Regulation of tumor antigen synthesis by simain virus 40 gene A.

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JOURNAL OFVIROLOGY,JUlY1975,P.168-178 Copyrighti 1975 AmericanSocietyforMicrobiology

Vol. 16,No. 1

Printed in U.SA.

Regulation

of Tumor

Antigen Synthesis by Simian

Virus 40

Gene

A

PETER TEGTMEYER,'* MICHAEL SCHWARTZ, JOHN K. COLLINS, AND KATHLEEN RUNDELL

DepartmentsofPharmacology,* Microbiology,andAnatomy, Case WesternReserveUniversity School of

Medicine, Cleveland, Ohio44106

Received forpublication 28February1975

Simianvirus 40geneA haspreviously been showntopromotethereplication of viral DNAand thetranscription of late viral RNA in productive infection andto

maintain the growthcharacteristics ofsometransformed cells. The presentstudy

examines the effect of the A functiononproteins synthesizedduring productive

andtransforming infections. Under restrictive conditions, temperature-sensitive A mutants induce the overproduction of a 100,000-dalton protein both in

productively infected monkey cells and in transformed rabbit cells. Immuno-precipitation of theinduced protein with antisera, prepared against simian virus 40-induced tumors in hamsters, was used to identify the induced protein as

tumor antigen. The same protein can be precipitated from extracts of cells infected by wild-type virus but not from uninfected cells. Furthermore, the mutant-induced protein is more rapidly degraded in vivo and is less tightly

boundto intranuclear components than theprotein induced bywild-type virus. The presenceofthesame virus-induced protein in infected cells from different

species and the altered behavior of that protein in mutant infection strongly

suggest that the protein is virus coded. Because the protein islarge enough to accountforthe entirecoding capacityintheearly region of the simian virus 40

genome, the 100,000-dalton proteinmaywell be the primary product of the only

earlygene identifiedby complementation studies, the Agene.Ifthe

100,000-dal-tonprotein that isoverproduced inmutantinfection is the Aprotein and the only early protein, then functionalwild-typeAproteinmustregulate itsownsynthesis

inboth productive and transforming infections.

In productive infection by simian virus 40

(SV40),

gene A functioncontinuously regulates

the initiation ofviral DNA replication (5, 21,

34) and transiently controls the synthesis of late

viralRNA(6, 21).Inrestrictive infection, theA

function is

required

to establish the stable

transformation ofcellsand alsotomaintain the

growth characteristicsof sometransformed cell

lines (3, 16, 22, 25, 35). Thus, it seems quite

possible that the A protein may directly interactwithspecific recognitionsites on either viral or cellular DNA to regulatethe replication or transcription of either DNA. A satisfactory

testingofthis hypothesiswill require the

iden-tification, isolation, and characterization of the

A protein and a study of its interaction with

different DNA molecules. These studies could

eventually lead to a better understanding of

growth control mechanisms in mammalian cells

atthe molecular level.

' Present address: Department of Microbiology, State Universityof New York atStony Brook, StonyBrook,N.Y. 11794.

168

A major obstacle to these studies has long

been the

difficulty

encountered in identifying

the A protein within the large

background

of

cellular proteins (1, 15, 38, 39). We reasoned

that the synthesis,

location,

orprocessing ofthe

A protein could be perturbed in infection by

temperature-sensitive A mutants and that the

resultant alteration might be useful in

identify-ing the A protein. Thus a study of in vivo

protein synthesis in productive and transform-ing infections by the A mutants was under-taken.

Each of the A mutants tested induced the

overproduction of a 100,000-dalton protein at

the restrictivetemperature inboth monkeyand

rabbit cells. The induced protein was

specifi-cally and efficiently precipitated from extracts

ofproductivelyinfected or transformed cellsby

antiserapreparedagainstSV40-inducedtumors

in hamsters. A similar protein could not be

identified in uninfected cells. This and other

studies (2, 9, 13, 20, 26, 27, 29, 30, 33) suggest

thatSV40-induced tumorantigen is virus

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ed. Weproposethat theoverproduced protein is

the A protein and that functional A protein

regulates itsown synthesis.

