DNA binding properties of simian virus 40 T-antigens synthesized in vivo and in vitro.

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0022-538X/80/02-0689/08$02.00/0

DNA Binding

Properties of

Simian

Virus 40 T-Antigens

Synthesized

In Vivo and In Vitro

CAROL PRIVES,t* YAFFA BECK, ANDHELEN SHURE

Virology Department, The Weizmann Institute of Science, Rehovot, Israel

Simian virus 40 large T- andsmall t-antigens have been shown previously to share immunological determinants and common sequences and to have roles in virus-induced cell transformation. However, only large T-antigen is a DNA binding protein. Under all conditions tested, small t-antigen did not interact with DNA. Large T-antigen synthesized in infected cells bound to both native calf thymus and simian virus 40 DNAs. As its binding efficiency was less than 100%, it is likely that there aredifferent forms of T-antigen which vary in their affinity for DNA. Large T-antigensynthesized in cell-free protein-synthesizing systems primed by simian virus 40 mRNA also bound toDNA-cellulose,whereas small t-antigen similarly synthesized in vitro did not. An 82,000-molecular-weight T-antigenpolypeptide synthesized in cell-free protein-synthesizing systems primed by simian virus 40complementary RNA transcribed in vitro from simian virus 40 DNAbyEscherichia coli RNApolymeraseboundefficiently to simian virus 40 DNA. Asthisproduct did not share sequences with the small t-antigen, it can be concluded that the amino-terminal portion of the T-antigen is not required for some ofits specific DNA binding properties.

Theearly region of the simian virus 40 (SV40) genome encodes twopolypeptides, thelarge T-and smallt-antigens (7,15, 23,38).Thelarge T-antigen,orviral A geneproduct,isrequiredfor initiation of viral DNA replication (35),viral L-strand RNA transcription (6), and autogenous control of itsownE-strand mRNAsynthesis (28, 38).Considerablebiologicalevidenceimplicates large T-antigen (4, 11, 13, 16, 36) and small

t-antigen (the F gene product) (3, 8, 14, 33) in virus-induced malignant cell transformation. These functions, directly and indirectly, imply

an interaction of the geneproductswith DNA. Biochemicalstudiesonpartiallyandhighly pu-rified T-antigen from different sources have shown that thisproduct bindsto DNA (5,31), withspecific affinityforsequencesatthe

origin

ofreplication onthe viralgenome (10, 27, 39). We have undertaken a study of some of the DNAbinding propertiesof theSV40 T- and

t-antigens synthesized in vivo and in vitro. The

experiments described in this paper were de-signedtoascertain thefollowing: (i)which

early

viralproteins isolated from infected cellsbindto DNA; (ii)which,if any, of thecell-freeproducts of E-strand mRNA bindtoDNA;and

(iii)

how efficientlytheearly productsbindtoDNA.

MATERIALS AND MErHODS

Cells andviruses. Thestandardwild-type SV40 usedwasstrain 777grownfromalimiting

dilution

in

tPresent address:DepartmentofBiological Sciences,

Co-lumbiaUniversity,NewYork,NY 10027.

theBSC-1 line of African green monkey kidneycells

previously described (12).

Labelingand extraction ofcelis.Extraction and

labelingof infectedcellsweredone according to

pub-lished procedures (24).Generally,cultures of 4 x 106

cellswerelabeled with[3S]methionine(40 ,uCi/ml) in

methionine-free medium from 46 to 48 h postinfection withSV40 or with

'Pi

(250,uCi/ml) in phosphate-free medium.Lysiswascarriedoutin 0.6mlof HIP buffer

(pH8.2) containing0.14MNaCl, 0.02 M

N-2-hydrox-yethylpiperazine-N'-2-ethanesulfonic acid (HEPES),

0.001M CaCl2,0.001M MgCl2,and0.5% Nonidet

P-40.Phenylmethylsulfonylfluoride and

L-1-tosylamide-2-phenylethyl chloromethylketone wereadded to a

final concentration of 0.25mg/mlofextract immedi-ately beforelysis. Nucleiwere removedby

centrifu-gationat2,000 rpm for2min, and theresulting

cyto-plasmicextracts werecentrifugedat30,000 rpmfor 40

min inaBeckman40 rotor at0°C.

