Simian virus 40 large tumor antigen: a "RNA binding protein"?

Article

Reference

Simian virus 40 large tumor antigen: a "RNA binding protein"?

KHANDJIAN, Edouard W., et al.

Abstract

Simian virus 40 large tumor antigen was isolated by immunoaffinity chromatography from monkey or mouse cell cultures undergoing lytic or transforming infection. RNase-treated gel-purified large tumor antigen, on hydrolysis with alkali, gave about equimolar amounts of AMP, GMP, CMP, and UMP. Furthermore, RNA fragments of approximately 45 nucleotides could be isolated from large tumor antigen purified by the same procedure. Mapping of the T1 oligonucleotides showed a high complexity, as indicated by the presence of unique sequences of 15-30 nucleotides and of poly(A). This is compatible with the hypothesis that these RNA fragments are derived from cellular pre-mRNAs or mRNAs. Our results suggest that Simian virus 40 large tumor antigen is a RNA-binding protein and might possibly be involved in regulation of synthesis, maturation, or translation of cellular mRNAs.

KHANDJIAN, Edouard W., et al. Simian virus 40 large tumor antigen: a "RNA binding protein"?

Proceedings of the National Academy of Sciences, 1982, vol. 79, no. 4, p. 1139-1143

DOI : 10.1073/pnas.79.4.1139

Available at:

http://archive-ouverte.unige.ch/unige:150437

Disclaimer: layout of this document may differ from the published version.

Proc.NatL Acad.Sci. USA

Vol. 79, pp. 1139-1143, February 1982 Cell Biology

Simian virus 40 large tumor antigen: A "RNA binding protein"?

(electrofocusing/messenger ribonucleoprotein/phosphoprotein/regulation of transcriptionandtranslation) E. W. KHANDJIAN*, M. LOCHE*, J.-L. DARLIX*, R. CRAMERt, H. TURLER*, ANDR. WEIL*

*DepartmentofMolecular Biology,UniversityofGeneva, 1211Geneva,Switzerland; and tSection deBiologie,Institutdu Radium,91405 Orsay, France

Communicated

by

V.Prelog, September21, 1981

ABSTRACT Simian virus 40large tumor antigen wasisolated by immunoaffinitychromatography from monkeyor mouse cell cultures undergoinglyticortransforming infection. RNase-treated gel-purified largetumor antigen, onhydrolysis with alkali, gave aboutequimolaramountsofAMP, GMP, CMP,andUMP.Fur- thermore, RNAfragmentsof -45nucleotidescould be isolated from largetumorantigenpurified bythe sameprocedure. Map- ping oftheT1 oligonucleotides showed a high complexity,as in- dicatedbythe presenceofunique sequencesof15-30nucleotides and ofpoly(A). Thisiscompatible with the hypothesisthatthese RNAfragmentsarederived from cellularpre-mRNAs or mRNAs.

Ourresults suggestthatSimian virus40largetumorantigen is a RNA-bindingproteinandmight possibly be involvedinregulation ofsynthesis,maturation, ortranslationof cellularmRNAs.

The early region of simian virus 40 (SV40) DNA codes two re- latedproteins,designatedlarge (T) and small (t) tumor antigens, ofapparent Mr 88,000and19,000,respectively(1-3). They can be isolated by immunoaffinitychromatography (4) from cells undergoinglytic or transforminginfection and from tumors in- ducedinanimalsbyinoculation with SV40. The primary struc- turesof theTand t antigens have been established (1, 5-7). T antigen is aphosphoprotein(8-11) that has at least two distinct phosphorylatedsites(4),andafractionof it is further modified bypoly(ADP-ribosylation) (12). Tantigen isrequiredfor initi- ationofviral DNAreplication(1, 13,14) and bindspreferentially to the origin ofreplication of SV40 DNA (15-19). SV40 T and tantigensplay aprimordial role in lytic andtransforming in- fectioninvitroandintumorformation in animals (1, 20).

Inthispaper, we showthat small RNAfragments,apparently derived from high molecular weight RNA, remain bound to RNase-treated gel-purified SV40 T antigen.