MATERIALS AND METHODS

Cellcultures.TheTC7clone(31)oftheCV-1 line of monkey kidney cells was grown in Eagle basal

medium containing 2 to 5% fetal bovine serum. Transformed lines were derived from New Zealand White rabbit kidney cells (Flow Laboratories) as previously described (35) and were cultivated in

mediumwith 10% fetal bovine serum.

Virus. The origin of the parental wild-type clone

(WT)ofSV40and mutants A7, A28, and A30 has been previouslydescribed(36, 37).Mutants A40, A47, A57, and A58 were recently isolated from stocks of WT virus exposed tohydroxylamine.A58 fails to produce any detectable progeny at 39C. All of the other A mutants are leaky at 39C but produce little or no progeny at 41 C. Virus stocks were grown at 33 C after inoculation ofTC7 monolayers at 0.1 PFU/cell. Vi-rions were assayed at 33 and 40 C as previously

described (36).

Productive infection. ConfluentTC7 monolayers were inoculated with input multiplicities of 10PFU/

cell. Mockinfection wascarried out in the same way withlysates from uninfected cells. After a 2-h adsorp-tion period at room temperature, the inoculum was replaced with medium containing 2% fetal bovine serum.

Radioactive labeling of proteins. Productively

infected or transformedcells,grown in 8-ounce(about 240-ml) prescription bottles (45-cm2 cell growing area), were radiolabeled with ['"SJmethionine (New

England Nuclear Corp.; 40 to 60 Ci/mmol). Short pulses ofradioisotope (1 h) were carried out in

me-thionine-free mediuminasmuchaspreliminary stud-ies had shown that protein synthesis continued at a constant ratefor 3h under these conditions. Chases

wereperformedwithmedium containing a200-fold ex-cessofunlabeled methionine. Long pulsesof radioiso-tope(24 h) were performedwith ["S

]methionine

in

completemedium to providean adequate supply of aminoacids to maintain proteinsynthesis.The precise conditions for the radioactivelabelingofproteins are indicated in each figure legend.

Fractionation and extraction of radiolabeled

proteins. Cells were extracted with 0.5% Nonidet P-40(NP-40)inphosphate-buffered saline at pH 6 or in Tris-buffered saline at pH 7 and 8 as previously

described(38). Thedisruptedcells were spun at 2,000 xg for 10 minat 4Ctoseparatethecytoplasmfrom the nuclearpellet.All extraction buffers contained 0.3 mg ofphenylmethylsulfonyl fluoride per ml to inhibit proteaseactivityand0.001Mdithioerythritol.

Gelelectrophoresis. Samples were heated for 10

minat100Cinelectrophoresissample buffer contain-ing 0.075 MTris-sulfate, 2% sodium dodecyl sulfate

(SDS), 2% 2-mercaptoethanol, 15% glycerol, and

0.001% bromophenol blue, pH 8.4, and were then dialyzed against sample buffer containing 0.2% SDS

overnight at 4C. Sampleswere analyzed by

discon-tinuous polyacrylamide gel electrophoresis using a

modification of the method described by Maurer and

Allen (23). The separating gel contained 0.375 M Tris-sulfate, 20% acrylamide, and 0.1% bisacrylam-ide. The well gel contained 0.075 M Tris-sulfate, 5% acrylamide, and 0.12% bisacrylamide. The 0.065 M Tris-borate tank buffer contained 0.2% SDS. The gels were prepared and run on a slab gel apparatus (Hoefer, San Francisco, Calif.). Electrophoresis was carriedout at room temperature for 7 h at 25 mA/gel. The gels were fixed and stained with Coomassie blue according to Fairbanks et al. (8), vacuumdried, and autoradiographed on Kodak Royal X-omat medical X-ray film. Stained gels or autoradiograms were scannedwith aJoyce-Loebl densitometer to estimate the relative quantities of protein or radiolabel in individual bands in the gel.

Immunoprecipitation. Cytoplasmic fractions of cellular extracts were spun at100,000 x g for 30 min. The supernatantfluids(0.3ml)wereincubated with either preimmunization control serum or hamster antitumor serum(0.01ml)preparedagainst virus-free

SV40-transformed hamster cells. After 90 min at 30C, rabbit anti-hamster globulin (0.05 to 0.15 ml) was added at equivalence for another 90 min. The samples were centrifuged at 2,000 x g for 10 min at 4C, and the pellet was washed three times with extraction buffer. The immunoprecipitate and

re-imainingsupernatant fractions were heatedin electro-phoresis sample buffer for 10 min at 100 C and

analyzedby gel electrophoresis.