Immunoprecipitations. Cellextracts orfractions

from DNA columns whichwereadjustedtoHIPbuffer conditionsatpH7.5werereacted with0.01to0.02ml of hamsteranti-SV40T-serum whichwas agiftfrom D. Gidoni. After incubationat roomtemperaturefor

1 h,the complexwasprecipitated bytheaddition of

eithergoatanti-hamster serum added atequivalence (24) or 50

pl

ofa10%suspension of heat-inactivated

formaldehyde-fixed Staphylococcus aureus(25).

Im-munoprecipitates were washed fourtimesin0.01 M

Tris(pH7.5)-0.15 MNaCl-0.01M EDTA with0.05% Nonidet P40 beforesuspensionin50

pi

of

electropho-resissamplebuffer(21).

Polyacrylamide gel electrophoresis. Reaction mixtures of immunoprecipitates in electrophoresis

sample buffer were heated at 100°C for 1 min and

appliedto10to20%gradientor10%simple

discontin-uousslab polyacrylamidegels prepared asdescribed

689

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previously (21).Electrophoresiswasgenerally carried

outfor 3 hat a constant 150V.Gelswerefixed and stained with Coomassiebrilliant blue and either

au-toradiographeddirectlyorfluorographedwithKodak

SB-5 film.

RNApreparationandtranslation.Cytoplasmic

polyadenylicacid-containingmRNAwasisolated from

SV40-infected BSC-1 cells at 48 h postinfection by

previously publishedprocedures (25). SV40

comple-mentaryRNA(cRNA)waspreparedby transcription

ofSV40form I DNA withhighly purifiedEscherichia coli RNApolymerasegenerouslyprovidedbyM. Go-recki asdescribed previously (23, 29).

Translation. Cell-free systemswerepreparedfrom wheat germ orreticulocytes.Thewheat germ system

wasessentiallythat describedby Roberts and

Pater-son(30) withpreviouslydescribedmodifications(24).

The reticulocyte system was prepared and used as

describedbyPelham andJackson(20).Ineither sys-tem,reactionvolumeswere25

pl

(ormultiples thereof)

containing10to20

pCi

of[3S]methionine,0.5to1,g

ofmRNA, and all other components as published previously.

DNA-cellulose. DNA-cellulose columnswere pre-paredby procedures describedbyAlberts etal. (1). SV40form I DNA(0.5mgin1mlof0.01M Tris[pH

7.5]-0.001M EDTA[Tris-EDTA])wasadded in

drop-wise fashionto 1gof washedcellulose(Whatman CF-11).The powderwasdriedat50°Cfor16h andthen

lyophilizedfor5 to 10h. The driedpowderwas

sus-pendedin 20volumes ofTris-EDTA,keptat4°Cfor

16h, and then washed three times with 10 volumes of cold Tris-EDTAtoremovefree DNA. The columns

werestoredat-20°CinTris-EDTAcontaining0.1M

NaCl. Thecolumns used in these experiments

con-tainedapproximately300,ugof DNA per gof cellulose. Native calfthymus DNA-celluloseobtainedfrom P-L

Biochemicalswasused atthesameconcentrationas

SV40DNA-cellulose.

DNAbinding.For thedifferential pHbinding

as-say,cellextracts orcell-freeproductswereadjustedto

pH 6.0 with

2-pl

samples of 1.0 M acetic acid and

appliedtonativeDNA-cellulose columns which were

preequilibratedwith buffercontaining 0.01 M sodium

phosphate, 0.14 M NaCl, 0.002 M

ethylenebis(oxy-ethylene-nitrolo)tetraacetic acid(EGTA),0.5%

Noni-det P-40, and 10% glycerol at pH 6.0, which was

essentiallytheapproach ofCarrolletal. (5). Proteins

elutedatpH6.0werecollected untilnofurther radio-active nonbound materialwasdetected.Thecolumns

wereeluted with the same bufferasdescribed above

atpH8.5and insomeexperiments with pH 8.5 buffer

containing 1.0 M NaCl. All collectedfractions were

adjustedtopH 7.0 and 0.15 M NaCl before immuno-precipitation with anti-T-serum.