MATERIALS ANDMETHODS

Confluent primary mousekidneycultures and cultures of CV- 1(Africangreenmonkey)cells were infected with SV40 or mock infectedunder the conditions described (21, 22). The cultures were labeled 21-25 hrafter infection with 100 ,uCi of[3S]- methionine(500-1000Ci/mmol;1 Ci=3.7X

1010 becT.uerels)

in 2ml ofmethionine-free medium, or 100 ,uCi of[5- H]- or [2-14C]uridine (540 mCi/mmol and 50 mCi/mmol, respec- tively)in 2mlofmedium, or100,uCi of32p;(carrierfree)in 2 ml ofphosphate-free medium. DNA synthesis was inhibited with

1-,8-D-arabinofuranosylcytosine

(araC; 20

jig/ml)

in the medium used to infect and label the cultures. T antigen was extracted with 0.5% Nonidet P40, pH 9, and isolated by im- munoaffinitychromatography (4, 23). The immune complexes were eluted with either NaDodSO4 sample buffer (65 mM TrisHCl,pH6.8/2%NaDodSO4/2%2-mercaptoethanol/0.01%

bromophenolblue/15% glycerol) (24) or urea lysis buffer [9.5

Murea/2% Nonidet P40/5% 2-mercaptoethanol/2% amphol- ytes (LKB-ProdukterAB, Sweden)] (25). Immunecomplexes, bound to proteinA-Sepharose, wereincubatedin0.5ml of10 mMTrisHCl,pH 7.0, or in 0.5ml of 10 mM NaOAc, pH 5.0, containing100

Ag

of RNase A, orin 0.5ml of10mMTris-HCl, pH 7.4/5 mM MgCl2 containing 100 ,ug ofDNase I (RNase free). RNase A and DNase I werepurchasedfromWorthington.

After incubation for 30 min at room temperature, thecomplexes werewashed andTantigen waselutedwithNaDodSO4orurea.

Isoelectric focusing in sucrose densitygradients [5-40% (wt/

vol)] containing 2% (wt/vol) ampholytes (pH 3-10) was done in23-ml columns(ISCO model212; Lincoln, NB) accordingto Nelson etaL(26). Thereafter, 0.25-ml fractions werecollected andradioactivity and pH values were measured. Tantigen was assayedinpooledfractionsby immunoaffinitychromatography followedbyelectrophoresisinNaDodSO/polyacrylamidegels andrevealed by fluorography (27). Two-dimensional gelelec- trophoresiswascarriedoutaccordingtoO'Farrell(25).

Forchemicalanalysisand forRNAextraction, T antigen was isolated from 10-12 cultures andpurified bypreparative elec- trophoresisin

NaDodSO4/7.5%

acrylamide gels.Thebandcon- tainingTantigenwas localizedin thegelsbyautoradiography orbystaining averticalgelstripwith Coomassie blue (orboth).

Tantigen waselutedfrom thefragmented gel by shakingin 2 ml of0.25 MNaHCOJ0. 1% NaDodSO4for 20 hrat37°C.The eluate wasthenlyophilized.Foranalysisofphosphateresidues, aliquots of the lyophilized material were subjected to either alkaline (0.3 MKOHfor 20 hr at 37°C) or acid (2M HC1for20 hr at120°C)hydrolysis.Thehydrolysateswereneutralizedand analyzed by high-voltagepaperelectrophoresisatpH 3.5[0.5%

pyridine/5%aceticacid(vol/vol)] (28). ForRNAextraction, the lyophilizedmaterial wasincubated in2mlof10mMTrisHCl, pH 7.0, containing500,gof proteinaseK(Merck, Darmstadt) for2hrat37°C. Thesolutionwasthenextracted with phenol and the RNA wasprecipitatedtwicewith ethanolin the pres- enceof0.3MNaOAc(pH5.0) asdescribed (29).Thepelletwas dissolvedin 10mM TrisHCl,pH 7.4, andincubatedinbuffer alone or in buffer containing 20 ,ug ofRNaseA/mlfor 30 min at22°C.TheRNAwasanalyzed byelectrophoresisin15%acryl- amide/7 M urea slab gels (30). Foroligonucleotide analysis, unlabeled RNAwasextracted asdescribedabove and thenin- cubated in10,ul of10 mMTris-HCl,pH7.4/1mMEDTA con- taining0.5

pAg

^{of T1RNase}(Worthington)for30min at37°C.

