Structure and biochemical functions of four simian virus 40 truncated large-T antigens.

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0022-538X/82/100054-13$02.00/0

Structure

and

Biochemical

Functions of

Four

Simian Virus

40 Truncated

Large-T

Antigens

FURZANACHAUDRY,ROBERT HARVEY, AND ALAN E. SMITH*

Biochemistry Division,National Institute for Medical Research, Mill Hill, London NW7 IAA, United

Kingdom

Received 30 April 1982/Accepted 22 June 1982

Thestructureof four abnormalTantigens whichare presentin differentsimian

virus 40 (SV40)-transformed mouse cell lines was studied by tryptic peptide

mapping, partialproteolysis fingerprinting, immunoprecipitationwith monoclonal

antibodies, and in vitro translation. The results obtained allowedus todeducethat

theseproteins, which haveapparent molecular weights of15,000, 22,000, 33,000

and45,000, aretruncatedforms of large-T antigen extendingtodifferent amounts into the aminoacidsequencesuniquetolarge-T.Theproteinsareall

phosphory-lated,probablyatasite between amino acids106and123.ThemRNAscoding for the proteins probably contain the normal large-T splice butare shorter than the normal transcripts of theSV40 early region. The truncated large-Ts weretested for the ability to bindto double-stranded DNA-cellulose. This showed that the 33,000- and45,000-molecular-weight polypeptidescontained sequences sufficient forbindingunder theconditions used, whereas the 15,000- and 22,000-molecular-weight forms did not. Together with published data, this allows the tentative mapping ofaregion ofSV40large-T between amino acids 109 and 272 that is

necessaryandmaybesufficient for the bindingtodouble-stranded DNA-cellulose

in vitro. None of the truncated large-T speciesformeda stable complex withthe

host cell protein referred to as nonviral T-antigen or p53, suggesting that the

carboxy-terminal sequencesoflarge-Tare necessaryforcomplexformation.

The early region of simian virus 40 (SV40) codes fortwoproteinscalledlarge-Tantigenand small-tantigen,withapparentmolecularweights of 94,000 (94K) and 17K (7, 26, 33). Large-T playsapivotalrole in thebiologyofthe virus. It is required for the initiation of viral DNA

syn-thesis, for the stimulation of cellularDNA

syn-thesis, and for the regulation of viral DNA transcription (10, 36, 38). Large-T has also been implicated in both the initiation and maintenance of transformation (10,36,38).Theroleof small-t in bothproductive infection and transformation

isless clear.

Several biochemical activities have been

as-cribed to large-T. It has the ability to bind to

double-strandedDNA(dsDNA) (3),particularly

to aspecific regionneartheorigin ofreplication

on SV40 DNA (20, 21, 35). Large-T also

inter-actswithahostcellphosphoprotein, referredto as nonviralT-antigen (NVT) or p53, toform a

high-molecular-weight complex (13, 15, 19). In

addition,anATPaseactivity of large-Thas been

demonstrated (37), and an associated protein kinaseactivity hasbeenreported (9).

Thesedata suggest thatlarge-T isa multifunc-tional protein and raise the possibility that the proteinmaybe composed ofanumberof

func-tional domains,eachassociatedwithaparticular activity. Suchaview is supported by work with

mutants which are temperature sensitive for

replication and which have lesions mapping in the early region. Under certain conditions it is possible to separate the transforming function from the temperature-sensitive lesion (27). To date it is not known which of the biochemical activities oflarge-Tand,inconsequence, which of theputativefunctional domainsare associat-ed withitsabilitytotransform cells. The results presented hereare anattempt todefine

function-aldomains onthe large-T molecule.

Immunoprecipitation ofextractsof cells

trans-formedby SV40 withserumfromtumor-bearing animals shows thatthecellsoftencontain

abnor-mal forms of large-T, in addition to large-T, small-t, andNVT(4, 12,18, 32,34).Oneclass of these abnormal T-antigens consists ofproteins considerably larger than normal large-T andare

referredto as super-T(4, 12, 18, 34;M. Lovett, C.E.Clayton,D.Murphy,P. W.J.Rigby,A. E.

Smith, and F. Chaudry, submitted for publica-tion). Another class is composed of truncated forms oflarge-T (18, 32, 34). In this study we

characterized four such truncatedlarge-T

mole-cules and showedthat they containthe normal

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SV40 TRUNCATED LARGE-T ANTIGENS 55

amino-terminal region of large-T and differing

amounts of the amino acid sequences uniqueto

large-T. We also measured the ability of the

truncated large-T molecules to bindtodsDNA in

vitro and assayed for their presence in a stable

complex with NVT. Inthiswaywe wereableto

deduce a tentative functional map of a DNA

binding domain on SV40 large-T and to locate

sequences necessary forNVT binding.