RESULTS

Wemodified theapproach ofCarrollet al. (5) to examine the affinities of early proteins for DNA. Extracts of [35S]methionine-labeled SV40-infected BSC-1 cells were prepared,

ad-justed topH 6.0, and applied toSV40 and calf

thymus DNA-cellulose columnswhich had been

preequilibrated with pH 6.0 buffer. Figure 1

a

b

c

ci

e

t

g

I_m

90-

U

1

7-FIG. 1. Interactions of T- and t-antigens with DNA-cellulose. Extracts of 4 x 106 infected cells labeled with[35S]methionine from 46 to 48 h postin-fection were adjusted to pH 6.0 and bound to and elutedfromDNA-cellulose.Samplesof the fractions were immunoprecipitated with SV40 anti-T-serum and thensubjectedtopolyacrylamide gel electropho-resis. Autoradiograms are of the following: (a) marker 90KT-antigen and 17K t-antigen; (b) T-an-tigens bound to and eluted fromcalf thymus DNA at pH 6.0; (c)T-antigen eluted fromcalfthymus DNA at pH 8.5; and (d) T-antigen eluted from calf thymus DNA atpH 8.5 inbuffercontaining1.0MNaCl. (e), (f), and(g) arethesame as(b), (c), and (d), respec-tively, except thatSV40 DNA was used.

shows the autoradiogram of the

anti-T-imnu-noprecipitate of nonbound(pH 6.0eluates) and

bound (pH 8.5 or pH 8.5 plus 1.0 M NaCl)

fractionsafter polyacrylamide gel

electrophore-sis. In this experimentthemajorityofthelarge

forms ofT-antigen (80 to 90K) were boundto

the columnatpH 6.0 andeluted atpH8.5.The

smaller forms of the largeT-antigen (80 to 90K)

have been consideredby various authors(24, 34,

37) tobe possibleartifacts as theirappearance

varies with the extractionprocedure(34,37)and

cell line used (24, 37). However, theirdiscrete

size and DNAbindingproperty may indicateas

yet undefined biologicalfunctions. The

promi-nent bandattheapproximate centerofthe gel

has an estimated molecularweight of45K and

may be SV40 VP-1 as it has been shown that

some SV40 anti-T-sera immunoprecipitate the

major capsid protein (34). Generally,

approxi-mately 10% of theradioactivelylabeled proteins

in the cell bind to DNA at pH 6.0, forming

unique components differing in size and

distri-bution from the nonbinding proteins (22). The

addition of 1.0M salt tothepH8.5 elutionbuffer

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resulted in the elution of a small amount of T-antigen from SV40DNA-cellulose but not from calf thymus DNA-cellulose, indicating that some T-antigen has a higher affinity for SV40 DNA sequences than for calf thymus DNAsequences. This isconsistent with previous studies demon-strating aspecific interaction of T-antigen with the SV40 origin of DNA replication sequences (27, 39). Thesmall 17K t-antigen does not bind totheDNA under theseconditions. It should be noted here that in the manytimes that we have repeated thisexperiment, there has been consid-erable variation in the proportion of the large T-antigen forms (80 to 90K) immunoprecipitated from the nonbound pH 6.0 and bound pH 8.5 fractions. This does not appear to be afunction of the amount of T-antigen. We have carried out severalkinds of experiments demonstrating that the DNAis in excess of the bindable T-antigen in the range of concentration ofcell extract and quantities of DNA-cellulose used (data not shown).

T-antigen binding efficiency. Because of thevariation in the interaction of the T-antigen with calf thymus DNA-cellulose, we examined theabilityof thebound and nonbound T-antigen formsto rebind. The pH 6.0 nonbound and pH 8.5 bound fractions were collected, the pH 8.5 fraction was readjusted to pH 6.0, and then both fractions were bound to second DNA-cellulose columns. Theresults of a typical experiment are shown in Fig. 2. Approximately 60% of the la-beled T-antigen bound to DNA in the first round. The bound T-antigen consistently re-bound to DNA with near 100% efficiency. The T-antigenoriginallyeluted at pH 6.0, however, didnot clearlybehave as a nonbinding class: it appeared to redistribute into bound and non-boundforms. It is not likely a question of satu-rationorthepresenceof other nonbinding pro-teins asaddition of equivalent quantities of un-labeled infected-cell extract to the originally bound form did not prevent its efficient rebind-ing (C.Prives,unpublisheddata).