The T1 oligonucleotides were 5'-end labeled with 50 ,uCi of [

y-32P]ATP

(500 mCi/mmol), usingT4polynucleotide kinase (31) and thenanalyzed bytwo-dimensionalgelelectrophoresis (32) followedby autoradiography.

RESULTS

Isolation of SV40 T Antigen. We isolated SV40 Tantigen synthesizedⁱⁿCV-1culturesorprimary mousekidneycultures

Abbreviations: SV40,simian virus40;Tandtantigen, SV40largeand smalltumorantigen, respectively; araC,1-,B3D-arabinofuranosylcytosine.

Thepublicationcostsofthis articleweredefrayedinpartbypagecharge payment. This article musttherefore be hereby marked "advertise- ment'inaccordance with18U. S.C.§1734 solelytoindicate this fact.

1140 CellBiology: Khandjian et al.

undergoing lytic or transforming infection, respectively (21).

Mock- andSV40-infected cultures were labeled21-25 hrafter infection with [3S]methionine, [3H]- or[14C]uridine, or

32pi.

Atthe time of extraction, the percentage of T-antigen-positive nucleihad reached its maximum, as determined by immuno- fluorescent staining,correspondingto -100%and50% inCV- 1and mouse kidney cultures, respectively. T antigen wasiso- lated by immunoaffinity chromatography. Samples to be used for electrophoresis in NaDodSO4/polyacrylamide gels were eluted from the immune complexes with NaDodSO4 sample buffer (24) and samples to be analyzed by isoelectric focusing wereeluted with urea lysis buffer containing 9.5 M urea (25).

The two elutionmethodsgavesimilarrecoveries of T antigen, as determined by electrophoresis in NaDoSO4/polyacryla- mide gels. The recovery of T antigen and the results reported belowwerethe same whether infection took place in the pres- enceorabsenceofaraC.

Isoelectric Focusing of T Antigen in Sucrose Density Gra- dients. All attempts to focus in sucrose density gradients (26) 3S-labeled urea-elutedT antigen isolated fromCV-1 (Fig. 1) ormousekidney cultures (not shown) were unsuccessful: T an- tigen,detectedbyimmunoaffinity chromatography followed by NaDodSOpolyacrylamide gel electrophoresis, was broadly distributed between pH 4.5 and pH 9 (Fig. 1A). The distri- bution was the same if thesamplewasincluded in thegradient and it wasindependentofthe polarity ofthe electric field. Con- sideringthepossibility that T antigen might be complexed to nucleicacids,wetreated the immune complexes, while bound toproteinA-Sepharose, with RNase A. Theresults show that RNase-treatedTantigenfocused in distinct bands at about pH 6.0, 6.5,and 7.0(Fig. 1B);asexpected, these bands were absent inextractsofmock-infectedcultures(not shown). RNase treat- mentdidnotchange the migrationofTantigen inNaDodSO4/

polyacrylamide gels.

AnalysisofTAntigenby Two-DimensionalGelElectropho- resis.35S-LabeledTantigen extracted from SV40-infectedCV- 1cultures(withorwithout araC)waseluted with urea and im- mediately analyzed by two-dimensional gel electrophoresis (25). ThebulkofTantigen failedtoentertheelectrofocusing gel(Fig.2A) whereas other proteins(e.g.,actin)formeddistinct spotsatthe expectedpositions. Similar results have been re- ported by others (33-35). In contrast, the bulk ofRNase A-

_ 41

i4

0 r₀3

w

2i

x 1

..

tn._{m 0}

I.

A

A *8i b c d e f g h

A

.A

^.

,

^,

20 40 60 80 100

Fraction

__1 _- _.

a b c d e f g h

Poolforsecond immunoprecipitation

treated T antigen entered theelectrofocusinggel, forming ma- jor spots at about pH 5.5, 6.0, 6.5, and 7.0 (Fig. 2B). During thesestudies, we noticedthat treatmentof the immune com- plexes with DNase I, storageofurea-eluted T antigen for a few days at-20TC,orheating the samples for 1 min at 60'Callowed avariable fraction of

'S-labeled

Tantigen to enter the electro- focusinggel, forminganextended streak withoutdistinct spots.

