Two-dimensional analysis of proteins sedimenting with simian virus 40 chromosomes.

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JOURNAL OF VIROLOGY, Sept. 1980, p.854-864

0022-538X/80/09-0854/11$02.00/0 Vol. 35, No. 3

Two-Dimensional

Analysis

of Proteins

Sedimenting

with

Simian

Virus 40

Chromosomes

BARRYI. MILAVETZ,* LORETTA D.SPOTILA, RICHARDTHOMAS, ANDJOEL A.HUBERMAN DepartmentofViralOncology,Roswell Park MemorialInstitute, Buffalo,New York 14263

Thenonhistone proteinssedimenting in low-saltglycerol gradients with simian virus 40 chromosomes were analyzed by two-dimensional gel electrophoresis, utilizing nonequilibrium pH gradientsasthefirstdimensionandsodium dodecyl

sulfate-gel electrophoresisasthe seconddimension.Bydensitometricquantitation

of the radiolabeled proteins present in each fraction of the gradients, it was

possibletoidentify proteins sedimenting with allor afraction of the simian virus 40chromosomes. VP-1 sedimented with simianvirus 40chromosomes;additional

evidence for its binding to chromosomes was obtained by immunochemical techniques. Four proteins (Mr 25,000, pI6.0;Mr32,000,pI 7.2;Mr35,000,pl8.5;

andMr80,000, pl 7.2)sedimented with specificsubsets of chromosomes.

Oneapproachtothe identification ofproteins involved ineucaryotic DNAreplicationinvolves theisolation ofreplicatingchromatinand phys-icalorenzymatic characterization of theproteins found in association with thereplicating DNA. Replicatingpapovavirus chromosomesmay

pro-vide a useful model for the identification of proteins involved in cellular DNA replication because these chromosomes (i) are easily iso-lated inaformstructurallysimilartoeucaryotic chromatin (2, 9, 11, 19, 20, 22-24, 28,32-34,36) and(ii) replicatelikecellularchromosomes (12, 14-16, 18,43, 49,50).

Theproteinssedimenting withpartially puri-fied chromosomes of simian virus40(SV40) (the papovavirus chosen for this study) have been analyzed by physical andenzymatic techniques withsome success. By utilizingsodiumdodecyl sulfategelelectrophoresistoidentify proteins by molecularweight, SV40 chromosomes have been showntosediment with VP-1(10, 17, 23,26,28,

35,38,51,52), VP-2 and VP-3(28,38,52), cellular histone-Hi (6, 13, 23, 28, 31, 38, 54, 55), cellular histonesH2a, H2b,H3, andH4 (6, 17, 22, 23, 26, 28, 31, 35,37, 51, 52,55), and Tantigen (35,45). Inadditiontothesemajorproteins,anumber of as-yet-unidentified minor proteins have also beenobservedonmany of thesegels (17, 28, 35, 38,52). Bycomparison, SV40virus containsonly VP-1, VP-2, VP-3,andthehistones H2A,H2b, H3, and H4 (1,6, 7, 17, 21, 23, 28, 31, 35).The

enzymatic activities sedimenting with SV40

chromosomesincludeDNApolymerases (13,41,

42, 53), RNA polymerase (28), DNA relaxing

enzyme (25,28,53),andasingle-strand-specific

DNA binding protein (42). The variable amounts of proteins and enzymes reported to copurify withSV40 chromosomes are probably

due to the different isolation and purification

systems used in thedifferentreports.

Unfortunately, the physical analyseshavenot

unambiguously identified proteins functionally

associated with SV40 chromosomes. This is in part due to aninherent limitation ofthe tech-nique used. One-dimensional gel systems lack

resolvingcapacity; thus, quantitation ofprotein

bands is subject to the uncertainty that each

band may contain more than one protein.

En-zymatic techniques are not appropriate for all proteins; they require the availability ofan

ap-propriate assay. In addition, neither of these approachesallowsproofofassociation beyonda

demonstrationofsedimentation ofthe proteins ofinterest withSV40 chromosomes.

One method ofovercoming these dffilculties is to use a two-step approach. The first step

consists of two-dimensional gel electrophoresis (19) rather than the one-dimensional method previouslyused to assay acrossgradientsor col-umns used to purify SV40 chromosomes. The increased resolution oftwo-dimensional gel elec-trophoresis allows recognition of proteins which would be overlooked by the one-dimensional method.Inaddition, because individual proteins

are generally resolved from other proteins of similar molecular weight by two-dimensional

electrophoresis, it is possible to legitimately quantitate the amount of eachprotein in each fraction of thegradientorcolumn used for

pu-rifying chromosomes and thus determine un-ambiguously whether each protein comigrates

with the chromosomes of interest.

The secondstep involves the use of theprotein purified on the two-dimensional gel (or larger quantities of the proteinpurified from cells by using two-dimensionalgelelectrophoresisasan

854

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assay) toinduce the formation of antibodies in

mice or rabbits. After appropriate demonstra-tion ofspecificity,theantibodies could be used

to test (i)whether theproteinreally is boundto SV40 chromosomesand(ii) whattherole of the

proteinmight beinthe variousfunctions carriod

outby SV40 chromosomes.

