Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells.

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0022-538X/79/02-0612/12$02.00/0

Intracellular

Forms of

Simian Virus

40 Nucleoprotein

Complexes

I. Methods of Isolation and Characterization

in CV-1

Cells

R. FERNANDEZ-MUNOZ,tM.COCA-PRADOS,ANDM.-T. HSU*

TheRockefeller University, New York,New York 10021

Received forpublication10August1978

Anewmethodwasdeveloped for isolation of intracellular forms of simian virus

40 (SV40) nucleoprotein complexes from SV40-infected CV-1 cells late in the

infectious cycle. Incontrast totheTriton extractionmethod, which yields onlya

60-70S complex, this newprocedure yielded three forms of SV40 nucleoprotein

complexes: complex I, complexII, and thematurevirion(V). The three

nucleo-protein complexes differed in physicalaswellasbiochemicalproperties.Complex

I, whichis onlya small portion of the total SV40 nucleoprotein complexes late

duringinfection, wasactive in synthesizing both SV40-specific DNA and RNA.

Pulse-labeling experimentssuggestthefollowingmetabolicpathway: I-+ II-+V.

Conversion ofcomplex ItoII occurredshortly after the completion of SV40 DNA

replication and resulted in the inactivation of thebiosynthetic activities ofI.

Simian virus 40 (SV40) chromatin isolated

fromlytically infectedcells exhibits many

prop-erties similar to those of eucaryotic cell

chro-matin. It iscomposedmainlyof a histone-DNA

complex arranged in the nucleosome structure

(8) and is associated with both DNA (19) and

RNA (6)polymerase activities.

We areinterested inusing SV40 chromatinas

a model system for studying the relation

be-tween the structure and function ofeucaryotic

chromatin. During our initial study of SV40 chromatin isolatedby the Tritonextraction

pro-cedure (7), we werepuzzledby thefactthat no

mature virions were everrecovered in the

ex-tract evenwhen mature virions could be isolated

by banding infected cell lysate in a CsCl

gra-dient. Furtherinvestigationshowed that virions

aswellasmanyintracellularnucleoprotein (NP) complexeswere disrupted byTriton treatment to yield a single species sedimenting at about

60S. This observationpromptedus todevelopa

newisolationprocedure which preserves the

in-tracellular forms of the SV40 NP complexes.

With the new procedure three forms ofSV40

NP complexes were isolated. A minor

compo-nent, complex I,sedimentingatabout70S,was

found to beactively synthesizing SV40RNA and

DNA. In addition, a more condensed form of

SV40 chromatin (complex II),which is derived

from complex Ishortlyafter the completion of DNA replication, was isolated as well as the

maturevirion.

tPresent address: Virologia (Microbiologia) Centro "Ra-monyCajal," Madrid-34,Spain.

In thepresent communication wedescribe the

method forisolating these intracellular forms of

SV40 NPcomplexes and the initial

characteri-zation of their physical and biochemical

prop-erties.

MATERIALS AND METHODS

Cell and virus. TheSVS strain ofSV40was used to infect CV-1 cells. The description of virus, the growth of cells and virus, and the infection procedure have beenreported (10).

Extraction of SV40 complexes. Two alternative methodswereusedfor extraction ofSV40 complexes. (i) For total cell extract,48hafterinfection, cells were washedoncewith TDbuffer (25 mM Tris-hydrochlo-ride, pH 7.4, 0.136 M NaCl, 7 mM KCl, 0.7 mM

Na2HPO4)andwerescraped off the plate into2mlof

cold hypotonic buffer (25 mM Tris, pH 7.9, 1 mM

MgCl2, 0.4 mM CaCl2, 0.5 mM dithiothreitol). The

cellswerebrokenby homogenization (30 strokes) in a tightly fittingglass Dounce homogenizer. The nuclei werespun downby centrifugation in an IEC PR-6000 centrifuge at 2,000 rpm for 5 min. The supernatant was thenlayeredon asucrose gradient for isolation of SV40 NP complexes. (ii) For nuclear extract, cells werewashedonce with TD bufferand were scraped into TD buffer at107 cells per ml. The cells were spun down(1,500rpm,3min) and resuspended inTDbuffer at 107 cells per ml, and the detergent Nonidet P-40 (Shell) wasadded to 0.5%. Afterbriefmixing, nuclei werespundown(2,000 rpm, 5min)and resuspended in 2mlof cold TDbuffer,and thenucleiwere homog-enizedbyDouncehomogenization (30strokes).Nuclei werespundown asbefore, and the nuclearextract was analyzed byusinga sucrosegradient.