Thesame results wereobtained with T antigen extracted from SV40-infectedmousekidneycultures (not shown).

Labelingof T Antigen withRadioactiveUridine or

32Pi.

To testwhetherpolyribonucleotides arecomplex'edto Tantigen, welabeled mock- orSV40-infectedCV-1ormousekidney cul- tures(withorwithoutaraC) with[3H]-or

[14C]uridine.

Aliquots ofTantigenboundtoproteinA-Sepharosewereincubatedwith RNase A or withbuffer alone. Extracts ofmock-infectedcul- turesweretreatedinthe same way. T antigen was eluted with NaDodSO4 sample buffer,boiledfor 90 sec,andthenanalyzed by electrophoresis in

NaDodSO4polyacrylamide

gels. In all samplesfrominfectedcultures, whether they had been treated with RNase or not, asingle bandofradioactivity wasobserved at the position of T antigen (Fig. 3); this band was absent in extractsofmock-infected cultures. Furthermore, thisbandwas absent when extracts ofinfectedcultures wereallowed to react with nonimmune hamster serum or with antiserum produced by inoculation of mice with syngeneic spontaneously trans- formedcells(T-AL/N;ref.36)provided byT.Rose(University ofGeneva). T-antigen preparations thathad not been treated with RNase also contained radioactivity in the stackinggel.

Radioactivity wascompletely removed bytreatmentofTanti- genpreparations with1 M KOHfor1hratroomtemperature beforegel electrophoresis (Fig. 3)orby incubatingthegelswith 5% trichloroacetic acid for30 min at 90'C (37) after electro- phoresis and beforefluorography (notshown). Tantigenfrom

32P-labeled

^{CV-1 or mouse} kidney cultures formed a single strongly labeledband(refs.4,8-11and Fig. 3).After treatment of the immunecomplexeswith RNase, no32pwasfoundinthe stacking gelsand-30% ofthe

32p

wasremovedfromTantigen.

Todetermine the chemicalnature of the 32P-labeled ^resi- dues,Tantigen wastreatedwith RNaseandsubjectedtoprep- arative NaDodSOJpolyacrylamide gel electrophoresis. T an- tigen was eluted from the gels and hydrolyzed with alkali or acid,and thehydrolysateswereanalyzed by high-voltagepaper

4 - 9

7

₅

4.L

_..

..4

... ^.

i..

a

b

c

d

e

f g h

^{5 Z}

3 3 88

2^F

20 40Fraction60

-., L _ _ ,L ^{-. -. - -}

a b c d e f g h i

Poolforsecondimmunoprecipitation

FIG. 1. Isoelectricfocusing of35S-labeled SV40Tantigeninsucrosedensitygradients.TantigenwasisolatedfromCV-1culturesbyimmu- noaffinitychromatography.The immunecomplexes^wereincubatedwithbufferalone(A)orwith buffercontainingRNase A(B). Tantigenwas

elutedwithurealysisbuffer(25)andsubjectedtoisoelectricfocusinginsucrosedensity gradients(26).RadioactivityandpHweremeasured in 0.25- mlfractions, whichwerethenpooledasindicated.Tantigenwasassayed byimmunoaffinity chromatographyfollowedbyNaDodSO4/polyacryl- amide(12.5%acrylamide)gelelectrophoresisandfluorographyfor 2 weeks(Insets).88,88-Kilodaltonposition.

Proc.Natl.Acad. Sci. USA 79

(1982)

Proc.Natl.Acad. Sci. USA 79(1982) 1141

---- ---IE

F

pH 5 6 7 8 9

IT- A

0 ^N

F Act--

in

z0

B

0.4 if) 0 0

Act-

z

FIG. 2. Analysis of 35S-labeled SV40 T antigen by two-dimen- sionalgel electrophoresis (25). T antigen^wasisolated fromCV-1 cul- tures.The immunecomplexeswereincubated withbuffer alone (A)or

withbuffer containing RNase (B). Urea-eluted T antigenwassubjected totwo-dimensionalgel electrophoresis. Fluorographywasfor 10 days.