MATERIALS ANDMETHODS

Cells. FourSV40-transformedmousecell lineswere

used in this study: MES 2006, obtained from W. C.

ToppatColdSpringHarborLaboratory, ColdSpring

Harbor, N.Y., and SWSV3T3, BALB/c 3T3C120, and

BALB/c 3T3 ClM, which have been described

previ-ously (5, 33, 34).

Antisera. The anti-SV40 tumor cell (anti-T) serum

used was apool of sera obtained fromtumor-bearing

golden hamsters as described previously (33). The

monoclonalantibodies to large-T and NVT werethe

kindgiftof Ed Harlow(11).

Labeling, extraction, andimmunoprecipitation.

Con-ditions for the growth, labelingwith

32Pi,

and

extrac-tion oftransformed cells and forimmunoprecipitation

have been described (33, 34). Sodiumdodecyl

sulfate-polyacrylamideelectrophoresiswas on10or15%gels.

Driedgelswereautoradiographedfor 2to3days.

RNA preparation andpurification withSV40

DNA-ceUlulose. Preparation of RNA from cells grown in

culture was exactly as previously described (22).

Polyadenylated RNA was partially purified on SV40

DNA-cellulose aspublished (23).

In vitro translation. The nuclease-treated

reticulo-cytelysatewaspreparedby the procedure of Pelham

and Jackson (24). Conditions for cell-free synthesis

andimmunoprecipitation were asdescribed(22).

Tryptic peptide mapping. [355]methionine-labeled

extracts were immunoprecipitated with anti-Tserum

andsubjectedtoelectrophoresisin12.5%

preparative-scalepolyacrylamide gels. Individual polypeptides in

unfixed dried gels were located by autoradiography.

Elution of polypeptides from gels and subsequent

tryptic digestion and two-dimensional fingerprinting

onthin-layercellulose sheets were aspreviously

de-scribed (33, 34).

Partialproteolysisanalysis.32P-labeledextracts were

immunoprecipitated, and theproteinswereseparated

on a10% polyacrylamide gel. Large-T and the

truncat-edlarge-Tmoleculeswerelocatedbyautoradiography

of the wet gels, excised, and eluted in 100 mM

ammoniumbicarbonate-0.1%sodiumdodecyl sulfate.

The eluted materialwasdiluted,toreduce the

concen-tration of sodiumdodecyl sulfate, and reprecipitated

asdescribed by Lane and Robbins (14). The washed

immunoprecipitatesweresuspended in 120 mM

NaCl-50 mMTris-hydrochloride (pH 8.0)-0.5%Nonidet

P-40and treated with limited amountsof trypsin at 0°C

for 60 minasdescribedby Schwyzeretal. (30). The

32P-labeled digestionproductswereseparatedby

elec-trophoresis and autoradiographed.

DNA-cellulose chromatography. Calf thymus

dsDNA-cellulose (native) was eitherpurchased from

PL Biochemicals or made by a modification of the

method of Albertsetal.(1). Thewetslurry (PL)orthe

dry DNA-cellulose powder was suspended

immediate-ly before use in pH6.2bufferwhich contained 5 mM

potassium phosphate (pH 6.2)-0.1 M NaCI-1 mM

dithiothreitol-0.5% Nonidet P-40-10% (vol/vol)

glyc-erol. The columnwaspoured ina5-mlsyringe (0.5ml,

packed volume) andequilibratedwith 5 volumes ofpH

6.2buffer (3).

32P-labeled extracts (500,ul)wereadjustedtopH 6

with 2 to 5 ,ulof 1 M acetic acid and applied to the

column.Nonboundmaterial wascollected, and

wash-ing was continued until no furtherradioactivity was

detected. The column was then eluted with pH 8.0

elution buffer containing 10 mM Tris-hydrochloride

(pH 8.0), 1 mMdithiothreitol, 10%(vol/vol)glycerol,

and 0.1, 0.3, or 1.0 M NaCl. All fractions were

adjusted to pH 8.0 and 0.15 MNaCl.Portions of each

fraction were takenfor determination of radioactivity,

and 0.5-ml samplesfrom the peaks of eluted material

wereimmunoprecipitated with anti-Tand control sera

and separated onapolyacrylamide gel.

Sedimentation analysis of cellextracts. Samples (0.4

ml)of32P-labeledcellextracts werecentrifuged ona

4.7-mllinear 5 to20%o sucrosegradientwitha0.4-ml

60%osucrosecushion under conditions similartothose

described byCarrolletal.(3).Thegradientscontained

10 mM Tris-hydrochloride (pH 8.0), 10 mM

dithio-threitol, and 0.14 MNaCl and were centrifuged for 15

h at25,000 rpmin an SW50.1 Beckman rotor at4°C.