Sedimentation velocity analysis of SV40 NP complexes.A5to20% sucrose gradientwasusedto 612

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analyze the properties of SV40 complexes. In later experiments we used a 5 to 40% sucrose gradient, which gives better resolution of different forms of SV40 complexes. The buffer usedisindicated in the figurelegends. The conditions for centrifugation were: SW40 rotor, 37,000 rpm,70min,4°C.

Glutaraldehyde fixation of NP complexes and determination of buoyant density. Fractions from asucrose gradient of SV40complex were fixed with glutaraldehyde according to the procedure of Balti-moreandHuang (1). Fixed samples were layered onto apreformedCsCl gradient (1.2 to 1.6 g/ml in 0.01 M Tris-hydrochloride, pH 7.9,0.01MEDTA, 0.1% Triton X-100) and centrifuged in an SW65 rotor at 32,000 rpmfor12hat20°C. Fractions, 100jl,werecollected intomicrotiterplates. A drop of mineral oil was then added to each fraction to prevent evaporation. The density of fractionswasdetermined by the refractive index.

Unfixedsampleswereanalyzed in thesamegradient exceptthata1-ml cushion ofa1.7-g/ml CsCl solution wasincluded. Unfixed samples were also analyzed in

a44%(wt/vol)metrizamide(Nyegaard)gradient in50

mM Tris-hydrochloride, pH 7.4.The gradients were spunat32,000 rpmfor60hat20°C inanSW65 rotor. Gradientswerefractionated asin the CsCl gradient, and the density (p) of metrizamide solutionwas deter-mined from therefractive index (n), using the formula:

p(5°C) =3.453 n(20°C) -3.601.

Gelelectrophoresis.Low-salt agarosegel

electro-phoresis foranalyzing the mobility of SV40 complex was performed according to Varshavsky et al. (20). SV40complexeswereelectrophoresedina0.4, 1.0,or 2%agarose tubegel (12 by0.7cm)containing 10mM Tris,pH 7.4,at 50V for9h. Afterelectrophoresis the gelswere eitherstained with0.5,ug of ethidium bro-mide per ml or0.2% Coomassie brilliant blueorthe

gels were sliced into 2-mm slices for analyzing the radioactivity. Agarose gelelectrophoresis for analyzing supercoiled DNAwasperformed ina2% agarose slab gel containing40 mM Tris-30mM NaH2PO4-1 mM EDTA,pH 7.8, according to theprocedure of Shure and Vinograd (17). SV40 DNA or DNA fragments

obtained after micrococcal nuclease digestion were analyzed in either 1.4or2.5% agarose underthe con-ditionsdescribedpreviously(10).

Protein samples were analyzed by electrophoresis

ona 14%polyacrylamide-sodium dodecylsulfate slab

gelasdescribedby Laemmli (13).

DNA, RNA, and protein labeling, extraction, and RNA-DNAhybridization.SV40DNA was iso-lated from cells infected at low multiplicity as de-scribed previously (10). The DNA was labeled with 200,iCi of['H]thymidine (NewEngland Nuclear, 20 Ci/mmol) perplate,and theradioactivitywaschased bywashingthecells with fresh medium andincubating them in the medium containing 100 mM thymidine (Calbiochem) for1h. Fordouble-labeling experiments, SV40DNA waslabeledwith 20,ICiof['4C]thymidine

(New England Nuclear,40to60mCi/mmol) per plate. Topreparepulse-labeled RNA, cellswereincubated with1mCiof[:3H]uridine(NewEngland Nuclear,>25 Ci/mmol) perml for1min andimmediately quenched onice with thesimultaneousaddition of 10 mlof ice-cold TDbuffer. The RNA was isolated fromSV40NP

complexes after sucrose gradient fractionation by ex-traction withphenol(saturated with buffer containing 0.1MNaCl,0.01MTris, pH 7.4, 0.01 M EDTA, 0.1% sodium dodecyl sulfate) and chloroform-iso-amyl alcohol (24:1). Conditions for the hybridization oflabeled RNAtotheSV40DNAfilteraredescribed inreference12.