IEF, isoelectric focusing;T,Tantigen; Act, actin.

FIG. 4. Analysis of 32P-labeled residues in SV40 T antigen. Im-

munecomplexes from CV-1 cultures labeled with32Pi^wereincubated with RNaseA. Tantigenwaseluted with NaDodSO4 sample buffer and purified by electrophoresis inNaDodSO4polyacrylamide(7.5%acryl- amide) gels. T antigenwaseluted from the geland lyophilized. Ali- quotswerehydrolyzedwith KOH (lane A)orHCl (laneC);onealiquot

wasnothydrolyzed (lane B). Samples^wereanalyzed by high-voltage

paperelectrophoresisatpH 3.5 (28) followed byautoradiography. P- Ser,phosphoserine; P-Thr, phosphothreonine.

electrophoresis. As shown by autoradiography, alkaline hy- drolysatesgavefourspotsof similarintensity,correspondingto UMP, GMP, AMP, andCMP(Fig.4). Inaccordance with the

Mr

xl-3

116- 97- 77^- 68-

ABOCD ^E

m

53

F G

-T

-H

FIG. 3. Labeling ofSV40 T antigen with [35S]methionine,32Pi,^or [3H]uridine. T antigen ^was isolated by immunoaffinity chromato- graphy from lysates ofSV40-infected CV-1 cultures and subjectedto electrophoresis inNaDodSO4/polyacrylamide(7.5%acrylamide) gels.

Lanes:AandB,Coomassie blue stainedmock-infected and SV40-in- fectedextracts,respectively; C and D, autoradiographs of 35 and 32p labeled T antigen, respectively, E-G; fluorographsof [3H]uridine-la- beledTantigen after incubation of immunecomplexes in buffer alone (E), with RNase (F),orwith 1 MKOH (G) for 60 min. T, SV40 T antigen;

H,gammaglobulin heavy chain.

resultsreported by others, acid hydrolysates contained phos- phoserine, phosphothreonine (8-11), and ^a third, unidenti- fied, component. Toexclude thepossibility that these results

wereduetofortuitouscontaminationwithRNA comigratingin

thegels withT antigen, wecarriedout thefollowing control experiments. Mock-infected CV-1 or mouse kidney cultures

werelabeled with32Piand lysed with NonidetP40, andthe ly-

sates were mixed with lysates from unlabeled SV40-infected CV-1or mousekidney cultures.Tantigenwasisolatedandsub- jected to electrophoresis in NaDodSOjpolyacrylamide gels.

NoradioactivitycomigratingwithT antigencould be detected.

Under the same electrophoresis conditions (7.5% acrylamide gels), mouse tRNA, 5S RNA, and 5.8S RNA migrated to the bottom ofthegel while Escherichia coli 16SrRNAremained inthestacking gel. Similarresults have beenreported byothers (37). When SV40-infectedCV-1^{or mouse}kidney cultureswere

labeled with[3H]thymidine,^noradioactivitycomigratingingels withTantigencouldbe detected byfluorography.

Small RNAFragments Are ComplexedtoTAntigen. SV40- infected CV-1^ormousekidney cultures (withorwithout^araC)

werelabeled with radioactive uridineor32Pi. RNase-treatedT antigenwas isolatedand subjected^topreparative electropho-

resis inNaDodSOjpolyacrylamide gels. T antigenwaseluted from the gel and digested withproteinase K. The digest^was extracted withphenol. The ethanolprecipitate was dissolved in10 mM TrisHCl,pH7.4;aliquots^wereincubated with buffer aloneorwithRNaseAandthenanalyzed by electrophoresisin 15%acrylamide/7Mureaslabgels.Fluorography of[3H]uridine- labeled samples showed ^adistinct band that had an electro- phoretic mobility correspondingtoapolyribonucleotideof =45 residuesand^wassensitivetoRNase;insomepreparations,this

B A

Pi- 0

C

Pi-

-P-Ser

UMP.-

-P-Thr

4

GMP-

AMP- CMP-

i

----U --- 40 ^- Origin Cell

Biology: Khandjian

etal.