Inthis report, we describe the identification of severalproteinswhich do anddonotsediment with SV40 chromosomes. In addition, we

pro-videimmunochemical evidence suggestingthat oneof theseproteins, VP-1,isactuallybound to

SV40chromosomes.

MATERIALS AND METHODS

Virusesand cells. SV40was agenerousgiftfrom PeterTegtmeyer andwaspropagatedat alow multi-plicity of infection in CV-1 cells (a gift from M. L.

DePamphilis). Cellsweregrownat37°Cina5%CO2

atmosphere inEagle modified minimal essential

me-dium (GIBCO Laboratories) supplemented with 5% fetal calf serum (GIBCO Laboratories). Virus was

plaqueassayedonBSC-1 cells. The virus hadatiter of109 PFU/ml.

Infection of cells. CV-1cellswereseeded into 10-cmplasticpetridishes and infectedduringlogarithmic

growth with2 ml of mediumcontaining2%fetal calf

serumand50PFUof virus per cellor novirus in the

case ofmock-infected cells. After 2 h ofadsorption

with shaking at 15-min intervals, 8 ml of medium containing 2% serum wasadded, and incubationwas

continuedat37°C.

Radiolabeling of SV40 chromosomes. Plates of infectedcellswereincubatedat40 to 42hpostinfection

with methionine-free medium for 15 min and then labeled for10 to 15minor 3h withamixture

contain-ing0.5 to 1.0mCi of[3S]methionine (specific activity,

600 to 800 Ci/mmol) and 250 yCi of[3H]thymidine

(specific activity, 400 Ci/mmol) in 2 ml of minimal essential mediumlackingmethionine. Identification of nonreplicating chromosomeswasaccomplished by

la-beling at 40 to 42h with0.2 ,uCi of['4C]thymidine

(specificactivity,40.1mCi/mmol)perplatefor3 hin

10ml of medium. The mediumwasremoved before cell harvesting, and, to identify replicating chromo-somes, 100,uCiof[3H]thymidine(specific activity,49.1 Ci/mmol)in2ml of fresh mediumwasaddedtoeach plate. After10minof incubationat37°C,the medium

wasremoved, and the ceUswerewashed with ice-cold

phosphate-buffered saline andkept oniceuntil har-vested. The[14C]thymidine labelwasfound incorpo-ratedprimarily into nonreplicating chromosomes by thistechnique, whereas the[3H]thymidinewas incor-poratedprimarilyintoreplicatingchromosomes.

Isolationof nuclei.Cellmonolayerswerewashed

twice with5ml of coldphosphate-buffered saline per plateorflask, followed by washing with5ml of buffer

A (5%glycerol, 2 mMHEPES [N-2-hydroxyethylpi-perazine-N'-2-ethanesulfonic acid], pH 7.5, 0.5 mM MgCl2,0.5mMdithiothreitol,0.5mM

phenylmethyl-sulfonyl fluoride). After 5 min ofswelling, the cells

weresuspended witharubberpoliceman and

centri-fuged at800 x gfor 10min. The packed cellswere

suspended in 0.2 ml of buffer A perplate,mixedwith 0.2ml of 0.5% Triton X-100 per plate, layeredonto 8

volumesofbufferA,andcentrifugedat800xgfor 10 min. Thepelletwaswashedby suspensionin 0.4 mlof bufferA perplate and centrifuged through buffer A,

asdescribed above.

Extractionof nuclei and glycerol gradient sed-imentation. Thenuclear pelletswereextracted with

1 ml of buffer B (10 mM HEPES, pH 7.5, 1 mM EDTA,0.5mM dithiothreitol,0.1 mM phenylmeth-ylsulfonyl fluoride) for2honice.After the removal of the nucleardebrisbycentrifugationat10,000xgfor 15 min, the supematant SV40 chromosome extract waslayeredonto 10ml of a 10 to 30%(vol/vol) glycerol gradient in buffer C (10 mM HEPES,pH 7.5,5mM

KCl,1mMEDTA,0.2mMMgCl2,0.5mM dithiothre-itol,0.1mMphenylmethylsulfonyl fluoride) and

cen-trifugedat40,000 rpmand4°C for135min inanSW41

rotor. After centrifugation, gradients were

fraction-ated, and samples were analyzed by liquid scintillation counting.