TheproportionsofT-antigen eluted at pH 6.0 and 8.5 did not depend upon the time it was

synthesized in the lytic cycle. T-antigen

ex-tractedintheearly phase,10hpostinfection, in the presence of cytosine arabinoside behaved very similarly to T-antigen extracted at 48 h postinfection, well into the late phase (Fig. 3). Furthermore, the distribution of 32P-labeled

T-antigenintoboundandnonboundfractions late in lytic infection was similar to that of the [3S]methionine-labeled T-antigen (Fig.3).

DNA binding

properties

of

T-antigens

synthesized

in vitro.We

investigated

whether

DNAbinding is anintrinsicproperty of

newly

a b c d e f g

90--

17-FIG. 2. Efficiency of T-antigen binding to calf thy-musDNA-cellulose. Extractspreparedasdescribed in thelegendto Fig. 1 were bound and rebound to

calfthymusDNA-cellulosecolumns.Autoradiograms

areofanti-T-immunoprecipitates ofthefollowing: (a)

cellextracts before bindingtoDNA-cellulose, show-ing T- andt-antigen markers; (b)extracts boundto

andelutedfrom DNA-cellulose atpH 6.0; and (c)

material elutedfromDNA-cellulose at pH 8.5. Non-bound materialfrom (b) wasreappliedto asecond calf thymus DNA-cellulose column atpH 6.0 and eluted atpH6.0(d)and atpH8.5 (e).Bound material

from(c) wasreadjustedtopH6.0andappliedtocalf

thymusDNA-cellulose and elutedatpH6.0(f)and

atpH8.5(g).

synthesizedandpresumably notfullymodified T-antigen. T-antigen was synthesized in vitro directedbymRNAisolated from infected cells. We usedcell-free protein-synthesizing systems bothfrom wheat germ extracts and from rabbit reticulocytelysates.Theformerisgenerally rel-ativelylessefficient than the latter in synthesiz-inglargeT-antigen.The wheatgermsystemwas

the first thatweusedtostudythe DNAbinding propertiesof invitro-synthesized T-antigen.We found that the 90K T-antigen synthesized in wheat

germ

extracts consistently bound

ex-tremely efficiently to calf thymus DNA-cellu-lose. The 17Ksmallt-antigen, even when syn-thesizedinconsiderableexcess over

large

T-an-tigen, still did not interact with DNA under theseconditions (Fig. 4).

Curiously,

the90K T-antigen synthesized in the

reticulocyte

lysate

system did not bind as efficiently to DNA. A

variable proportion of it was eluted with the

nonbound proteins at pH 6.0. Premixing the

labeled reticulocyte cell-free

products

with the

same amount of infected-cell extract

normally

usedin a DNAbinding

experiment,

representing

a great excess over that

synthesized

in the in

vitro systems, did not alter the

proportion

of labeledcell-free

synthesized T-antigen

elutedat

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692 PRIVES, BECK, AND SHURE

i:]t-J G d +~~f:~<

.I

-

E

r

C -.

FIG. 3. Proportion of boundandnonbound T-an-tigens. Cellextracts wereprepared asdescribedin

thelegendtoFig.1undervarious labeling conditions, adjustedtopH 6.0, andboundtoand eluted fromcalf

thymus DNA-cellulose. (i) Autoradiograms are of

anti-T-immunoprecipitates ofextractsofcells labeled

with [35S]methionine from 46 to48 hpostinfection

and thenlysedwith Nonidet P-40 buffer. (a) Bound

T-antigen elutedatpH8.5; (b)nonboundT-antigen

elutedatpH6.0.(ii) Sameas(i)exceptthe cellswere

labeledwith32Pafterprestarvation of cells for12h inphosphate-freemediumanddialyzedserumfrom

46to48 hpostinfection. (c) Bound T-antigen eluted

atpH 8.5; (d)nonboundT-antigenelutedatpH6.0.

