhibition with p30 antigen fromNRK(MSV-lb) grown at either 39 or 33 C. This suggested that qualitatively similar anti-genic determinants are present in the cells grown ateither 39 or 33 C. However, to achieve half-maximal saturation (28% inhibition),
NRK(MSV-lb) cells grown at 33 C required 50
;g of
protein,
whereas cells grown at 39Crequired 120 MAg of protein, indicating that cells atthenonpermissive temperature contained 2.4
80
_-
20-z 40 C
20 0 6 Bo 00 10
COMPETING PROTEIN(pg)
FIG. 3. Radioimmunoassay inhibition of murine
intraspecies and interspecies p30 antigen in
NRK(MSV-l b)cells grown at 33C(0) and 39 C (0).
(A) Immunoassay of intraspeciesp30 with anti-M-MSV-MuLV p30 serum. (B) Immunoassay of in-terspecies p30 with anti-FeLV serum.
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[image:5.498.277.431.412.598.2]timesmoreintraspecies p30antigen proteinper cell proteinthan cells grown at the permissive temperature. Similarly, using the anti-FeLV
serum to measure interspecies reactivity, maxi-mal saturation was obtained at 85 to 87% inhibition forp30 antigen from NRK(MSV-lb)
cells grown at either permissive or
nonpermis-sive temperatures. However, cells grown at the nonpermissive temperature required 50 ,ug of protein to attain half-maximal saturation,
whereas cells grownatthe permissive tempera-ture required 145
;tg
of protein to reachhalf-maximal saturation(Fig.3B). Thus,theresults
indicate thatNRK(MSV-1b) cells grown at the nonpermissive temperature contain approxi-mately threefold more interspeciesp30antigen percell protein than cells grown at the permis-sive temperature.
Inspection ofthe inhibition curves in Fig. 3
revealed that extracts of NRK(MSV-1b) cells
exhibited 85 to 87% competition with labeled
antigen for interspecies antibodybindingsites (Fig. 3B), whereastheextentofcompetition for intraspecies antibody binding sites was only 56% (Fig. 3A). The increased competition for interspecies p30 antibody binding sites may
reflect the activation of cross-reacting RaLV
p30antigen.Sinceit was shownthat extracts of productively infectedcellsexhibited 90 to100% competition with labeled antigen forantibody
bindingsites (seeFig. 2), the results suggested
that nonproducerNRK(MSV-lb)cellseither (i)
lack certain antigens which are normally
ex-pressedin productively infected cells, (ii) con-tain material in thecell extractthat interferes withantigen-antibody binding, or (iii) contain masked p30 antigenic determinants which are unavailable forantibody binding.
DISCUSSION
We examined by
radioimmunoassay
theex-pression of the murine intraspecies and
in-terspecies antigensinanumberofcelllineswith varyingdegrees ofviral geneexpression. Based
on our comparative survey, uninfected NRK cells contained no detectable MuLV p30 or
interspecies p30 antigen. There is conflicting evidence for theexpression of interspecies
anti-gen in NRK cells. Stephenson and Aaronson (35) reported undetectable levels of interspecies antigen in NRK cell extracts by radioim-munoassay, whereas Lieber et al. (20) were able
todetect small amounts of interspecies antigen
in NRK cell extracts by radioimmunoassay. Our data agree with those of Stephenson and Aaronson (35), but it is conceivable that with further passage of NRK cells or with higher
concentrations of cell protein an endogenous
interspecies antigen might be detected. NIH/3T3 cells of mouse origin contained low levels of murine intraspecies and interspecies p30 antigen, confirming the accumulating evi-dence that all mouse cell cultures express some endogenous viral information (27, 35). The expression ofinterspecies p30 antigens in the XC and HT-1 cell lines deserves mention. XC cells contained undetectable levels of murine intraspeciesp30butreadilydetectable levels of interspeciesp30antigen.Thisdemonstrates, as
previously shownbyothers (18), that XC cells express endogenous type C viral information,
presumably of rat origin. Neither murine in-traspecies nor interspecies antigens were
de-tected in extracts of HT-1 cells, although the
cells contain a rescuable MSV genome. Al-though HT-1 cells do not express the p30 antigens,theydo apparently express additional murinevirus-induced proteins (15).