Two-dimensional electrophoresis. Samplesfor electrophoresiswereprepared by adding to each gra-dientfraction20

jig

of bovineserumalbumin, 20,tgof RNase A(Worthington Biochemicals Corp.) and, after

a 10 minincubationat4°C, cold trichloroacetic acid (final concentration, 10%); this wasfollowedby cen-trifugationat10,000xgfor10min.Precipitates were washed once with cold acetone and centrifuged at 10,000xgfor10min.Electrophoresis conditions were

essentiallythesame asdescribed previously (40), with minorexceptions. Nonequilibrium pH gradient elec-trophoresiswasinaBio-Rad model 220 apparatus at

400Vfor3.25hinslabgels containing 2% ampholines (LKB), pH3.5-to 10.After electrophoresis, lanes were

cut out andtransferred to the second-dimension

so-dium dodecylsulfate-gel (10% acrylamide) in a Bio-Radmodel221apparatusandelectrophoresedat80 V overnight. The acrylamide stock solution contained

30%o acrylamide and 2.25%diallyltartardiamide. Gels weredriedon aBio-Rad model 224 slab gel dryer and exposed for varyingperiodsof timetoKodak XR-1 X-rayfilm.Development was in a Kodak model MCA-N RPX-Omat processor.

Identification ofproteins.Anti-SV40 and

preim-mune sera were purchased from Flow Laboratories

andwereusedat1:10dilutions in 0.67% agarose inan

immuneoverlay(47). Actinwaspurchased fromSigma Chemical Co. Tubulin was a gift from Joanna B.

Olmsted, and CV-1 nuclear matrix wasprepared by Ronald Berezny (3). Digestions were performed by the method of Clevelandetal. (8).

Electronmicroscopy.Sampleswerepreparedfor electron microscopy by fixation with formaldehyde andglutaraldehyde, using the method ofGriffithand Christiansen(24). Beforeadsorptiontocarbon-coated, glow-dischargedgrids,thesampleswere made 0.15 M

with NaCl.Gridswerethenwashed, dried,androtary shadowed at an 8° angle with tungsten. Microscopy

wasperformedwithaHitachiHU-11Aelectron micro-scopeat 50kV.

RNase Adigestions.RNaseA(Worthington Bio-chemicals) was heatedto 95°C for10 min and then spun at 10,000 xg for 15 min. Crude chromosomes

were divided intotwofractions, andone of the

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856 MILAVETZ ET AL.

tions was digested at 0°C for 10 min with 1 yg of RNase perml. The other fractionwasincubated with-out RNase. This RNase concentration (1,ug/ml) di-gested labeledcellular RNA fromSV40-infectedcells but not DNA. Thesampleswerecentrifugedon

glyc-erol gradients, as described above.

Quantitationofproteins.Forquantitationof la-beledproteins,allsamplesfromasingle gradientwere

prepared atthe sametime, as described above. Elec-trophoresis conditionswerekeptconstant. When all fractionshad been electrophoresed, the driedgels were exposedto the same lot ofX-ray film for the same

periodof time and developed atthesametime inan

automatic developer. Protein spotsof interest were

scannedon aComing model750scanning densitome-ter,sequentially oneachgel. Scanningwasdone

lin-early throughthecenterof eachprotein spot. Molec-ularweightcalibrations werecarriedout by electro-phoresis ofprotein markers (bovine serum albumin,

ovalbumin, chymotrypsinogen,andcytochrome c) in alaneadjacenttothefirst dimension during electro-phoresis in the second dimensionorby identification of the positions within the two-dimensional gel of bovineserumalbumin(addedascarrier),tubulin, and actin.

Effect of antibody on the sedimentation of SV40 chromosomes. Pooled nonreplicating SV40 chromosomes were divided into three portions and thenincubated withorwithoutanti-SV40 horse serum for10minat0°C.Onecontrolwasincubated without additions, and the secondwasincubated with 10

pLl

of

preimmune horse serum (Flow Laboratories). The thirdportion wasincubated with 10 llofanti-SV40 horse serum (FlowLaboratories). The portions were centrifuged as described above, and the position of the DNAlabel in each gradient was determined.

RESULTS

Extraction and partial purification of SV40 chromosomes. SV40 chromosomeswere

isolated from infected CV-1 cells byaprocedure which maximized the yield of SV40

chromo-somes capable of in vitro DNA synthesis (53). Infected cells were treated with low-ionic-strength buffer containing

Mg2".

After a brief Triton X-100 treatment to release nuclei and afterwashing of the nuclei withbuffer,the chro-mosomes wereextracted from the nuclei without Dounce homogenization by leaching into low-ionic-strength buffer containing EDTA. The SV40 chromosomeswereseparatedfromsoluble proteinsandcellulardebrisbycentrifugationon low-ionic-strength, 10 to30%glycerol gradients.

Aspreviously described, thismethod also pro-duces a partial separation ofreplicating SV40

chromosomes from nonreplicating SV40 chro-mosomes (10,13, 16, 17, 42, 50,53,54).