Immunofluorescentassay. The presence of

SV40-induced tumor antigen in infected cells was deter-mined by the indirect immunofluorescence tech-nique (27) with the same hamster antiserum used for theimmunoprecipitation studies.

RESULTS

Overproduction of a specific protein in

infection by A mutants. The patterns of pro-teinsynthesis in cells infected by WTor mutant

virus were compared to detect any significant perturbance that might provide clues to the function of theA gene orthe identity of the A

protein. The cells were separated into nuclear

andcytoplasmic fractions primarily to increase

resolution but also to comparethe distribution

ofproteinswithin cells. Thepatternsof protein

synthesis in uninfected cells varied depending

onthetemperature ofincubation, the lengthof

theperiodofradiolabeling, and thegrowthstate

of the cells. Thus, extracts of mock-infected cells were included in every experiment.

Figure 1 compares the patterns of proteins

labeled byashort pulse(1 h)with [35S

]methio-nine in cellsinfectedbyWTvirus or A30 at the permissive temperature (33 C), at the restric-tivetemperature (41 C),orafterashift from 33 to 41 C. WT virus induced the synthesis of virion

piroteins

VP1 (46,000 daltons), VP2

(40,000 daltons), and VP3 (28,000 daltons)

equally well under each set of temperature

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conditions. Most of the capsid proteins were

found in the nuclear fraction ofthe cells even

though approximately 80% ofthe total labeled

protein was present in the cytoplasmic fraction

of the cells. Cells infected by A30 produced

virion proteins during continuous incubation at

33C but failed to produce capsid proteins

during continuous incubation at 41 C. In

con-trast,when cellsinfected byA30 for 72 h at 33 C were shifted to 41 C for 24 h and then labeled

with [35S]methionine for 1 h, capsid proteins

were synthesized at the same rate as in cells

infected by WT virus under the same

condi-tions. These findings support previous studies

(6, 21) showing that the A function is only

transiently requiredforthe transcription of late

viral RNA.

The most significant finding in infection by A30 at 41 C was the overproduction of a

100,000-dalton (1OOK)protein.This proteinhad

noapparentcounterpart inuninfectedcellsand

wasdifficult toidentify in cells infected by WT

virus. After extraction at pH 7, most of the

A-induced proteinwasfoundinthecytoplasmic

fraction of cells, but smaller quantities were

present in the nuclear fraction as well. The

overproduction ofthe.100KproteinbyA30 was

not the resultof a high multiplicityof infection

because the same protein was not present in

increased quantities in infection by the same

stockofvirus at 33 C. Nor wasthe

overproduc-tion of the protein caused by an absence of

capsid proteins incells infected byA mutants,

since the protein is also overproduced in cells

synthesizing capsid proteins after a shift from

33 to41 C (Fig. 1).Further,the excess synthesis

ofthe A-induced protein is a general

phenome-nonininfectionbyA mutants at 41 C inasmuch as each of seven independently isolated mu-tants (A7, A28, A30, A40, A47, A57, A58)

induced the

synthesis

ofthesame protein to a

similar extent. The excess

synthesis

is not the

result of a nonspecific inhibition ofviral DNA

synthesis because inhibition ofthe replication

of DNA

by

150,ug ofcytosinearabinosideperml

in cells infected by WT virus does not cause

overproduction

oftheprotein. The 100K protein

isalso madein excess 24and72haswellas48h

after infection by theA mutants at 41 C. This

finding excludes the possibility that the lOOK

protein isproduced in similar quantities in WT

and mutant infection but with analtered tem-poral sequence.

Identification of the A-induced protein as

tumor antigen. Cellular extracts, known to

contain the lOOK protein induced by the A

mutants, were exposed either to serum from hamsters bearing SV40-induced tumors or to serum taken from the same hamsters before immunization. Antigen-antibody complexes

werethenprecipitated by the additionofrabbit

immunoglobulin prepared against hamster

globulin. Theprecipitated andnon-precipitated

proteins were analyzed by SDS-gel electropho-resis (Fig. 2).TheA-inducedprotein was specif-ically and efficientlyprecipitated byantitumor

antibody but not to a significant extent

by

preimmunization control serum. Further, the

same protein could also be identified

unam-biguously in immunoprecipitates of cells

in-fected byWT virus butnot inuninfected cells.