(iii)Extractsofcells whichwereinfected in the pres-enceof cytosinearabinoside (20pg/ml) andlabeled

with[35S]methionine from 10to 12hpostinfection

and then extracted with Nonidet P-40 buffer. (e) BoundT-antigen elutedatpH8.5; (f)nonbound

T-antigenelutedatpH6.0.

pH 6.0 and 8.5 (Fig. 5). At presentwe do not

knowwhether thereisadifferenceinT-antigen

itselfsynthesized in thetwosystemsorinother

newly synthesized proteins or in the extracts

themselves which is responsible for the

differ-ence inbinding efficiency.

The invitrotranslationsystemhas provided

an opportunity to ask another question about

the binding of T-antigen to DNA. The entire

sequenceofSV40 DNA has beenestablished (9,

26), and termination codons in the T-antigen

readingframe have been mapped between0.54

and 0.59 map unitson the genome. To obtain

thelarge form of T-antigen,the mRNA directly

transcribed from DNA in vivomustbesubjected

to one or moreinternalsplicing events in that

regiontodeletethe terminatorcodons. Evidence

for these eventshas been derivedfrom studies

onviralmRNA(2) andsequencedataonthe

T-antigens (15, 18, 19). SV40 cRNA preparedby in

vitro transcription of SV40 DNA by purifiedE.

coli RNA polymerase is unmodifed and un-spliced and, therefore, contains the terminator codons. Invitro translation of cRNA yields au-thentic 17Kt-antigen (18;C.

Prives,

unpublished

data), aswell as aseries ofpolypeptideslarger than 17Kwhicharespecifically immunoprecip-itable withanti-T-serum, with sizesrangingup to82K (17, 23,29). These >17K cRNA

products

mostlikelyinitiatedistaltothe 17Kterminator codons. The 82Kformprobablyinitiatesatthe first AUG codon after the 0.54 spliced region andpresumablyterminatesat0.17mapunit,as doeslargeT-antigen. This suggestion is borne

outby the observationthatthe trypticpeptide

mapsof the60to80K cRNA

products

shareno uniquetryptic peptideswith the 17K

t-antigen

(17;C.Prives,unpublished data).This allowsus

toaskwhether thesequencesinlarge

T-antigen

thatare commontosmallt-antigen,i.e.,theNH2

portion of themolecule, arerequired for DNA binding. The cRNA used in these

experiments

hadabroad size distribution

predominantly

of 26Suponanalysis bysucrosegradient sedimen-tation(Fig. 6A), equivalentto a

genome-length

A

B

-9

0

6,'p ...

FIG. 4. Bindingof T-antigen synthesized in wheat germextractstoDNA-cellulose.Autoradiogramsare

ofanti-T-immunoprecipitates of

[35SJmethionine-la-beled T- andt-antigens synthesized in wheat germ cell-freeextracts(0.5ml) primed by20pgof mRNA from SV40-infected cells and then adjusted to pH 6.0. Anti-T-immunoprecipitates are of products synthe-sized in vitrobefore addition to DNA-cellulose (A), elutedfrom DNA-celluloseatpH6.0(B), and eluted from DNA-celluloseatpH8.5(C).

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a b c d e f

17-FIG. 5. Binding of T-antigensynthesizedin

retic-ulocyte lysates to DNA-cellulose. Autoradiograms

areofanti-T-immunoprecipitates of

[5S]methionine-labeled T- and t-antigenssynthesizedinthe

reticu-locytelysate sy8tem(0.5ml)primed by20pgofmRNA

fromSV40-infectedcellsandthen adjustedtopH6.0.

Productssynthesizedin vitrobefore bindingtocalf

thymus DNA-cellulose (a), eluted from DNA-cellulose

atpH 6.0 (b), and eluted from DNA-cellulose atpH 8.5(c).(d), (e), and (f)arethesameas(a),(b),and (c),

respectively, butwerecarriedoutin thepresenceof

anunlabeledextractfrom4x106infectedcells.

transcript ofthe viral DNA. When cRNA was

translated in the reticulocyte system, its

prod-ucts varied with the preparation: generally,

when the yield of 26S RNA washigh, we

ob-served products as large as 82K. When these

products were bound to SV40 DNA-cellulose,

weobserved thatonly thelargest cRNA product,

the 82Kprotein, boundtoDNAanddidsovery

efficiently (Fig. 6B). Thissame product bound

considerably less efficiently to calf thymus

DNA-cellulose.