[36S]methionine-labeled SV40

chromo-somes. To characterize the

proteins

cosedi-menting with SV40 chromosomes and also to monitor contaminating cellularproteins, itwas

necessary toradiolabel infected cells. The best

labeling conditions consisted of starving cells

with methionine-free medium for 15 min,

fol-lowed by labeling with 0.5 to 1.0mCi of [35S]-methionine per plate of infected cells in

suffi-cient mediumtokeepthecellsmoist. With this

procedure itwaspossibleto obtainproteins

la-beled sufficiently foranalysis ofindividual

gra-dient fractions. When nuclear extracts of

in-fected cells labeled for 15min or 3 h or

mock-infected cells labeledfor 3 h with [35S]methio-nine and[3H]thymidine weresedimented on 10 to30%glycerolgradients, the patterns of radio-activity shown inFig. 1A, B, and C, respectively,

were obtained. DNAwhich was labeledfor 15 minusually showedabiphasicdistribution (Fig.

1A). Because 15 minisapproximately the time

necessaryfor oneround ofreplication by SV40 chromosomes, the two peaks corresponded to

replicating chromosomes and to newly

com-pleted chromosomes. Therewasmuchless DNA in the mock-infected nuclearextracts, and the littleDNA presentsedimentedmoreslowly(Fig.

1C). The patterns of labeled protein were

sub-stantially the same in all cases. There was a

peak at the top of the gradient, a small peak sedimentingslightlymoreslowly than the SV40 chromosomes, andanaccumulation atthe

bot-tom.

Two-dimensionalelectrophoresis of

pro-teins sedimenting with SV40

chromo-somes. Individual fractions from glycerol

gra-dientslike those showninFig. 1were analyzed bytwo-dimensionalgel electrophoresis,utilizing

anonequilibrium pH gradientasthefirst

dimen-sion and sodiumdodecyl sulfate-gel

electropho-resis as the second dimension (40). The

none-quilibriumpH gradientwaschosen for the first dimension in ordertoobservebasic proteins as

wellasacidicproteins. The conditions whichwe

usedwere optimized for theseparation of

non-histone proteins (that is, acidic to moderately basic proteinswith molecularweights between 15,000 and 100,000). Figure 2 shows

autoradi-ogramsof theproteins from fractions containing replicating and nonreplicating chromosomes which were obtained after labeling with

[35S]_

methionine for 3 h (Fig.2A) or for 10 min (Fig. 2B).Figure 2Cshowsacomparablefractionfrom

a mock-infected, gradient-purified nuclear

ex-tractlabeledfor3h. It isapparent thatallthree

autoradiograrns

containedanumber ofproteins

in common (for example, actin; see below for

protein identifications). The autoradiograms

from SV40-infected cells contained a group of

proteins greatly enhanced in intensity relative totheproteinsinmock-infectedcells, including VP-1, P-1 through P-4 (easily identifiable pro-teins which werequantitatedand shown to sed-iment with SV40 chromosomes), and a large J. VIROL.

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groupof other proteins less intensely labeled. In the shorter pulse, these proteins were less in-tensely labeled relative to actin and tubulin. Although thepatternof labeled proteins and the relative intensity of label in each protein were similar in various preparations of SV40 chro-mosomes, the absolute intensity of each spot and its absolute position dependedonthe label-ing conditions used andonthe batch ofreagen;s used forelectrophoresis. Small variations in po-sition (compare actin and VP-1 in Fig. 2A and B) and in intensity were observed frequently. Many of the less intensely labeled proteinswere

not apparentinthe photographic reproductions. To observe them, itwas necessarytoinspect the original autoradiograms. Although Fig. 2

in-Q

9

I

cludes the histone regionof theautoradiogram,

individual histones are notapparent. This was probably duetoaselective loss of histones dur-ing sample preparation in 10% trichloroacetic acid, a relative absence of methionine in

his-tones,andalack of resolutionattheverybasic low-molecular-weightrangeof thegel.

When chromosomes were prepared from

in-fected BSC-1 cells instead of CV-1 cells, the sameproteinspotswerefoundasinFig. 2 (data

notshown).

Identification of proteins. VP-1 was iden-tifiedby the following three criteria: (i)presence in infected cellular extracts and absence in mock-infectedextracts; (ii)presenceinfractions ofpartially purified virus; and (iii) positive

re-a

a

5 t0 IS 20 2! FRCTIO NUMBER

b

a

iI

FIG. 1. Glycerol gradient sedimentation ofnuclear extracts obtainedfrom [35S]methionine- and

[3H]-thymidine-labeledCV-1 cells. Nuclearextractswereobtainedfrom infectedcells labelcdfor15min (A)and

3 h(B)andfrom mock-infectedcells labeledfor3 h(C)andpurifiedasdescribed in thetext.Samples (25 ,Il) from each fractionwerecounted.Symbols:0,[35S]methionine; U,[3H]thymidine.RCand NRC indicate the

position of replicatingandnonreplicating ch-omosomes, respectively.