The amount ofradioactivityinthe lOOKbands ingels ofthe supernatantand pellet fractionsof

the immunoprecipitation mixture was

com-pared byexcision oftheappropriate bands and

liquid scintillation counting. More than80% of

the lOOK proteinwas

precipitated by

antitumor

serumwhereasless than2% was

precipitated

by

the control

preimmunization

serum. A similar

quantitation can be seen indensitometer

trac-ings of an autoradiogram of the same gel (Fig.

3). Several other proteins with molecular

weights ranging from 66,000 to 88,000 daltons

were also immunoprecipitated in small

quan-tities from extracts of WT or

mutant-infected

cellsbutnotfrom extractsofcontrol cells.

Intracellular localization of the A-induced

protein. Carrolletal. have recently shown that

"T"antigen bindstoDNA in vitroatpH 6but

elutesatpH8(4). On thebasis of this

informa-tion, the intracellular localization and in vivo

binding

characteristics of tumor antigen and

the 100K proteinwerecomparedininfection

by

WT or mutant virus. The location of tumor antigen wasdetermined by immunofluorescent

staining (Fig. 4). Intact cells showed a more

FIG. 1. Alteredpatternsofprotein synthesisin cells infected bywild-type (WT) virus andA30at33C,at 41 C, and afterashift from33 to 41 C. The cultures were labeled with50uCiof [35S]methionineper mlof

methionine-free medium70 to 72hafterinfectionat33C,47 to 48hafterinfectionat 41 C,and23to24hafter ashift from33C(72-hpreincubation) to 41 C.SDS-polyacrylamide

(20%o)

gelautoradiogramsoffractionated

cellproteinsare shown. Thesample order is: (a) control cells,33 C; (b) control cells,41 C; (c) controlcells,

temperatureshift; (d)WT-infected cells,33C; (e)WT-infectedcells,41C; (f) WT-infected cells,temperature shift; (g) A30-infected cells,33C; (h) A30-infected cells,41 C; and (i) A30-infected cells, temperatureshift.The upper and lowerpanels show thecytoplasmicand nuclear extracts, respectively.

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172 TEGTMEYER ETAL.

Supernate

a

b

c

d

Precipitate

a

b

c

d

e

f

e

t

A58

FIG. 2. Virus-inducedproteinsspecifically precipitated byantitumorserum. Cultureswerelabeled with 30

'UCiOf [35SjImethioninepermlof methionine-freemedium 47to48hafterinfection.Solubleproteinsextracted fromcells with 0.5%NP-40atpH7wereprecipitatedwithserumfromhamstersbearingS V40-inducedtumors

orwithpreimmunizationcontrolserumusingtheindirectimmunoprecipitation techniquedescribed in thetext. The precipitated proteins were identified by SDS-polyacrylamide (20%) gel electrophoresis. Both the

supernatant and the pelleted fractions of the immunoprecipitation reactions are shown in the slab gel autoradiogram. Thesample order is:(a) controlcells, controlserum;(b) WT-infected cells, controlserum;(c) A58-infected cells, controlserum; (d)controlcells, antitumorserum;(e) WVT-infectedcells, antitumorserum; and (f) A58-infected cells, antitumor serum. The side wells are whole cytoplasmic extracts of WT- and A58-infected cells. Thearrows indicate theposition oftheoverproduced 100,000-daltonprotein.

uniformstainingofnucleiinWTinfectionthan

in rnrlant infection. Further, the cytoplasm of

cellsinfectedby A58, butnotbyWTvirus, was

distinctly immunofluroescent in most but not allinfectedcells. Extraction with NP-40atpH6

removed most of the tumor antigen from the nuclei ofcells infectedby A58 butnotfrom the nuclei infectedby WT virus. Extraction at pH 8, however, efficiently removed tumor antigen from the nuclei infected by either WT or

mu-tantvirus.

The location of the lOOK protein was

deter-mineddirectly by the electrophoresis of proteins from cellularextracts (Fig. 5). After extraction atpH 6,mostof the lOOK proteinwaspresentin thecytoplasmicextractofcells infectedby A58

but inthe nuclei ofcells infected by WT virus. AfterextractionatpH 8, the lOOK protein could

no longer be identified in the nuclei of cells

infected by either virus. The dataareconsistent

with the interpretation that the WT-induced protein binds to intranuclear DNA with a greateraffinitythan the A-induced protein.