The gradient fractions (0.24 ml) were collected and

immunoprecipitatedasdescribed(18, 19).

RESULTS

We(18, 34; Lovettetal.,submittedfor

publi-cation) and others (4, 12, 32) have previously

shownthatcells transformed bySV40contain a

numberof proteins that arespecifically

immuno-precipitatedby SV40 anti-T serum. One class of

theseproteinsaresmaller thanlarge-T and have

beenshown to be related to it byanalysisof their

methionine tryptic fingerprints. These proteins

weretentatively identifiedastruncatedformsof large-T (32, 34). Here we analyze the structure offourtruncatedlarge-T antigens inmoredetail. Immunoprecipitationof truncatedlarge-T

anti-gens. Figure 1 shows the phosphoproteins

spe-cifically immunoprecipitated by anti-T serum

from32P-labeledextractsof four differentmouse

cell lines transformed by SV40. In addition to

super-T(140K), large-T(94K), and NVT (53K),

which have all been characterized previously (12, 15, 17, 18, 34; Lovett et al., submitted for publication), a number of other polypeptides

withmolecular weights in the range from 45K to

15K are specifically immunoprecipitated. Four

such proteins with apparent molecular weights of15K, 22K, 33K, and 45K, each of which is synthesized in adifferent cell line,were investi-gated further.Theproteinsarereferredto asthe

15K, 22K, 33K, and 45K species, respectively.

Two-dimensional tryptic fingerprint analysis.

Tocompare the structureoftheseproteins with

thoseoflarge-Tandsmall-t,they wereanalyzed

by two-dimensional tryptic peptide

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0

Super -

e

Lz-arit: -r--NV

---451K

-3K

15K-s

FIG. 1. Immunoprecipitat tracts of fourmouse celllini

Theextractswereseparated

gel after immunoprecipitati(

serum (C) and hamster anti

BALB/c 3T3CIM;2,BALB/I

4, SWSV3T3, M,molecular

ing. Previous work has s

of large-T and small-t

[35S]methionine-labeled

that each has additionalp

(23, 33). This occurs bec

early region ofSV40DN)

alternative ways, resultir

large-T and small-t. Smal

contiguous sequenceofS'

large-T mRNA is spliced

coding for the carboxy-te

including the termination

peptides common to smE

natefromtheirshared am

tides unique to small-t ar

spliced from the large-T

tides unique to large-T

quences downstream frc

region (23, 34).

The SV40-transformed

with [35S]methionine, anc

tation ofcell extracts, th

ratedon preparative-scal

Theproteinswereeluted:

with performic acid, dige

gerprinted by electrophc

finally chromatographed

Figure 2 shows the f

large-T and small-t togel

22Kandthe 45Kproteins

the common N-terminal I

Asp-Lys) (18) as well as all the otherpeptides

shared between large-T and small-t. The 22K

protein contains none of the peptides unique to

either small-t or large-T, nor any additional

peptidesnot normallypresentintheT-antigens.

r* -ir><- The 45K

protein

contains several

peptides

with

mobility identical to those of large-T unique

iss^ peptides, including a peptidepreviously referred

to as peptide B (18, 34).

Wehave already shown that the fingerprint of

the 33K polypeptide contains the peptides

sharedbetween large-T and small-t and at least

two large-T unique peptides (34). It does not

containpeptide B. These data establish that the

33K and 45K polypeptides are truncated forms

of large-T, since the three proteins share

com-monpeptides.

As the 22K protein only contains methionine

peptides sharedby bothlarge-Tand small-t,it is

not possible to conclude on the basis of the

tion

3of2

Plabele

fingerprint

data that it contains sequences from

son

of P-labeled ex- thelarge-T unique region. This was not

surpris-os

transf

olrmed bylam40ing,

asthe sequence of SV40 DNA

predicts

that

onf

with normal hamster few

if

any soluble,

methionine-containing

tryptic

i-T

serum (T). Lanes: 1, peptides are coded for by the region of the

'c 3T3C120; 3, MES2006; mRNA fromimmediatelyafter thesplice.We do

weight size markers. know, however, that the 22K polypeptide

con-tains neither small-t unique peptides (23) nor a

methionine-rich tryptic peptide coded in an

al-,hown that fingerprints ternative reading frame in the sequences around

share a number of the splice junction and present in an abnormal

tryptic peptides and small-t coded for by the deletion mutant d12122

eptidesthat are unique (39; M. Kress and F. Chaudry, unpublished

ause transcripts of the data).

kcan be spliced in two To examine further the structure of the 22K

ng in the mRNAs for polypeptide, the

32P-labeled

degradation

prod-l-t is translated from a ucts generated by partialproteolysis ofthe 22K

V40DNA,whereasthe protein were compared with those of

normal-to remove sequences sized large-T.