Labeled protein was obtained from cells labeled

with [3H]lysine (New England Nuclear, 60 to 80

Ci/mmol),50,uCi/ml,for24to 48hpostinfection. To isolate CV-1cell histone proteins, monolayer cell cul-tures werewashed twice with10ml of TD buffer, and the cellswerescraped off the plates into TD buffer andcollected by low-speed centrifugation. The histone proteinwasextracted from thecellpellet with0.25N H2SO4at4°Cfollowed by further extraction with 0.4 NH2SO4. The two extracts were combined,10volumes of coldacetonewasadded, and the protein was precip-itated at-20°C. (Calf thymus histones were a gift of G. Vidali.)

In vitro assayof RNA polymerase activities. Endogenous RNA polymerase activities associated with SV40 complexes were assayed according to the conditions of Green and Brooks (6). In vitro transcrip-tionof SV40complexes, with Escherichia coli RNA polymerase (purified according to the procedure of Burgess and Jendrisak[3]),wasperformed in40mM Tris (pH 7.9), 10mM MgCl2, 150 mMKCl, 0.4 mM

K2HPO4, 0.1 mM dithiothreitol, 0.15mM each ATP,

GTP, and CTP, and10,iCiof[3H]UTP(NewEngland Nuclear, 35 to50Ci/mmol) per mlat37°C for30min. Micrococcalnucleasedigestion. SV40 complexes isolated after sucrose gradientfractionation were

di-gestedwith500U ofmicrococcal nuclease

(Worthing-ton) perml for10min inbuffercontaining25mMTris (pH 7.5)-50mM KCl-10 mM NaCl-10mM

mercap-toethanol-10 mM MgCl2-1 mM CaCl2-0.15 mM spermine-0.5 mM spermidine. The reaction was stoppedby the addition of sodium dodecylsulfateto 0.5% and EDTA to 25 mM, and the DNA was ex-tracted with phenol-chloroform-isoamyl alcohol (24: 1)andprecipitated with2volumes of ethanol.

Electronmicroscopy.Adropofsolution contain-ingSV40complexes in TD bufferorin0.1M Tris(pH 7.4) was placedon a sheetofParafilm. Immediately after the drop was placed on Parafilm, grids coated with pallodion were touched to the drop for5 sand washed in distilledwaterfor5s.Alternatively,adrop ofsolutioncontainingcomplexeswasplacedon agrid for5 s, and theexcess liquidwasremoved withfilter

paper.Gridswereshadowed withplatinum-palladium

(80:20) with orwithout priorstainingin 5 x 10-5 M uranylacetatein90%ethanol. The method for spread-ing of SV40 DNA in 50% formamide or the SV40 complexesin 50 to70%formamide has been described (11).

RESULTS

Isolation of SV40 DNA-protein

com-plexes.Previously,the isolation of intracellular

SV40 NPcomplexeshasbeen achievedbyusing

Triton extraction method (7). This extraction

methodyields asingle SV40DNAprotein

com-plexsedimentingatabout 60-70S in thesucrose

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gradient(Fig. 1). However, repeated efforts with thismethodto recover maturevirions fromcells late after infection failed, even when a large

amountof virion could be isolated fromsimilar

cultures afterCsCl gradient fractionation of

ex-tracts obtained from cells lysed by repeated

freeze-thawing. The absence ofany mature vir-ion in the Tritonextractstrongly suggeststhat the intracellular forms ofSV40 NP complexes

are disrupted during isolation. Further investi-gation showed that40to50% of [3H]thymidine-labeled virions purified on aCsCl gradient are

converted into a species sedimenting at 60S

whencoextracted with unlabeled SV40-infected cells by the Triton method. The disruption is dependent on the presence of Triton-treated cellularmaterial becauseno disruptionwas ob-served whenpurified virus alonewas extracted with Triton. These observations indicate that the 60-70S complex extracted by the Triton method could be derived from many different forms of NP complexes, includingmature

viri-ons.To avoid thedisruption of intracellular NP complexes,a newisolationprocedurewas devel-oped. Briefly, SV40-infected cells in hypotonic buffer or nuclei in isotonic buffer obtained by Nonidet P-40lysis ofSV40-infected cells were

homogenizedin atightly fitting Dounce homog-enizer (see Materials andMethods).SV40

com-plexesselectivelyleakedoutfromthenucleiand

could be separated from cellular chromatin by low-speed centrifugation. SV40 complexes

ex-15

E 10_ i

I

'

tracted bythis new procedure exhibitvery

dif-ferentprofilesinthesucrosegradient fromthose

obtained by the Triton method (Fig. 1 and 2).