Temperature-sensitive degradation of the

lOOK protein. The lOOK proteins induced by WTvirusorA58andradiolabeledbya1-hpulse

with [35S ]methionine were compared in the same slab gel both by staining with Coomassie

blue andautoradiography (Fig. 6).Although the lOOK protein ofthe Amutantwas moreheavily

radiolabeled than the corresponding protein induced by WT virus in immunoprecipitated

WT

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-T

D

E

F

MIGRATI ON

FIG. 3. Densitometertracings ofselectedsamples

of thegel autoradiogram shown inFig.2tocompare the relative quantities of proteins precipitated by

antitumor serum. The sample order is: (A)

A58-infected cell extracts without immunoprecipitation; (B)A58precipitate,antitumorserum;(C) WTprecip-itate, antitumor serum; (D) control cell precipitate, antitumorserum;(E)A58precipitate, controlserum;

(F) WTprecipitate, control serum; and (G) control

cellprecipitate, controlserum.

material, theprotein from cells infected by WT

virus was moredensely stainedwith Coomassie

blue.

The stained gels and autoradiograms of the same gels were traced with adensitometer

and the relative amounts of accumulated,

stained protein and newly synthesized,

pulse-labeledprotein were quantitated bymeasuring

the appropriate areas in the tracings. The

mutant-induced protein contained 8.2 times as

much radiolabel but only 0.6 times as much

stain as the WT-induced protein. Thus, the

mutant-induced proteinhad a specific activity

(radioactivity/stain)

more than 10-fold greater than the WT-induced protein. These

observa-tionsindicate that the A-induced proteinis not

only more rapidly synthesized than the

WT-inducedproteinbut alsomorerapidly degraded.

To confirm these findings, the stability of the

lOOK protein was determined by pulse-chase

radiolabeling techniques. After the lOOK

pro-teinwaslabeled with [35S ]methionine for 1hat

41 C, infected cultures were incubated in

me-dium containinga 200-fold excess ofunlabeled

methionine for 12 h at 41 C. The 100K protein

was extracted with 0.5% NP-40 at pH 8.0,

precipitated with antitumor serum, and

quan-titated by SDS-gel electrophoresis (Fig. 7).

After a short pulse, radiolabeled,

mutant-induced 100K protein was degraded more

rap-idly than the WT-inducedproteinduringa12-h

chase at 41 C. A 24-h pulse with

[35S]methio-nine without a subsequent chase resulted in

approximately

the same

degree

of

labeling

of

the 100K protein ininfection

by

WTormutant

virus. Thesefindings strongly supportthe

con-clusion that the induced protein is both

over-produced and more rapidly turned over in

infection by theA mutants and that the 100K

protein istemperaturesensitivein its behavior

andstability invivo.

Tumor antigen in transformed cells. After

stable transformation ofrabbit cells by WT or

mutantvirus at 33C, transformed and

untrans-formed control cultureswereshiftedto 41 Cfor

24 h and thenradiolabeled foreither 1or 24h.

When proteins were extracted at pH 8 and

examined

directly

by electrophoresis,

no 100K

protein could be identified in the transformed

cells.

After immunoprecipitation, however, the

100K

protein could be easily identified in

trans-formed cells butnotincontrolrabbit cells (Fig.

8). As in productive infection, the

mutant-induced protein was more heavilylabeled after

a short but not after a long pulse with

[35S]methionine.

These findings indicate that the same protein is induced by SV40 in two distinct species of host cells and that the A function regulates the production of tumor bg

T

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174 TEGTMEYER ET AL.

a

b

C

.A

f

h

FIG. 4. Intracellular localization ofSV40-induced tumor antigen. Intact cells or nuclei of cells were stained either with toluidine blue or with antitumor serum using the indirect immunofluorescent technique. The cytoplasmwasremoved from the nuclei with 0.5% NP-40 at either pH 6.0 or 8.0. During extraction with NP-40 without agitation, most of the nuclei remained adherent to the glass surface so that morphological and

fluorescentantibodystudiesonextracted nuclei could beperformed in situoncoverslips.Thesampleorder is: (a)intactuninfectedcells, toluidine blue; (b) uninfected nuclei extracted at pH 6, toluidine blue; (c) uninfected nuclei extracted atpH 8, toluidine blue; (d) intact WT-infected cells,immunofluorescence; (e) WT-infected nucleiextractedatpH 6.0, immunofluorescence;