1142 Cell Biology: KhandjianetaL

A B CD

n

*

75-

32-

I0-~

,i.

FIG. 5. Electrophoresis of T-antigen-associated RNA. CV-1 cul- tureswerelabeledwith[3H]uridineor32Pi.T antigen was isolated and purified,and RNA was extracted. Aliquots were incubated in buffer alone (lanesAand C)or inbuffercontainingRNaseA(lanesBand D).

Sampleswereanalyzed by electrophoresis in polyacrylamide gels con- taining15%acrylamide/7M urea (30). Lanes: AandB,[3H]uridine- labeled material, fluorography for12weeks; C andD,32P-labeled^ma- terial, autoradiographyfor 2 weeks. Migration markers weretRNA [75nucleotides(n)],xylene cyanol (32 n) and bromophenol blue (10 n).

band formedadoublet (Fig. 5).Autoradiographyofgelsloaded with32P-labeledsamples showed severalbands. In all samples that had been incubated with RNase, the bandcorresponding to that observed by fluorographywas absent. In 2 out of 10 preparations, a32P-labeledband having a mobility correspond- ing to 15nucleotideswasalsoremovedbyRNase.The chem- ical nature of this and the other RNase-resistant 32P-labeled band remains unknown.

Infurther experiments, weisolated T antigen from unlabeled

CdCD cL~

-

_0

0

(\J

SV40-infected CV-1 or mouse kidney cultures 24hr after in- fection(with or without araC) and extracted theTantigen-as- sociated RNA-as described above. The RNA was digested with T1 RNase, and the resultingoligonucleotides were phosphor- ylated by using T4 polynucleotide kinase and [y-32P]ATP and analyzed by two-dimensional gel electrophoresis followed by autoradiography. The patterns show the presence of TI oligo- nucleotides with 15-30 residues and poly(A), indicating that the RNAhas a rather highcomplexity (Fig. 6). Fig. 6 A and B also shows thatthe patterns obtained from T antigen-associatedRNA isolated from CV-1 or mouse kidney cultures are similar but not identical. The results were closely similar whether infection took place in the absence or presence of araC. As controls, we used gel strips excised from the same gels 1 cm above and below theT-antigenbandand alsogelstripsfromparallel gelsloaded withextracts from mock-infected CV-1 or mouse kidney cul- tures. In all controls, only trace amounts of oligonucleotides with 2-8residues were observed (Fig. 6C).

Theseresultsindicatethat SV40 T antigen protects RNAfrag- mentswith =45 residues fromdigestionby RNase A. The pro- tected RNA sequences are heterogeneous and may originate from pre-mRNAs or mRNAs as suggested by the presence of uniquelarge T1 oligonucleotides and of poly(A).

DISCUSSION

SV40Tantigenisolated fromSV40-infected monkeyormouse kidney cultures labeled with radioactive uridine always con- tained small amounts of RNase-resistant alkali-sensitive radio- activity. Furthermore, alkaline hydrolysates of RNase-treated gel-purified Tantigen containedaboutequimolaramountsof AMP, GMP, CMP,and UMP. These observations and the be- havior ofT antigenduringisoelectricfocusingsuggestedthat itmightbecomplexedwith RNA. Directevidence for thisas- sumption wasobtained bytheisolation ofRNAfragmentscom- prising ==45nucleotides from RNase-treatedgel-purifiedTan- tigen. Mapping of the 32P-end-labeled T1 oligonucleotides showed ahighsequencecomplexity,which wasemphasized by the presence ofunique T1oligonucleotideswith 15-30 residues and poly(A). ThedegreeofcomplexityofT-antigen-associated RNA wascomparableforTantigensynthesizedinlyticortrans- forming infection and was independent of cellular and viral DNAreplication. The results arecompatiblewith thehypoth-

C

b I

..~~~~~

^{. 4}

-m ^..II.t_

x

I10f acrylamide pH3.5/7Murea

FIG. 6; T1oligonucleotidemapsofT-antigen-associatedRNA.Tantigenwasisolated from CV-1 (A)or mousekidney cultures (B)infectedwith SV40 for25hr.T-antigen-associatedRNAwasextractedanddigestedwithT1 RNase, andtheresulting oligonucleotideswere5'-endlabeled with