DISCUSSION

Theaffinities of theSV40T-antigens

synthe-sizedin vivo andin vitro forDNA-cellulosehave

beenanalyzedwithanassayin which the

pro-teinsarebound toDNA atpH6.0 and eluted at

pH 8.5. Thegeneralresultsof these studiesare

summarized in Table 1. Inallof thecases

stud-ied,thelargeT-antigenbound toDNA, although

withvaryingefficiency,whereas thesmall

t-an-tigendid not.These data agreewith the known

biological functionsof these proteins, i.e., that

thepresenceofthe smallt-antigenisapparently

notrequiredinthelytic cycle (32), whereasthe

presenceofafunctioningA geneproduct (or

T-antigen) is essential for viral DNA replication

(35). One other conclusion drawn from these

experiments is that the general DNA binding

propertyof T-antigenisnot dueto ahostcellular modification ofthe proteinasT-antigen

synthe-sized intwodifferent translationsystems primed

by viral mRNA also bindsto DNA.However, as

T-antigensynthesizedinthe reticulocyte lysate

systembindslessefficiently thanthesame

prod-uct synthesized in the wheat germ system, we

cannot rule out the influence of

post-transla-tionalmodification of the protein on some

as-pectsof DNA binding. It is quitereasonable, for

example, that the crude reticulocytelysates used

in these experiments havethe ability to

phos-phorylateorotherwisemodify the newly

synthe-sizedT-antigenproduct and thus affect itsDNA

affinities.

Thevarying efficiency with whichthe 90K

T-antigen binds to DNA can be due to several

causes. As all experiments to check that the

amountofbindable DNA is in considerable

ex-cesshaveprovedpositive, it ispossible that T-antigen can exist informs which vary in their affinity for DNA. Whenextracts ofinfectedcells

were subjected to sucrose gradient sedimenta-tion and thegradient fractionswere immunopre-cipitated with anti-SV40 T-serum, it was ob-served that therearetwoforms ofT-antigen in lyticallyinfected cells whichapproximately

co-sediment with5and18S rRNA markers. Itwas

found that the 5S form binds to DNA much moreefficientlythatdoes the heavier16to18S form (D. Gidoni and C. Prives, manuscript in preparation).Thus, theoligomerizationof T-an-tigen may affect its DNA binding properties. However, the matteris somewhatcomplicated by our repeated observations that T-antigen synthesizedin thereticulocyte lysate translation

systems doesnot bind efficientlyto DNA,

be-cause we have also observed that thecell-free

product sedimnents only as the 5S form, even

when mixed with considerable quantitiesof

T-antigenfrominfected cells (Gidoniand Prives, in preparation). Thus, there may be different modes by which the binding of T-antigen to

DNA isaffected.Phosphorylationisonepossible modification which could affect the affinityof this protein for DNA. We did not detect any

marked difference in the relativeproportion of the 32P-labeled in vivo T-antigen in the non-bound and non-bound fractions when

compared

with

the [tS]methionine-labeled fractions.

Further-more, both 5Sand 16to 18S in vivo T-antigen

formsarephosphorylated(GidoniandPrives,in preparation). This does not rule out possible differences inthe extentof

phosphorylation

of

individual T-antigen molecules or turnover of phosphate moieties, either ofwhich wouldnot havebeen detected in these experiments. One relevantobservation thatwehave made

repeat-edly is that the newly

synthesized

T-antigen

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694 PRIVES, BECK, AND SHURE J

28S

2)

IiA

!-.C.