-rminal half of small-t, Partial proteolysis analysis. Partial protdolysis

codon (7). Thus, the fingerprints of SV40 large-T have previously

all-t and large-T origi- been generated by methods devised by Cleve-iino terminus, the pep- land et al. (6, 18, 33) and bySchwyzeretal.(30).

re coded by the region The latter method is particularly useful for the

mRNA, and the pep- analysis of32P-labeled large-T molecules, since

are coded for by se- the major proteolysis product is a 17K

N-termi-)m the small-t coding nal fragment which hasbeenshownbysequence

analysistoresultfrom the cleavage oflarge-Tat

celllines were labeled a lysine-rich region ending at amino acid 131

J afterimmunoprecipi- (30). Thus, the presence of the authentic 17K

e products were sepa- fragment in alarge-T-related molecule indicates

e polyacrylamide gels. that the protein is normalforatleast thefirst131

from the gels, oxidized amino acids. Since the sequences coding for

-sted withtrypsin, fin- these amino acids span the splice joint in the

)resis at pH 2.1, and large-T mRNA (30), the presence of the

frag-(33). ment also impliesthat the mRNAcoding forthe

fingerprints of normal proteinhas been spliced normally.

ther with those of the Figure 3 shows the partialproteolysisanalysis

,.Both proteins contain of the 33K and 22Kpolypeptides. In both cases

peptide (N-acetyl-Met- a major

32P-labeled

fragment which comigrated

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A

B

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*^

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D

C

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FIG. 2. Comparisonofmethionine tryptic peptidemapsoflarge-T and small-t withthose of 22K and 45K

polypeptides. Peptideswereseparated byelectrophoresisatpH2.1andascendingchromatographyin

butanol-aceticacid-water-pyridine. (A) Large-TfromBALB/c 3T3CIM; (B)45KfromBALB/c 3T3C120;(C)22Kfrom

MES2006;(D)small-tfrom BALB/c 3T3 CIM. Thearrowsindicate the amino-terminalpeptide

(N-acetyl-Met-Asp-Lys), and BindicatespeptideB.

with the 17K peptide from normal large-T was

produced. This resultimplies that although we

did not detect any methionine-labeled tryptic peptides unique to large-T in the fingerprint of the 22K protein, it is nevertheless a truncated

large-T moleculeand it is normal at leastuntil

amino acid131.Theresultalso indicatesthat the

mRNAfor the 33K protein is spliced normally

and that the protein is a contiguous, truncated large-T molecule.

The15Kprotein was notanalyzedbytryptic fingerprinting, since wecouldnotprepare suffi-cient methionine-labeled material. It could not be analyzedby partial proteolysis because it is

smaller thanthe 17Kfragment. Toinvestigateits

structureweusedmonoclonal antibodies to

im-munoprecipitate the protein.

Immunoprecipitationwith monoclonal

antibod-ies. A number of monoclonal antibodies that

reactwith SV40large-T have beenisolated,and

the regions of the molecule with which they

interact havebeenmapped (11).Wehave used a

series ofsuchmonoclonal antibodies isolatedby Harlow et al. (11) to immunoprecipitate 32p_ labeledextractsof the transformedcells.

Figure4Ais anexampleof the immunoprecip-itationof the15Kpolypeptide. Antibodiesto the

amino-terminal region of large-T recognize the

VOL.44, 1982

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2 3 4 5

L -lrge T-_

33.R----m

-A*

S

M

94

*-3

*-53

0 ---0

.A4,

7K

fragmrlet

_:0

S

-c--21

[image:5.496.108.394.74.305.2]

cm --*- 14

FIG. 3. Comparison of the partialproteolysisproducts of large-T, 33K, and 22K polypeptides. 32P-labeled

extractsoftransformed cells wereimmunoprecipitatedandseparated on15% polyacrylamide gels. The proteins

wereelutedfrom the gel,re-immunoprecipitated, and digested with trypsin. The fragments generated (+) were

analyzed alongsideuntreated material (-) on a 15% polyacrylamide gel. Lanes: 1, Large-T from SV40-infected

CV1 cells;2,large-Tfrom SWSV3T3;3, 33Kfrom SWSV3T3;4,large-T from MES 2006; 5, 22K from MES

2006; M, molecularweightsize markers.

15K protein, but those against determinants mappingin the carboxy-terminalend oflarge-T (seeFig. 4B) do not. Experiments onthe other proteins yielded similar results; L19 interacted strongly with theproteins, L16 interacted weak-ly,and all other monoclonals tested(L5,L6, L7, L9)were negative.