[y?'PIATP

^andT4polynucleotidekinaseandanalyzed'bytwo-dimensionalgel electrophoresis(32) followedbyautoradiography for1day. (C)Ma- terial frommock-infectedCV-1 cultureswasisolated andprocessedinparallel.Extractsfrommock-infectedmousekidneycultures gaveessentially thesamepatternasshowninC.Markers; x,xylene cyanol (32 nucleotides); b, bromophenolblue(10nucleotides).

Proc.NadAcad.Sci. USA 79

(1982)

i:,;

.4

CProc.NatiAcad. Sci. USA 79(1982) 1143 esisthat theT-antigen-associated RNAfragments arederived

from cellular pre-mRNAs or mRNAs. The electrophoretic mi- gration of proteins in

NaDodSO4polyacrylamide

gels is not necessarily changed by boundRNAfragments (37); e.g., p19 of Rous sarcoma virushasthesame migration withorwithout acrosslinked viralRNAfragment of30-40nucleotides(unpub- lished results).

Theseresults and therecentobservationthatT-antigenmol- ecules are complexed to purified cellular messenger ribonu- cleoproteins isolated from polyribosomes of SV40-infected monkey or mouse cell cultures (ref. 38; M. R. Michel and M.

Schwyzer, personal communication), suggest that SV40 T an-

*tigen is a "RNA-binding protein." Earlier observations had shown that treatment of fixed cells with RNase A removed in- tranuclear SV40 T antigen,judged. by the immunofluorescence reaction, suggesting an association ofTantigenwithRNA(39).

Stimulation of nucleoplasmic transcription and increased synthesis ofcellular mRNAs and proteins are observed inboth lytic and transforming infection shortly after onset of SV40 or polyomaT-antigensynthesis (ref. 22; unpublished data). As a working hypothesis, we assume that T antigens induce this stim- ulation by modulating synthesis, maturation, and translation (or combinations thereof) of specific cellular mRNAs. This mode of action might account for the phenotypic reprogram- ming of the host cell observed in lytic and transforming infection.

WethankProf. L.PhilipsonandDr.A. J.Shatkinfor criticalreading ofthemanuscript, Mr. N. Bensemanneforpreparingthe cell cultures, andMr. 0.Jennifordrawingthefigures. Thisworkwassupported by Grants 3.474.79(toR.W.)and3.664.80(toP.-F.Spahr) from theSwiss NationalScienceFoundation andshort-termfellowships from theSo- cieteAcademiqueGeneveandtheInternationalUnionAgainstCancer (toM.L.).

1. Acheson, N. H. (1980)inMolecularBiology ofTumorViruses, ed.Tooze, J.(ColdSpringHarborLaboratory,Cold SpringHar- bor,NewYork),part2,pp. 125-204.

2. Ahmad-Zadeh, C., Allet, B., Greenblatt, J. &Weil, R. (1976) Proc. Natl Acad.Sci. USA 73, 1097-1101.

3. Crawford, L. V., Cole, C. N., Smith,A. E., Paucha, E., Tegt- meyer, P., Rundell, K. &Berg, P. (1978)Proc.Natl, Acad.Sci.

USA75,117-121.

4. Schwyzer, M., Weil, R., Frank, G. &Zuber, H. (1980)J. Biol Chem. 255,5627-5634.

5. Fiers, W., Contreras, R., Haegeman, G., Rogiers, R., Van de Voorde, A., Van Heuverswyn, H., Van Herreweghe, J., Vol- ckaert, G. &Ysebaert,M. (1978)Nature(London)273,113-120.

6. Reddy,V.B.,Thimmappaya,B.,Dhar, R.,Subramanian,K. N., Zain,B. S., Pan, J., Ghosh, P. K.,Celma, M. L. &Weissman, S. M. (1978)Science200,494-502.