1?s

B

A

--.-3

a

-d

-_ -e=-f.__-I

g

--0

9 -D 17-.h_

fractlon lUrnber

FIG. 6. (A) SucrosegradientsedimentationofSV40 cRNA. SV40 cRNAwasprepared by using[3H]uridine

triphosphateas aradioactive precursor.Approximately105 cpmweresubjectedtosucrosegradient sedimen-tationthrougha10to30%(wt/vol)sucrosegradientin sodiumdodecyl sulfate buffer (0.1 MNaCl,0.01 MTris

[pH 7.5], 0.001MEDTA,0.5% sodium dodecyl sulfate)in anSW27rotorfor22hat24,000rpm.Fractions

werecountedbyliquidscintillation. 28 and 18S32P-labeledrRNA internal markers werefirstcounted by

Cerenkov radiation. (Fig.6B)DNAbindingof SV40cRNAcell-free products. (Left)Autoradiogramsareof

thefollowing: (A) anti-T-immunoprecipitatesof[35S]methionine-labeledcellextractpreparedasdescribed in

the legendtoFig.1and(B)[35S]methionine-labeledcell-freeproducts (5 ,ud) of2 pgofSV40 cRNA translated in thereticulocytelysatesystem. The 95K(a)and 47K(g)productsaresynthesizedin thereticulocytelysate

systemwithout exogenous mRNAadded,andallofthe otherproductsaresynthesizedspecificallyin response

tocRNA andarespecificallyimmunoreactive withanti-SV40 T-serum. (Right) Autoradiograms areofthe

following: (C) SV40cRNA cell-free products (5 yI) asin (B);200,lofsame batchofcRNAproducts was

adjustedtopH6.0and boundtoSV40DNA-cellulose: (D) pH6.0nonbound cRNAproducts;and(E)pH8.5

bound and eluted cRNAproducts.Estimated molecularweightsofproductsa,b, c, d, e,f, g, h, and i, derived

fromrelativemobilitytoknownproteinmarkers,are95,82, 78, 72, 60, 56, 47, 30, and17K,respectively.

TABLE 1. DNAbindingproperties ofSV40 T-antigenssynthesized in vivo and in vitro

DNA T-antigen

SV40 Calfthymus

90KT-antigeninvivo + +

90K T-antigen invitro + +

17Kt-antigen invivo + +

17K t-antigen invitro -

-82K cRNAproduct +

<82KcRNA products

bindsmoreefficiently than do the accumulated

orolderpopulations of this protein (M. Oren, E.

Winocour, andC. Prives, Proc. Natl. Acad. Sci.

U.S.A., inpress; C. Prives, Y. Beck, D. Gidoni,

M. Oren, and H. Shure, Cold Spring Harbor

Symp. Quant.Biol., in press).

The products of SV40 cRNA are a useful

subset ofT-antigen molecules whichcanprovide information about functional domains of the molecule. Paucha et al. (17) have shown that cRNA directs thesynthesis ofauthenticsmall

t-antigen and a series oflarger polypeptides,

in-cluding a predominant 60K polypeptide,which mustmap distal to the small t-antigen coding

sequences and that it islikely that the cRNA

products greater than 17K share common

C-rather thanN-termini (19).Thus,partial

T-an-tigenproductscontaining the NH2 terminal (the

smallt-antigen) and lacking the NH2 terminal

but containingsequences distal to the small

t-antigen coding region can be produced in

re-sponse tocRNA.From theSV40DNAsequence

the largest possible product that can initiate

distal to 0.54 map unit on thegenome is

approx-imately 82K. In an efficienttranslation system such as the micrococcalnuclease-treated

retic-PF L

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ulocyte lysatesystem,small quantitiesof an 82K

productaresynthesized. Wehavealso observed that the more prominent cRNA 60K product

described by Paucha et al. (17) and the 82K

products share common tryptic peptides with

each other butnotwithsmall t-antigen (Prives,

unpublished data). According to our

experi-ments, only the 82K cRNA product binds to

DNA and binds far more efficiently to SV40 DNA thantocalfthymus DNA-cellulose.It can

be deduced from the SV40 DNA sequence (9,

26) that 18 amino acids from thefirst AUG in

thisregion, i.e., after0.54 mapunit,isfound an

unusual and highly basic amino acid sequence (Pro-Pro-Lys-Lys-Lys-Arg-Lys) which couldbe akey inconferringspecific binding properties in theT-antigen. The amino acidsequencein this region ishighly conserved in BK virus (I. Seif and R. Dhar, personal communication) and partly conserved in polyoma virus (B. Griffin, personal communication). Whether the entire post-small t-antigen coding region mapping to