(f)

WT-infectednuclei extractedatpH 8.0, immunofluores-cence; (g) intact A58-infected cells, immunofluorescence; (h) A58-infected nuclei extracted at pH 6.0, immunofluorescence;and(i) A58-infected nuclei extracted at pH 8, immunofluorescence.

antigen in

transforming

aswellasin

productive

infection.

DISCUSSION

At the restrictive temperature, the tempera-ture-sensitive A mutants induce the

overpro-ductionofatemperature-sensitive protein with

an altered intracellular distribution. Further-more, theproteinextractedfromeitherinfected

monkey ortransformed rabbit cellsreacts

effi-ciently with antisera obtained from hamsters

bearing SV40-induced tumors. These findings

strongly suggestthat the 100Kprotein isaviral

protein. Recent directevidenceto bepresented

elsewhere confirms that the structure of 100K

tumor antigen is determined by the viral ge-nome. The induced protein isasingle

polypep-tide chain that cannot be converted into

sub-units by reduction and alkylation. The protein

is, therefore, large enough to represent the

entire

coding

potential of the early region of

SV40 DNA (14). This observation is also

con-sistentwithaccumulating genetic evidence that

SV40 hasasingle earlygene, the A gene.

Ifthe 100K protein is the A

protein

and the

only earlyprotein, then it mustregulate its own

synthesis directly or indirectly. The rate of

synthesisofthe mutant-inducedprotein is5- to

10-fold greater than that of the WT-induced protein. This overproduction is striking when

cnnsidered in terms ofgene dosage. In

produc-tiveinfection, the replicationof mutantDNA is

completely blocked atthe restrictive

tempera-ture, whereas thousands ofcopies of WTDNA

d

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per cell are present late in infection. Although

d e f these findings provide the first evidence for

t I or autogenous regulation in animal virus systems,

examples of this kind of regulation have been identified in bacteriophage systems (10). The f

"^F lambdarepressor acts asapositive regulator of

1OOK-1

!.

its own structural gene

(11, 17).

Functional

productsofgenes32 and 43 ofbacteriophageT4

inhibit expression of theirrespective structural

MM -

i+

genes

(18, 32).

In thecase ofSV40,

self-regula-tion may represent the only possibility for the

control ofgene function in view of the limited

VPi

-4,

C

"

content

of its

genome.

Thus, theA function initiates DNA

replica-tion and late transcription but apparently

in-hibits the synthesis of early protein. Further

studies willbe required to determine the

molec-VP3

- ular mechanism for theA

regulatory

function,

but the mechanism probably requires binding of

the A protein to viral DNA at a specific site.

Presentevidence is consistent with the idea that

viral DNA replication and early and late

tran-scriptionare initiatedinthe same region of the

viralgenome (14, 24). By binding to a single site

FIG. 5. Intracellular localization of the

100,000-dalton virus-induced protein. The cultures were la-beled with 30 uCi of [35S]methionine per ml of

methionine-freemedium47to48 hafter infectionat

*B_ ~~~S

a

b

41 C. Nuclear and cytoplasmic proteins were ex-tracted with 0.5% NP-40 at pH6. SDS-polyacryla-mide (20%) gel autoradiograms of fractionated cell

proteins are shown. The sample orderis: (a)

unin-fected cells, cytoplasm; (b) WT-inunin-fected cells,

cyto-plasm; (c) A58-infected cells, cytoplasm; (d)

unin-fected cells, nuclei; (e) WT-inunin-fected cells, nuclei;and (f)A58-infected cells,nuclei.