.

e.0

FIG. 2. Two-dimensionalgel electrophoresis of [35S]methionine-labeledproteins sedimenting with SV40

chromosomes.Sampleswereprepared and analyzedasdescribed in thetext. (A) 3-h label,infected cells. (B)

10-min label, infectedcells. (C) 3-h label, mock-infected cells. The numbersonthe left sides of the gelsare

molecularweights.

w f _ §~~~~~00

50 *5 20 2

'20 50

* I5

to / 2

5 10 15- L205 FRACT1O NUMBEt

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858 MILAVETZ ET AL.

action in the immune overlay technique (47) whenanti-SV40serum wasused.Proteins

react-ing with the antiserum showed a halo around theoriginalposition of thetestedproteininthe two-dimensional gel (Fig. 3). Twoforms of VP-1wereobserved,namely,VP-1, the majorform, andVP-1*,aminor form whoseintensityvaried from preparation to preparation. No reaction

wasobserved withpreimmuneserum.Actinand

tubulinwere identifiedby comigration of unla-beled authenticsamples with labeled SV40 chro-mosome-containing gradientfractions,followed by protease digestion of the individual spots,

using the method of Cleveland et al. (8). Al-though tubulin containstwo subunits, onlyone

largespot wasobservedinthissystem.Thiswas

perhaps due to alack ofresolution atthevery

acidic end of thegel. The nuclear matrix protein MTXa was identified by comigration with

au-thenticsamples preparedby the method of

Ber-ezny andCoffey (3).Figure4showsaschematic

representationofsomeof theproteins sediment-ing withSV40 chromosomes. The identified

pro-teins(VP-1,actin,tubulin,andMTX-a),aswell as additional proteins sedimenting with SV40 chromosomes (P-1 through P-4),werelabeled.

A

J. VIROL.

Electron microscopic analysis of gra-dient fractions. Fractions ofgradients obtained during purification ofSV40 chromosomeswere

analyzed by electron microscopy to determine

the morphologyof the SV40chromosomes and to monitor the contamination of the

chromo-somesby cellular debris. As previously reported, nonreplicating chromosomes appearedas beads onstrings (11) (Fig. 5C).Inaddition, therewere

othermorphological forms similar tothose

ob-tained frommock-infected cells (Fig. 5D).There were novirus-like particlesintheregion of

non-replicating chromosomes or replicating

chro-mosomes(Fig.5B), but suchparticles appeared

in the bottom fractions of the gradients (Fig.

5A). Although there wereparticles in fractions enriched forreplicatingchromosomes (Fig. 5B) which appeared similar in shape to compact chromosomes (9, 55), additionalevidencewould beneeded toidentify these particlesascompact

chromosomes. However, the lack of obvious

beadedchromosomes in theregion of replicating chromosomes suggests that some of the larger particles present were compact chromosomes. We can deduce from the specific activitiesand

distributions of14C and 3H inthe gradients la-beled for nonreplicating and replicating SV40 chromosomes, respectively, thateveningradient regions enriched for replicating chromosomes

ACIDIC

70,000H

B

55,000

42,000H

FIG. 3. Immune overlay identification of VP-1.

Two-dimensional gels of SV40 chromosomes were

obtained, andtheregionof interestwasenclosed in arectangularform (4 by8cm)(47). Then,anti-SV40

serum (1:10dilution) (in0.67%oagarose) at56'Cwas

poured intotheform. Thegel and overlay were

in-cubatedat37°C inamoist atmosphere for4 h, and

the agarose overlaywasremoved and washed in a

0.1Mphosphate buffer(pH 7)containing0.1 MNaCl for18h with buffer changes. The agarose overlays

werethen driedandautoradiographed. (A) Autora-diogram of duplicate oforiginal gel. (B)

Autoradi-ogramof immune overlay.

MTX-o-0

BASIC

* -P4

-Tubulin---Actin

VPi

VP1 P2

P3

.-Pi

FIG. 4. Schematic representation ofthe most

in-tensely labeled proteinssedimentingwithSV40

chro-mosomes. The identified proteins and proteins of

interest are labeled. The numbers on the left are

molecularweights.

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859

FIG. 5. Analysis of glycerol gradient fractionsby

electronmicroscopy.(A)Fraction frombottom of

gra-dient containing virus-like particles. (B) Gradient fraction containing replicating chromosomes. (C)

Gradient fraction containing nonreplicating

chro-mosomes. (D) Gradient fraction comparable to (C)

frommock-infectedcells. Bar=150nm.

(Fig. 5B), the majority of chromosomespresent werenotreplicating.

RNase A digestion of SV40 chromo-somes.Both the two-dimensional gels and the electron micrographs suggested that the chro-mosome-containing gradientfractionswere

con-taminated with cellular material. Labeling with

[35S]methionine

(Fig. 1) and with [3H]uridine (data not shown) suggested that the contami-nation included proteins and RNAs,

respec-tively. To exclude the possibility that the heavily

labeled proteins detected on two-dimensional gelswereassociated withRNA, both crude

nu-clearextracts andpartially purified SV40 chro-mosomes weretreatedwithRNaseAandfurther purified by glycerol gradient centrifugation. Analysesofthe fractionscontaining SV40 chro-mosomes by two-dimensional gels showed that theheavilylabeledproteins(with the exception of P-2andP-3, whichweredetectablyreduced) werenotappreciably reduced and, in thecaseof VP-1, actin, and tubulin,actuallyincreased(data

not shown). Electron microscopy showed that the increase in the amountof VP-1 mayhave been duetothe presenceofvirus-likeparticles inthe fractions containingchromosomes,which wereapparently freedby RNase fromaggregates

normally found atthe bottom of the gradient. The increase in the structural proteins actin and tubulinwasalsoprobablyaresult ofdigestion of aggregates.