7. Reddy,V. B., Ghosh, P. K.,Lebowitz, P.,Piatak, M. &Weiss- man, S. M. (1979)J.Virot 30,279-296.

8. Tegtmeyer, P., Rundell, K. &Collins, J. K. (1977)J. Virol 21, 647-657.

9. Edwards, C. A. F.,Khoury, G. &Martin, R. G. (1979)J.Virol 29, 753-762.

10. McCormick, F., Chaudry, F., Harvey, R., Smith,R., Rigby, P.

W.,Paucha,E. &Smith,A. E.(1979)ColdSpringHarbor Symp.

Quant.Biol44, 171-178.

11. Walter, G. &Flory, P. J., Jr. (1979)ColdSpringHarbor Symp.

Quant.Biol44, 165-169.

12. Goldman,N.,'Brown, M.'&Khoury,G.(1981) Cell24, 567-572.

13. Tegtmeyer, P.(1972)J.Virol 10, 591-598.

14. Lai, C.-J. & Nathans, D. (1974) Cold Spring Harbor Symp.

Quant.Biol39, 53-60.

15. Reed, S. I., Ferguson,J., Davis, R. W. & Stark, G. R. (1975) Proc.NatLAcad.Sci. USA72, 1605-1609.

16. Jessel,D., Landau,T.,Hudson,J., Lalor,T., Tenen, D. & Liv- ingston, D. M. (1976)Cell8,535-545.

17. Tjian, R. (1978)Cell13,165-179.

18. Reiser, J., Renart, J., Crawford, L. V. & Stark, G. R. (1980)1.

Virot33,78-87.

19. McKay, R. D. G. (1981)J.Mol BioL-145,471-488.

20. Weil, R.(1978)Biochim.Biophys.Acta516,301-388.

21. Weil, R., Salomon, C., May, E. & May, P. (1974) ColdSpring HarborSymp. Quant.Biol.39,381-395.

22. Khandjian, E. W., Matter, J. M.,Leonard,N. &Weil,R.(1980) Proc.NatlAcad.Sci.USA77, 1476-1480.

23. Schwyzer, M. (1977) Colloq. Inst. NatL Sante Rech. Med. 69, 63-68.

24. Laemmli,U. K. (1970)Nature(London)227,680-685.

25. O'Farrell, P. H.(1975)J. BiolChem.250,4007-4021.

26. Nelson,J. W.,Allington,W. B. &Aron, C. G. (1974)ISCO Ap- plications ResearchBulletinNo. 15,pp. 1-14.

27. Laskey, R. A. & Mills, A. D. (1975) Eur. J. Biochem. 56, 335-341.

28. Brownlee, G. G.(1972)inLaboratoryTechniquesinBiochemis- tryand MolecularBiology, eds. Work,T.S. &Work, E.(North- Holland,Amsterdam),Vol. 3, pp. 1-265.

29. Scherrer,K. (1969)inFundamental TechniquesinVirology, eds.

Habel,K.&Salzman,N. P.(Academic,NewYork),pp. 413-432.

30. Reijnders,L.,Sloof,P.,Sival,J. & Borst, P.(1973) Biochim.Bio-

phys.Acta324,320-333.

31. Richardson,C. C.(1971)inProceduresinNucelic AcidResearch, eds. Cantoni,G. L.&Davies,D. R.(Harper&Row,NewYork), Vol. 2, pp.815-828.

32. DeWachter, R. &Fiers, W.(1972) AnaL Biochem.49, 184-197.

33. Crawford, L. V. &O'Farrell, P. Z.(1979)J.Virot29, 587-596.

34. Greenspan,"D. S. &Carroll, R. B. (1979)Virology99,413-416.

35. Palme,K. &Henning, R. (1980)FEBSLett.118,229-232.

36. Smith,R. W.,Morganroth,J.&Mora, P.T.(1970)Nature(Lon- don)227, 141-145.

37. Bhorjee,J.S. &Pederson,T.(1976)AnaLBiochem.71,393-404.

38. Michel, M. R. & Schwyzer, M. (1981) Experientia 37, 654 (abstr.).

39. Gilden, R. V., Carp, R. I., Taguchi, F. & Defendi, V. (1965) Proc.NatLAcad.Sci. USA53,684-692.

Cell

Biology: Khandjian

^etal.