Themonoclonal data, therefore, provide evi-dence that the 15K protein contains sequences

relatedtothe amino terminus of SV40large-T.

In vitro translation. Toestablish whether the

truncatedlarge-Tantigenswereprimary transla-tionproducts ordegradation products of other forms oflarge-T, we studied their synthesis in

vitro. mRNA was isolatedfrom each of the cell

lines and partially purified by chromatography

onSV40 DNA-cellulose(23). Figure5shows the proteins immunoprecipitated from a nuclease-treated rabbit reticulocyte lysate programmed withthepartially purifiedmRNAsfromBALB/c 3T3C120andClM.Proteins withmobility

identi-cal tothatof the45Kand15Kpolypeptideswere

detected, indicating that both are synthesized directly from virus-codedmRNA. The 33Kand

22Kproteins werealso detectedinthe cell-free translation products ofpartially purified mRNA

fromtheirrespective cell lines(datanotshown).

ThemRNAscodingforthepolypeptideswere

fractionated on formamide-containing sucrose

gradients, and samples from the different

frac-tions were translated in vitro.Figure 6 shows the

gradient analysis of thepartially purified mRNA

from MES 2006 cells. The mRNA coding for the 22K polypeptide sedimented at approximately 11 to12S, whereas the mRNA for normal

large-T sedimented at about 18S. Similar analysis of the mRNAs forthe45K, 33K,and 15K

polypep-tides showed that they sedimented in therange

11 to 16S. This result indicated that the four

truncated large-T antigens studied here are all synthesized fromtruncated mRNAs.

Predicted structure of the truncated large-T

molecules. Taken together, the data presented

hereallowusto deduce atentative structurefor the 15K, 22K,33K, and45Kpolypeptides (sum-marized inFig. 7). There isstrongevidence that theproteins consist of the normal amino-termi-nal region of SV40 large-T, and in three cases

there isgood evidence thatthe slicejunctionis

normal.

The methioninetrypticfingerprintofthe 45K

truncatedlarge-T containspeptideB.This

pep-tide isduplicatedin the 115Kform(16, 17)and

the 145Kform (18; Lovettet al., submitted for

publication) of super-T. Since the duplicated region ofthese molecules has been sequenced,

we can deduce that peptide B corresponds to

one of the two predicted methionine tryptic

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FIG. 4. Immunoprecipitation of 32P-labeledextractof BALB/c3T3 CIM cells. Extracts(50 jil)weremixed

with approximately 20 ±II of tissue culture supernatant from cultures of secreting hybridoma cells (11).

Immunoprecipitateswerecollected,and thepolypeptideswereseparatedon a15%polyacrylamide gel.(A) M,

Molecularweightsizemarker;C, normal hamsterserum;T,hamster anti-Tserum.Theremaining sampleswere

immunoprecipitatedwith the Lseries of monoclonal antibodiesasindicated.(B)Location of the determinants

recognized bythe monoclonal antibodies(takenfrom reference11).

A

Large

r'

u0

B

L5

L6

Li

L9

L16

L19

L31

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60 AND

130--

40-1 2 3

M

FCTIrCT

CTI

f.w

'N

b*

21-

-FIG. 5. Cell-free synthesis of T-ai

tidesfrom different cell lines. The in vi

products made inresponseto(1) mRN

CV1cells, (2) purified mRNA from BW

cells, or (3) purified mRNA from BA

cells were immunoprecipitated with r

serum(C) and anti-Tserum(T). M, M

size markers.

peptides, which end at amino acii

Thissuggests thatthe45K species

the unique region of large-T at h

amino acid 281. This has been

recent sequencing data on the int

template for the 45K protein. TI

quencehas been shown toextend 3713 (correspondingtoaminoacid

pointthere isapresumedvirus-ho

termination codon isreached after

tional codons (C. E. Clayton, D.

Lovett,and P. W. J.Rigby, Nature press).

The fingerprint data indicate I protein is atruncated form ofthe

since they share at least two la methionine tryptic peptides. Alth( notidentified each ofthe labeled I ent in the fingerprints, tocode fo

methionine tryptic peptides woulc

the 33Kspecies includesequences asamino acid236.

The partial proteolysis data pr( 22K polypeptide must extend t(

cleavage siteatamino acid131, bi

no large-T unique methionine pel

not extend as far as the predi

endingatamino acid 178.

Thereisnodirect evidencethatI encodingthe 15Kproteinextend

siteinto theunique portionoflarg

tentative datasupporting this inte the immunoprecipitation of the p

L16 and L31 monoclonals, which

sites that may map in this region (11), and the findingthattheprotein isphosphorylated.