Resedimentation ofSV40 chromosomes.

Inanattempttopurify SV40chromosomes

fur-therandtoremove contaminating proteins, in-dividual chromosome-containing fractions

which had been partially purified by gradient centrifugation wererecentrifuged on 10to 30%

glycerol gradients. Interestingly,

resedimenta-tion had little effect on the S values of the

replicating and nonreplicating chromosomes (data not shown). Although there were minor

lossesof someprotein, all of the major proteins

whichsedimented withreplicating or nonrepli-cating chromosomes were found after

resedi-mentation (datanotshown).

Quantitationofproteins in gradient frac-tions. One way of testing for association of

proteins withSV40chromosomes isto measure the amount of aproteinofinterest andcompare

its sedimentation with that of the SV40

chro-mosomes.Thisprocedure, whichisanalogousto anenzyme assay, does notrequire the

identifi-cation of anenzymatic activity. The amountof

eachlabeledprotein of interest in each fraction of thegradientswasmeasuredbydensitometry

ofthe autoradiogram for thatprotein,

normal-ized to the peak intensity of VP-1, and finally plotted against its fraction number. The calcu-lations for normalized intensitieswerebasedon

densitometric peak heights obtained by linear

scans with a 5-mm slit throughthe centers of two-dimensionalspots.Peakheights obtained in this way may not have been linearly

propor-tional to the totaldensity of label in the spot.

Unfortunately, we didnothave theequipment required to measuretotaldensity accurately (a microscanning densitometer). Proteins which

werestudied aftera3-hlabel anda10-minlabel included structuralproteins (actin, tubulin, and MTXa)and otherproteinsof interest(VP-1, P-1,P-2, P-3, and P-4). Aftera3-hlabel(Fig. 6A), VP-1 was distributed in several peaksthrough thegradient; thesewereatthebottom, ahead of thereplicating chromosomes, near the

nonrep-licating chromosomes, and near the top. The distribution of VP-1 after a 10-min label was

similar (Fig. 6B),exceptthat thepeaknearthe

top wasmuchlarger.

Of theproteins which sedimented in the

chro-mosome region, P-1 (approximate molecular weight, 25,000; approximate pI, 6.0) sedimented in the region ofreplicating chromosomes after both 3-h and 10-min labels (Fig. 6A and B). Although proteins P-1 throughP-4 didnot ap-pear aslargepeakswhennormalizedagainstthe intensityofVP-1,the differences between their peak intensities and background were between

one and two orders ofmagnitude. It was not

possibleto measuretheamountofP-1atthetop ofthegradientbecause of thecomplexityof the

autoradiograms.Incontrast, aftera3-hlabel, P-2(molecularweight,35,000;approximatepI,8.5)

and P-3 (molecularweight, 32,000;approximate pI,7.2)sedimentednearthenonreplicating

chro-mosomes(Fig.6A andC, respectively).In addi-tion, therewas apeak of eachproteinnear the top of thegradients. After a 10-min label (Fig.

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860 MILAVETZ ET AL.

B

NRC

RC ₁₂₀

( 100

z w

I-Z 80 0.

> 60

o 20 -0 0

FRACTION NUMBER

NRC

Re

2 4 6 8 10 12 14 820 FRACTION NUMBER

24 28

NRC

D__

NRC RC

,~~~~~~~

-. . t

--2 4 6 8 10 1--2 14 161820

FRACTION NUMBER

120r

z

a a.

x

44

a2

100

80

60 40~ 20

24 _{2 4 6 8} _{10 12 14} I820 24 28

FRACTIN NUMBER

FIG. 6. Quantitation of radiolabeled proteins in glycerolgradients. The intensitiesof the proteins were

measuredasdescribed inthetext. (A) 3-h label.Symbols:0, VP-I;A,P-i; ,P-2. (B)10-minlabel.Symbols:

*, VP-I;A,P-i; ,P-2. (C)3-hlabel.Symbols:0,P-3;A,P-4. (D) 10-min label. Symbols:@,P-3;A,P-4.RC

andNRCindicate thepositions ofreplicating and nonreplicating chromosomes, respectively.

6BandD) thepatternwassimilar, but the peak near the top wassubstantially increased in in-tensity relativeto thepeakcosedimenting with the chromosomes. A portion of P-4 (molecular weight,80,000; approximate pl, 7.2) sedimented with SV40 chromosomes aftera 3-hlabel (Fig. 6C), but after a 10-min label there wasa peak onlyatthetopof thegradient (Fig. 6D).