[image:8.498.44.236.81.375.2]

c

d

FIG. 6. Comparison of the synthesisandaccumulation of the100Kprotein in infectionby WT virus and A58. Cultures werelabeledwith 30gCiof [35S]methionine47to48h afterinfection at41 C. Afterextraction with 0.5% NP-40 at pH 8.0, the soluble proteins were precipitated with antitumor serum and analyzed by

SDS-polyacrylamide (20%) gel electrophoresis. Theupperpanelshowsanautoradiogram of thegel; thelower

panelisaCoomassie bluestain of thesamegel. The sampleorderis: (a) control cell immunoprecipitate; (b)

WT-infected cell immunoprecipitate; (c) A58-infected cell immunoprecipitate; and (d) whole cytoplasmic

extractof A58-infectedcells. Theareaof the gel containing the 100K protein is shown by thearrow.

*.Or. 4

*,#z

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a

b

c

d

e

f

-*

1OOK

FIG. 7. Temperature lability of the virus-induced 100,000-dalton protein as determined by pulse chase

studies. Cultureswerelabeled with30/Ci of[3S6S]methioninepermlofmethionine-free medium47to48hafter infectionat41C(shortpulse). Some cultureswerecollectedafter the shortpulse;otherswerechased for12hat 41 C in thepresence ofa 200-fold excess of unlabeled methionine. Alternatively cultureswere labeled with

[ 5S]methionine from 48to72h afterinfection (long pulse). Thesampleswereextractedwith 0.5%NP-40at

pH8.Solubleproteinswereprecipitated with antitumorserumandanalyzed bySDS-polyacrylamide (20%) gel

electrophoresis and autoradiography. Thesample orderis:(a) control cells, shortpulse; (b) WT-infected cells,

shortpulse; (c) A58-infected cells, short pulse; (d) control cells, pulse chase; (e) WT-infected cells,pulse chase; (f) A58-infected cells, pulse chase; (g) control cells, long pulse; (h) WT-infected cells, long pulse; and (i) A58-infected cells, long pulse.

onviral DNA, theA proteincould bothrepress

earlytranscription and induce DNA replication

orlatetranscription.

Antitumor sera precipitated at least four proteins from infected cells in addition to the

virus-induced lOOK protein.The sameproteins were not precipitated from extracts of unin-fected cells. These proteins, with molecular

weights ranging from 66,000 to 88,000 daltons,

could be cellular proteins complexed with the 100K protein, independent tumor antigens, or

products of the lOOK protein. Characterization

ofeachprotein with monospecific antisera

pre-pared against pure proteinsandby

fingerprint-ing of tryptic digests should determine the origin and interrelation of these proteins. Whether these proteins are viral or cellular in

origin, theymaybeoffunctional importance in

infection by SV40. Clearly, the findings

re-ported here and by other laboratories (4, 7, 17, 19, 28) indicate that the isolation, purification, andcharacterization of SV40tumorantigen and associatedproteinsare nowpossible.

Finally, it isespecially important to note the implications of the overproduction of the A-induced protein in cells transformed by the A

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SIMIAN VIRUS TUMORANTIGEN 177

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ML 40

FIG. 8. Overproduction of the 100 K protein in rabbit cells transformed by A mutants. After stable transformation of the rabbit cells by WTormutant virusat33 C, transformedand untransformed control

cultures were shifted to41 C. After 24 h ofincubation at 41 C, the cultures were labeled with 30 uCiof ['5Sjmethioninepermlforeither1h(short pulse)or24h(long pulse). Afterextractionwith 0.5%NP-40atpH 8, the soluble proteinswereprecipitatedwithantitumorserumandanalyzed bySDS-polyacrylamide(20%) gel

electrophoresis andautoradiography. Thesampleorder is: (a) control cells,shortpulse; (b) WT-transformed

cells, short pulse; (c)A28-transformedcells,shortpulse; (d)A58-transformedcells,shortpulse; (e)controlcells, long pulse; (f) WT-transformed cells, long pulse; (g) A28-transformed cells, long pulse; and (h)

A58-trans-formedcells, long pulse.

mutants. First, these studies show that the A

protein can regulate the production of tumor

antigen when the viral genome is in the

inte-grated state. Second, they strongly confirm previous findings showing that the A protein

may have a direct effect on the physiological

stateoftransformed cells (3, 16, 22, 25, 35).

ACKNOWLEDGMENTS

ThisinvestigationwassupportedbygrantPRA-113 from theAmerican Cancer Society, grant 1256 from the Damon

Runyon Fund, and Public Health Service grantCA 16497 from theNational Cancer Institute.

We are grateful for the skillful assistance of Judith Kohout.

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