Phosphoaminoacid analysis of the 17K frag--

--Super

T ment

generated by partial proteolysis

shows that

~-LargeT it contains predominantly phosphoserine with

lesser amounts of phosphothreonine (data not

shown).This is in agreementwith the results of Schwyzer et al. (30) and allows the tentative 45K mapping of at least one phosphorylation site in SV40large-T. Small-t isnotphosphorylated,nor

is an 8K truncated T-antigen characterized by Spangleretal.(32). Ifweassumetherefore that the phosphorylation site is not one of the two

serine residues in the sequences shared by small-t, large-T, and the 8K protein but that it is

Sm

al5

t presentin the

large-T unique

region,a

phosphor-ylationsitecanbemapped tentativelytoone or

ntigen polypep- moreof thefive serine residues between amino itro-synthesized acids 106 and 123 in

SV40

large-T.

Afrominfected If the 15K truncated large-T species is phos-kLB/c 3T3 C120 phorylated at the normal site, we can predict iLB/c 3T3 CIM that theprotein extends into the large-T unique normal hamster region atleast as far as amino acid 106. lolecularweight Since it is clear that the

15K

to 45K

polypep-tidesform afamily of truncated forms of large-T, we used them to study properties associated id 281 or 364. with large-T, with the hope that this would allow sextends into the mapping of functional domains on the pro-east as far as tein.

confirmed by DNA-cellulosechromatography. Large-T binds :egrated DNA to calfthymus dsDNAimmobilizedon cellulose he coding se- andcan be eluted by buffers ofincreased pH and tonucleotide ionic strength (3, 21). To test the ability of the 368), atwhich truncated forms of large-T to bind to DNA we stjunction. A subjected

32P-labeled

extracts of the trans-onlysix addi- formed cells to DNA-cellulose chromatography Murphy, M. andimmunoprecipitated the fractions obtained.

D[London],in Figure 8 shows the elution profiles obtained

with the extracts from MES 2006 cells and SW that the 33K SV3T3 cells containing the 22K and 33K

trun-- 45K protein cated large-T antigens, respectively. Figure 8A

rge-T unique shows that all of the 22K proteinis present in the Dugh we have unbound fraction, whereas a large proportion of

peptides

pres- large-T is present in thefractionseluting atpH

or two soluble 8.0 and pH 8.0 plus 1.0 M NaCl. Figure 8B

d require that shows that the 33K protein is present in the atleastasfar bound fraction and its presence parallels that of large-T. It has been argued that the different edict that the conditionsrequiredtoelute the various fractions

D the trypsin of large-T from

dsDNA-celiulose

reflect the

ut since ithas presence of more than one population oflarge-T ptides it may molecules andthateachpopulationhas a differ-cted peptide, ent affinity for dsDNA (3, 21). It is noticeable that the 33Kpolypeptidehas thesamecomplex thesequences elution profile as large-T.

past

thesplice Similaranalysisof the 15K and 45K truncated

;e-T.

Theonly large-T proteins showed that the 15K protein rpretation are does not bind, whereas the 45K protein does. rotein by the These data therefore suggest that 33K truncated have binding large-T contains sequences that are necessary

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62 CHAUDRY, HARVEY, AND SMITH

5 X100 200 300 400 500 690 7Q0

Large T

f

15K

m<>R

106

22K

A131

33K

236

45K

-_

FIG. 7. Structures deduced for the truncated large-T antigens. The shaded areas indicate regions where there is evidence that the amino acid sequences are coded for by SV40 DNA; the unshaded areas indicate regions for

which there is no direct evidence. The scale is amino acid number in normal large-T; othernumbers are referred

to in thetext.

4 6 b)

A,

FIIFT7IKIIFT

1K7IKVF7F7m

A

83-

aw7- a4 e e al^--Large-:

94

--_-

53--40--.-i

_~- h4

21----'-

1~~

* £9

1 2 3 4 5 6 A 8 9 1Q 1 1 1 1K3 14 1to V16

F~~~~~~~.-TT.r

_----33K

FIG. 8. Binding ofSV40 large-T and truncated large-Tmolecules (22K and 33K) to native calf thymus

DNA-cellulose. 32P-labeledextracts were bound to and eluted from aDNA-cellulose column. Selected fractions were

immunoprecipitated with anti-T (T) and normal (C) sera and analyzed bypolyacrylamide gel electrophoresis. (A)

MES2006: M,molecular weight sizemarkers; 1, starting material at pH 8.0; 2, starting material adjusted to pH

6.0; 3and4, flow-through fractions; 5 through 7, material eluted at pH 8.0 plus 0.1 M NaCl; 8 through 11,

material elutedatpH 8.0plus 1 MNaCl.(B) SWSV3T3: 1, starting material at pH 8.0; 2 through 4, flow-through

fractions;5through 8, material eluted at pH 8.0 plus 0.1 MNaCI;9through 11, material eluted at pH 8.0 plus 0.3

MNaCI; 12through 16, material eluted at pH 8.0plus 1.0 MNaCl.