Although structural proteinswerepresent in thesamefractions asSV40 chromosomes after a 10-min or3-h label (Fig. 7A and B), a 10- to 100-foldexcessofeach proteinsedimentedmore slowly than the SV40chromosomes. Therewas no indication ofa specific association ofanyof

theseproteins with SV40chromosomes. It isof

interest that thepatternsofsedimentation after

3-hand10-min labelswereverysimilar. Antibody to VP-1 bound SV40 chromo-somes. The evidence presented abovesuggests

thatVP-1andseveralotherproteins sedimented

withSV40 chromosomes. To determine whether

VP-Iwasactually boundtoSV40chromosomes, the effect of anti-SV40 serum on the sedimen-tation of SV40 chromosomes was tested. The antiserum utilized had been shown previously by immuneoverlayto reactwith VP-1 (Fig.3). The effectsofantiserum, preimmuneserum,and notreatment onthesedimentation of nonrepli-cating chromosomes are shown in Fig. 8. Al-though the sedimentation of the chromosomes wasaffectedslightly by preimmuneserum,there was a much greater shift after treatment with anti-SV40serum.Themorphologiesof the chro-mosomes which werereacted with either anti-serum orpreimmune serum werenot apprecia-bly different (datanotshown)from those ofthe

untreated control (Fig. 5).A similar effectwas observed when replicating chromosomes were

used.Thus, VP-1isprobablyassociated with at

leastafraction ofthenonreplicating and repli-cating SV40 chromosomes isolatedatlowionic

strength. Because the specificity ofthis antise-A

z

J. VIROL.

c

120 100 80 60 40 20 0_

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I-x 4x 2

at

M16 I

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11

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A

120

i-z

u,

z

-a.

x 4

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0

at

NRC RC'

1001

80 60

201

4 6 8101214161820 24 28 FRACTION NUMBER

B 120

100

z

Z 80

a-> 60

X 40

4 &. 20

0

at,

NRC RC

FRACTION NUMBER

FIG. 7. Quantitation of radiolabeled structural proteinsinglycerol gradients. The intensities ofthe

proteinsweremeasuredasdescribed in thetext. (A)

10-min label. Symbols:0,actin;A,tubulin; U,

MTX-a. (B) 3-h label. Symbols: 0, actin; A, tubulin; U,

MTX-a. RC and NRCindicate thepositions of

repli-catingandnonreplicating chromosomes,respectively.

rumfor VP-2orVP-3 hasnotbeendetermined, it ispossible thataportion of the antibodymay bindtotheseproteins also.

DISCUSSION

The nonhistone proteins cosedimenting with partially purified SV40 chromosomes, which have been isolated by a low-ionic-strength method that maximizes the yield of chromo-somescapable of invitroDNAreplication (53), showasubstantialheterogeneity when analyzed by two-dimensional gel electrophoresis. This is due partlyto the increasedresolution inherent in the two-dimensionalsystemandpartlytothe increased number ofproteins retained by SV40 chromosomesatlowionicstrength.

I 2 4 21 1 02 42

x 10

us

6-4

2

2 46 8

I0

21416IS2022 2426

FRACTION NUMBER

FIG. 8. Effect ofanti-SV40antibodyon sedimen-tation ofnonreplicating SV40 chromosomes. Par-tiallypurified nonreplicatingSV40 chromosomes la-beled with[3H]thymidinefor3hwereincubatedat 40Cwith noantibody (A), a 1:50dilutionof

preim-mune serum(A),or a1:50dilutionofanti-SV40serum

(0) for10min and thensedimentedon a 10 to30% glycerolgradient.

Amongtheproteins identifiedaschromosome associatedare twoforms ofVP-1.Multiple forms ofVP-1 have beenobservedpreviously both by one-dimensional analysis (44) and by

two-di-mensional analysis (39). The second form of VP-1 (VP-1*)isfound in variableamounts, and it is notclear whether it isadegradation product,as

reported previously (44), orit has a functional significance.

Byquantitating theamountof labeledprotein in eachgradientfraction,it waspossibleto

dem-onstrate partial or complete overlap between peaksofnonreplicatingorreplicatingSV40

chro-mosomes andpeaks of VP-1 (majorform) and induced proteins P-1 through P-4. In contrast,

the structural proteins studied (actin, tubulin,

and theprincipal matrix protein MTX-a) donot have separatepeaks which sediment with SV40 chromosomes, suggestingthe absenceof associ-ation. The quantitation technique, although

veryuseful,was notsufficiently sensitive inour hands to detect reproducibly minor changes in

protein concentration. The maximumintensity

of theproteinsdiscussed here is 10- to 100-fold greater than the minimum intensity. The sedi-mentation positions of all of these proteins do not appeartobeappreciablysensitive to RNase A treatment or to resedimentation. Although quantitationhas notbeen utilizedpreviouslyto

show cosedimentation ofVP-1 and SV40

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862 MILAVETZ ET AL.