0.17 mapunit isrequired for DNA binding isnot

yet clear as Rundell et al. (31) have demon-strated that the dl-1001 mutantT-antigen which

mapsapproximatelybetween0.67and0.37map

units can bind to DNA. An assay has been

developedrecentlywhichdistinguishesbetween

thespecific (tight)andnonspecific (weak)

bind-ing ofT-antigentoDNA-cellulosebycomparing

thesalt-sensitive affinities of T-antigen for

non-viral DNA versus DNA multiply enriched for viral origin sequences (Oren et al., in press).

With thismethodwe arecurrently analyzing the relative affinities of T-antigen synthesized in

vivo andin vitrofor viral andcellular DNAs.

ACKNIOWLEDGMENTS

We thank Moshe Oren foruseful discussionsduringthe courseofsomeof these studies and Gilbert Jay and Neil Goldmanforcritical appraisalof themanuscript.

Thisworkwassupported byagranttoC.Prives from the U.S.-Israel Binational ScienceFoundation.

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Figure

FIG. 1.pHpHfectionDNADNA-cellulose.elutedresis.markertively,tigensandlabeledwere(f), Interactions of T- and t-antigens with Extracts of 4 x 106 infected cells with [35S]methionine from 46 to 48 h postin- were adjusted to pH 6.0 and bound to and from DNA-ce
FIG. 1.pHpHfectionDNADNA-cellulose.elutedresis.markertively,tigensandlabeledwere(f), Interactions of T- and t-antigens with Extracts of 4 x 106 infected cells with [35S]methionine from 46 to 48 h postin- were adjusted to pH 6.0 and bound to and from DNA-ce p.2
FIG. 2.muscalfinarecellmaterialingandfromcalfelutedboundthymusat the Efficiency of T-antigen binding to calf thy- DNA-cellulose
FIG. 2.muscalfinarecellmaterialingandfromcalfelutedboundthymusat the Efficiency of T-antigen binding to calf thy- DNA-cellulose p.3
FIG. 3.tigens.the46thymusadjustedanti-T-immunoprecipitateselutedwithandlabeledBoundinT-antigenatencewithandantigen(iii) phosphate-free pH to Proportion of bound and nonbound T-an- Cell extracts were prepared as described in legend to Fig
FIG. 3.tigens.the46thymusadjustedanti-T-immunoprecipitateselutedwithandlabeledBoundinT-antigenatencewithandantigen(iii) phosphate-free pH to Proportion of bound and nonbound T-an- Cell extracts were prepared as described in legend to Fig p.4
FIG. 4.germAnti-T-immunoprecipitatesfromfromofelutedsizedcell-freebeled anti-T-immunoprecipitates Binding of T-antigen synthesized in wheat extracts to DNA-cellulose
FIG. 4.germAnti-T-immunoprecipitatesfromfromofelutedsizedcell-freebeled anti-T-immunoprecipitates Binding of T-antigen synthesized in wheat extracts to DNA-cellulose p.4
FIG. 5.fromrespectively,Products8.5atanthymusulocytearelabeledlocyte pH Binding of T-antigen synthesized in retic- lysates to DNA-cellulose
FIG. 5.fromrespectively,Products8.5atanthymusulocytearelabeledlocyte pH Binding of T-antigen synthesized in retic- lysates to DNA-cellulose p.5
FIG. 6.tation[pHtriphosphateweresystemCerenkovthethetoinfollowing:fromadjustedbound cRNA the (A) Sucrose gradient sedimentation ofSV40 cRNA
FIG. 6.tation[pHtriphosphateweresystemCerenkovthethetoinfollowing:fromadjustedbound cRNA the (A) Sucrose gradient sedimentation ofSV40 cRNA p.6
TABLE 1. DNA binding properties of SV40 T-antigens synthesized in vivo and in vitro

TABLE 1.

DNA binding properties of SV40 T-antigens synthesized in vivo and in vitro p.6