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VOL.44, 1982

and sufficient for binding to dsDNA, whereas the closely related 22K truncated large-T lacks these sequences.

Association with NVT. It has previously been shown that the complex formed between large-T and NVTin mouse cells transformed bySV40 is stable and can be readily detected by sucrose gradientanalysis (18, 19). The bulk of NVT and the fraction oflarge-T associated with it sedi-ment between 23 and 25S. Other multimeric forms of the complex can also be detected in the 16-18Srange (19).

Figure 9 shows the gradient analysis of32p_

288 18S

labeled extracts of the transformed cells after centrifugation and immunoprecipitation. The 15K and the 33Ktruncated large-Tantigens both remainnear the top of thegradients, whereas the large-T-NVT complexes sedimentrapidly.Both truncated large-T species sediment more slowly than the bulk of free large-T molecules. This may suggest that they are sedimenting in a monomeric form and do not associate stably with themselves, large-T, or NVT. Similar re-sults were obtained when the 22K and 45K species were analyzed on sucrose gradients (data not shown).

V-A

4 - - LargeT

"-NVT

_ 15K

18S 4S

B

4--'- LargeT

[image:10.496.107.394.220.620.2]

_4 33K

FIG. 9. Sucrosegradient sedimentation oflarge-Tandtruncatedlarge-T molecules. 32P-labeledextractsof

BALB/c 3T3 CIM(A)and SWSV3T3 (B)cellswere subjectedtosucrosegradient sedimentation.A totalof 23

fractionswerecollected from eachgradientandimmunoprecipitatedwith anti-Tserumbeforepolyacrylamide gel

electrophoresis.The Svalueswereobtained from 32P-labeled rRNA markersruninaparallel gradient.Trackson

the left endof eachgel show the productsimmunoprecipitated fromanunfractionatedextractwith anti-T(T) and normal(C)sera.

28S

CT I

f.w

A4.

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An independent analysis of the possible asso-ciation between NVT and the truncated large-T

specieswas made by using monoclonal

antibod-ies directed against NVT. Although the

mono-clonal antibody only recognizes NVT, it immu-noprecipitates large-T because of its association

with NVT (13, 15, 19). However, none of the

truncated large-T molecules coprecipitated with NVT, inagreementwith the results of the

gradi-entanalysis.

Taken together, all these results indicate that

truncated large-T species extending as far as

amino acid 368 in large-T lacksequencesthatare

required for stable association with NVT.

DISCUSSION

The results presented here partially define the

structure of four species of truncated large-T. The analysis is summarized in Table 1, and the

structures deduced are presented in Fig. 7.

There is no doubt that the proteins share a

common amino-terminal amino acid sequence.

They extend tovarying degrees into the large-T

unique region. However, the data presented do

not allow us to identify precisely the carboxy

termini of the protein. Such information could

beobtained by characterizing the template DNA

for the polypeptides. This has only been done

for the 45K species (Clayton etal., in press).

No methionine tryptic peptides other than

thosepresentin normal large-Tweredetected in

thetruncated large-T species analyzed. In

addi-tion, the amount ofSV40-coded sequence that

each protein was predicted to contain

corre-sponded roughly with the apparent molecular

weights of the proteins. This suggests that the

proteins contain relatively fewnon-SV40-coded

amino acids. Indeed, DNA sequence data

pre-dict that the 45K polypeptide contains only six

host-coded amino acids (Clayton et al., in

press). Assuming that the non-SV40

carboxy-terminal sequencesof the other truncated

large-T antigens are notextensive and that the

addi-tional sequences have no deleterious effect on

the activity of the proteins, the truncated large-T

moleculescanbe used for the tentative mapping

of functional domains on large-T, as discussed

below.

Several mechanismscanbeenvisaged for the

generation of truncated large-T molecules from

integrated viral DNA, e.g., point mutations in

the viral DNA resulting in the generation of a

termination codon, rearrangements of the viral

DNA,orinterruption of the viral DNA by fusion

with host cell sequences. Examples of the last

twomechanisms have been described for

poly-oma virus (H. E. Ruley, F. Chaudry, and M.

Fried, Nucleic Acids Res., in press) and SV40

(5, 29). The mRNAs coding for the truncated

large-T species described here are all smaller

than thenormal large-T mRNA, indicating that

they probably arise by eitherrearrangement or

fusion.

DNAbinding siteon large-T. The DNA

bind-ing data obtained with the truncated large-T molecules is summarized in Table 1 and

indi-cates that the 33K polypeptide has all the

se-quences necessaryandsufficient for the binding

of SV40 large-T to dsDNA-cellulose, whereas

the 22Kspecies lacks someof thesesequences.