mosomes, VP-1 hasbeen observedpreviouslyin

gradient fractions in which SV40 chromosomes arefound (10, 17, 23, 26, 28, 35, 38, 51,52). The pattern of VP-1 sedimentation observed here, withlargeramountsofVP-1cosedimenting with SV40 chromosomes ofhigher S value instead of with the nonreplicating chromosome peak, is consistent with theproposal that SV40 virus is formed by thegradualassociation of 1,

VP-2, and VP-3 with SV40 chromosomes (9). Be-cause ofthe potential for SV40 virusto be de-graded during the isolation of SV40 chromo-somes (46), it is not possible to exclude the possibility thatsome of the VP-1 observedwas

duetothe presence ofpartially degraded virus

or virus precursor particles. However, it is

un-likely thatdegradationwasamajor contributor

to the VP-1sedimentation pattern because the pattern was observed after a 10-min pulse, a

time tooshort for virustobeencapsidated and extensively degraded; and degradation was not adynamicprocessafter isolation since the rela-tive amount ofVP-1 and the position of

chro-mosomes was not changed after resedimenta-tion. Third, our isolation conditions yielded

amountsof chromosomes andVP-1 whichwere very similar to those obtained by conditions which minimize virus disruption (17). It does

seem clear from experiments with antiserum against VP-1 that VP-1 is actually associated withSV40 chromosomes and doesnotsediment with them for other reasons. The question of

whether the VP-1observed is due tomaturing

ordegrading SV40 virus is under further inves-tigation.

Of the other proteins which sediment with SV40 chromosomes, P-1 (molecular weight,

25,000;pI6.0)maybe associatedwith replicating chromosomes. In contrast, P-2 (molecular weight, 35,000; pI 8.5), P-3 (molecular weight,

32,000; pI 7.2), and P-4 (molecular weight,

80,000;pI7.2)appear tosediment with

nonrep-licating chromosomes. The functions of these proteins have not yet been determined. How-ever, the similarity among P-2, P-3, and the

minorspots found between P-2 and P-3 in terms of bothmolecular weightsandisoelectricpoints

(Fig. 2) and thesimilarity of the peptides after protease digestion suggest that these proteins

may belong to one of the principal groups of

proteinsassociated with heterogeneousnuclear RNA (4). The fact that they cosediment with

nonreplicatingSV40 chromosomes is consistent with their association with newly transcribed RNAstill linked to SV40 chromosomes (28; H. J.Edenberg,unpublisheddata). Thishypothesis is also underinvestigation.

Just asenzyme assays have shown the

induc-tion ofenzymes (29) and the sedimentation of DNA polymerases (13, 41, 42, 53) and DNA

relaxingenzyme(25,28,53) with SV40

chromo-somes,themethods described here demonstrate

asimilar induction and cosedimentation of

pro-teins based on their physical characteristics. However, thefollowingtwoproblemsremainto

be solved: the identification of additional

pro-teins which maybe associated with SV40

chro-mosomesandcharacterization of the function of all associated proteins. The first problemmay

be made simpler by obtaining chromosomes of

greaterpurity byusing additional techniques for the purification of the isolated chromosomes, such as agarose gel electrophoresis (17, 55) or agarose gel

filtration

(53).

To test functional association between

pro-teins of interestandSV40 chromosomes,we are

utilizing three approaches. First, we are using

mutants ofSV40temperaturesensitive for

rep-licationor maturation, suchas tsA58 or

tsBil.

It should be possible to identify the proteins whicharereduced after a temperature shiftto

restrictivetemperature. These experimentsare

in progress. Second, immunological techniques also havepotentialforanalyzingthefunctional associations ofproteins (27, 48). Specific anti-bodiestoparticularhistones havebeen usedto show thatparticularhistonesarepresentin

nu-cleosomes(5).Antibodytohistonewasshownto

cause a shift in the sedimentation of

nucleo-somes (asreportedhere foranti-VP-1) andwas also identified by electron microscopy on the nucleosome (5). The sedimentationshiftwhich

we observed with preimmune serum was also reportedpreviously andwasduetononspecific binding (5). Thiswassubstantiallyreduced after purification of the immunoglobulin fraction. It is obvious that this approach requires highly purified,

high-affinity

immunoglobulin. The

as-sociation ofTantigen with SV40chromosomes

has also beensuccessfully probed immunochem-ically (45). We are presently preparing anti-bodiestoproteins purified by two-dimensional gels. In addition, proteins cosedimenting with

SV40 chromosomes are being used to obtain

antibodies by the hybridoma technique (30).

Finally,theproteins alreadyidentified arebeing

purified

to determine their effects on in vitro DNAreplication.

ACKNOWLEDGMENTS

This work wassupported by Public HealthService 1F32-CA-06012-03 from theNationalCancerInstitute and by insti-tutional researchgrantIN-54R16 from the AmericanCancer Society (bothtoB.I.M.) and by grant PCM-7714451from the NationalScience Foundation to J.A.H.

Wethank EllenGrucza, Carolyn Reilly, andMaryEllen Armosinoforexcellenttechnicalassistance.

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