The significant finding derived from this survey was thedemonstration that MSV-trans-formednonproducerratcells contained detecta-ble levels of MuLV intraspecies and interspecies antigens. This finding contradicts earlier re-ports whichconcludedthatnonproducer MSV-transformed cells do not express detectable levelsofp30 antigen(2-4, 7, 19, 27, 30, 41). It is
unlikely that the differences can be explained by assay conditions alone, since radioim-munoassay methods withcomparable sensitivi-ties were used by workers who were unable to
detect p30 antigen in cell extracts. It seems morelikelythat MSV-transformednonproducer cells exist with variabledegreesofviral genome expression. Furthermore, it is likely that the expression of certainviral genes is under viral as well as cellular control, similartothe
morpho-logicalrevertants ofMSV-orRSV-transformed
cells (8, 23, 36), thus permitting a wide spec-trum of viral gene expression in transformed
cells.Inthisregard,it isinterestingtonotethat twoof theMSV-transformed nonproducerNRK cell lines
[NRK(MSV-la)
andNRK(MSV-lb)] have quite dissimilar levelsofmurine intraspe-cies p30 antigen but comparable levels ofthe interspeciesp30antigen. The origin of these two nonproducer cell lines is somewhat different (31, 32), and it is conceivable that phenotypic repression or the loss of MSV genetic informa-tion from cell lines in the absence of any selective pressure for the transformed pheno-type, as in the NRK(MSV-la) cell line, could account for the observed differences. Support for thishypothesiswasobtainedby the analysis of clones derived from NRK(MSV-la) for the presence of a rescuable MSV genome after M-MuLV superinfection. Nineteen NRK-1250on November 10, 2019 by guest
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VOL.14,1974 POLYPEPTIDEEXPRESSIONINMSV-TRANSFORMEDCELLS
(MSV-la) clones which exhibited transformed morphology weretested, andnoneyieldedMSV upon superinfection with M-MuLV (K. 0. Somers, unpublished data). In contrast, all of the five NRK(MSV-lb) clones tested were
found to contain a rescuable MSV genome. It
is conceivable that the NRK(MSV-la) clones are either spontaneous transformants or con-tain a defective MSV genome which cannot be
complemented by M-MuLV. These same cells might also be deficient in the information for murine intraspecies p30 antigen expression. Future experiments will attempt to determine whether viral-specific information is present in these clones. The expression of interspecies p30 antigen in NRK(MSV-la) cells mightalso beexplained by the activation ofRaLVp30 af-ter transformation by MSV. Interspecies p30 antigen expression inthenonproducer ratcells may represent in some cases a combination of murine and ratintraspeciesp30, both of which would contain cross-reacting interspecies anti-genic determinants.
Murineintraspecies andinterspeciesantigens
were detected in NRK(MSV-1b) cells when grownatthe permissiveornonpermissive
tem-perature. Atthepermissive temperature, NRK cells transformed by the cold-sensitive MSV
mutant exhibit a transformed morphologyand
the abilitytogrow ascolonies onmonolayersof NRK cells (32), increased 2-deoxy-D-glucose
transport (22), and altered
fucosylglycolipid
synthesis (34).These phenotypicmarkers asso-ciated with transformed cells are not
expressed
when the cells aregrown atthe nonpermissive
temperature. Theability todetect p30antigen
expression inNRK(MSV-lb) cells grownatthe nonpermissive temperature indicates that p30
antigen expression is not correlated with the maintenance of the transformed
phenotype.
Furthermore,theobservedincrease ofp30 anti-gen levels at the nonpermissive temperature supports this argument. Although the
mech-anism(s) responsible fortheincreased levels of
p30 antigen at the nonpermissive temperature is not understood, similar increases in group-specific antigen levels at the nonpermissive temperature were found in chick cells trans-formed by a temperature-sensitive Rous sar-coma virus mutant (9). Studiesin progressare
directed at examining the nature of the p30
antigen incells transformed byMSV.
Radioimmunoassay inhibition revealed that extracts from MSV-transformed nonproducer NRKcellsonlypartially competedwithlabeled
p30forantibodybinding sites,whereasextracts of cells productively infected with M-MSV-MuLV completely inhibited antibody binding
of 125I-labeled p30. Since it is known that p30 contains at least three classes of antigenic
determinants, classified as intraspecies, in-terspecies, and type specific (37, 39), it seems likely that certain competing cross-reacting de-terminants are either not present, present at noncompetitive levels, or are present but un-available for antibody binding sites in MSV-transformed nonproducer NRK cells. The masking of theseantigenic determinants could result from subcellular components in the cell extractthat form complexes or aggregates with the antigen and thereby interfere with antibody binding, or alternatively, may represent a con-formational alteration of the p30 antigen.
Al-though the data presented demonstrate that extractsfrom nonproducerNRK(MSV-lb)cells
only partially competed with labeled p30 for antibody, recent preliminary experiments indi-cate that increased competition for p30
anti-bodycanbeachieved withcell extractspartially purified bySephadex gel filtration orsubjected tomore extensivedisruption procedures. Simi-lar observations were reported by Parks et al. (26), who demonstrated that mild denaturing
conditions exposed additional reactive determi-nants on the major polypeptide of murine mammarytumor virus.
Finally,althoughthere maybenocorrelation
between p30 antigen and maintenance of the transformed state, it is important to recognize
that cellswhichbyanumberofcriteria appear
phenotypically normal continue to express a viral function, p30 antigen, which is a useful marker for viral gene expression in mammalian andpossibly human cells.
ACKNOWLEDGMENTS
Thisinvestigationwasaidedby Robert A. Welch Founda-tion grantsQ-163andQ-435,by Public Health Service grants
CA-06656-12andCA-10893from the National Cancer Insti-tute,andbyPublic Health Service grant 1-K6-2352-12 from theNational Institute of Allergy and Infectious Diseases.
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