Studies witha33Kfragment of large-T coded for

by theSV40deletionmutantdlO001 showed that

it also bound to dsDNA-cellulose (28). Other

work with fragments ofSV40 large-T generated

by in vitro translation ofSV40 cRNA showed

that an 82K fragment containing sequences

ex-clusively from the large-T unique region also

bound to dsDNA-cellulose, but that a related

70Kfragment didnotbind(25).

Taken together, these data indicate that a

region ofSV40large-T representing the overlap

between the 33K truncated large-T

character-ized here and the 82K cRNA translationproduct

containssequencesthatarenecessaryand

[image:11.496.60.452.539.664.2]

possi-bly sufficient for binding to dsDNA. If it is

TABLE 1. Structural features and biochemicalproperties ofthefour truncatedlarge-Tpolypeptides Structural features Biochemicalproperties

Size of Immunopre- Shared large- 17

Cell line truncated cipitation T and small-t Large-T d17,00 DNA binding Association T-antigen with N-ter- methionine unique haltoh (calfthymus with host

minal trypticpep- peptides

pepopdh

dsDNA) NVT(p53)

monoclonal tides

antibodies

BALB/c3T3 15K + NDa ND -

-CIM

MES2006 22K + + 0 + -

-SWSV3T3 33K + + 2 + +

-BALB/c 3T3 45K + + 2 + pep- ND +

Cl20 tideB

aND, Not done.

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Large

T

w

22K

178

,---33K

6 dl 1001

(33K)

82K

70K

272

109

186

dsDNAbinding regin

(serine

FIG. 10. A tentative mapof aphosphorylation site and a DNAbinding regionon SV40large-T, predicted

fromthe presenteddata.

assumed that the 82K protein results from the initiation ofprotein synthesis at the first AUG codon in the unique portion oflarge-T (21, 23) (corresponding to amino acid 109 in the whole molecule) and that the 33K truncated large-T extends no further than the dllO01 coding

se-quence(i.e.,toamino acid 272), thebinding site

canbe definedasbeing between amino acids109 and272(Fig. 10).

It must be emphasized, however, that this

resultrelatesonlytothebinding of SV40 large-T

to dsDNA-cellulose in vitro. No attempt has

beenmade to measureaccurately theaffinityof

suchbinding, noritsspecificity. Itisquitelikely that these activities would require additional large-T sequences.

Indeed,

recent results char-acterizing large-Trevertantsthat haveanaltered specificity ofbinding to the SV40 origin show that the mutations map within the sequences

between nucleotides 3459 and 3985, which are

outside the site defined here (31).

The influence of the secondary structure of large-T and the association between large-T and

NVT orbetween large-T molecules themselves

has also not been investigated in the studies

reported here. Such interactionsmay also influ-ence the affinity and specificity of binding.

In-deed, arecentreportsuggests thatlarge-Tmust

aggregate before it is able to bindto the origin

region ofSV40 DNA (2). We believe,however,

thatthe truncatedlarge-T moleculesthatbind to

dsDNA(thatis, the 45Kand 33Kspecies) bind

in their own right and not as a result of an

association with full-size large-T, because the

gradient analysis indicated that there was little detectableinteraction between thetwo.In addi-tion, several monoclonal antibodies that effi-ciently immunoprecipitated full-size large-T via carboxy-terminal determinants failed to coprecipitate the truncated large-T molecules.

NVTbinding site on large-T.Sincenoneof the

truncatedlarge-Tspecies studied here interacted stably withNVT it is notpossible todefine the region on large-T required for complex forma-tion. The results do show that the

amino-termi-nal sequences as faras amino acid 368 are not

sufficient for binding, implying that some

se-quencesfrom thecarboxy-terminalend mustbe

required. Since thelarge-T species coded for by the SV40 deletion mutants d11263 and d1265 form a complex with NVT, the extreme

car-boxy-terminal end of large-T wouldappear tobe

unnecessary(8). The results presented here

pre-dict that thesequencesbetweenamino acids 387

and662playanessential roleincomplex forma-tion with NVT. However, a more extensive study is necessary to determine whether all of

thisregion isrequired and whether it issufficient

fortheassociation with NVT.

ACKNOWLEDGMENTS

We thank Ed Harlow for the kind gift of monoclonal

antibodies; Peter Rigby forallowing us to see manuscripts

beforepublication;BenOostra, DanKalderon,andGraham Belsham for critical reading of the manuscript; and Lydia

Pearsonfor secretarial assistance.

F.C. is supported by an Imperial Cancer Rsearch Fund Bursarship.

4?0 590 690 700

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