When analyzed ina 5 to20% sucrose gradient,

major components were observed sedimenting

at70S (NP-I) and 180-200S (Fig.1). The heavier

speciescanbe furtherresolved intotwo

compo-nentswhen thecomplexesarepulse-labeledand

analyzed in5 to40%sucrosegradients (Fig. 2).

The material sedimenting at about 200S is

mostly SV40 virionasdefinedbythecriteriaof

electron microscopic morphology and CsCl

gra-dientbanding (virion bandsat a density of1.35

g/ml). The heterogeneousmaterial sedimenting

betweenNP-Iand SV40 virions (V) was opera-tionallydesignatedNP-II.

Thepossibility that SV40 complexes obtained by thenew procedure are due toartifacts

gen-erated during the isolation procedure was

ex-amined in a series of control experiments. 3H-labeled NP-I, NP-II, or virions were added to unlabeled SV40-infected cells and extracted by using the newprocedure. If NP-I orNP-IIwas

derived from virions during homogenization, then coextraction of labeled virion with unla-beled infected cells shouldyield labeled material sedimentingatNP-Iand NP-II positions. Such resultswere notobtained intwotrials.Similarly,

NP-II could be derived from NP-I by

aggrega-tion with itselforwithother cellularcomponents

during extraction. Coextraction of labeledNP-I withunlabeled infectedcells demonstrated that

Pellet

61,212 cpm

3

0 2"x E

IL

Fraction number

FIG. 1. Sucrosegradientanalysis of SV40chromatinisolatedby the Tritonextraction method(0) and by thenew method asdescribed in the text (0). Cells infected withSV40werelabeledwith 200IuCiof

PH]-thymidineper 100-mmdishofcellsfrom36 to48hpostinfection. SV40chromatinwasextractedandanalyzed

ina5 to20%1csucrosegradient containing50mMTris, pH 7.9,inanSW40rotor(37,000rpm, 60min,4°C).

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NPI NPfl V

f-11 r-N

0 20 40 60 80 100 120

Fraction No.

FIG. 2. Five to 40%o sucrosegradient analysis of

SV40 DNAprotein complexes extractedby the new

method(see text). SV40-infectedcellswere

pulse-la-beled with [3H]thymidine for (a)30min, (b) 90min,

or (c) 24h, with the endpoint of labeling at48 h postinfection. SV40 complexes were extracted from infectedcellsbyDouncehomogenizationinhypotonic solution andanalyzedina5to40%osucrosegradient

containing 2 mM Tris, pH 7.4, in an SW40 rotor

(37,000 rpm, 70min, 40C). Gradients were

fraction-atedfrom the top into100-julfractions. Similar

pro-files were obtained when the buffer in the sucrose

gradientwasisotonic TDbuffer. In TDbuffer NP-I

sediments slightly faster, whereas virions sediment slightlyslower.

this is notthe case. Similarargumentsindicate

that NP-I couldnotbe derived fromNP-II

dur-ing extraction. These experimentsdemonstrate

thatthe isolated NPcomplexesretain their

char-acteristic sedimentation properties upon

reex-tractionand are not artifacts of the extraction

procedure; therefore, they probably represent

genuineintracellular forms ofSV40NPs.

The efficiency ofextracting SV40 complexes

by the newprocedurewasestimatedasfollows. SV40-infected cellswerelabeled for 24h (24 to

48 h postinfection) with[3H]thymidine. The

ra-dioactive label present in the three classes of

SV40 complexes was compared with the label

present in the Hirt extract of the same nuclei

remaining after prior extraction ofcomplexes.

The ratio of label present in theisolated

com-plexes to that in the Hirt extraction is 1.1 x 106

cpm/1.0 x 106 cpm or about 1, indicating that

theextraction efficiency is about 50%. Analysis of the material present in the two extracts

showed that more than 98% of the material

present in the threeclasses of complexesisSV40

DNA, whereas, at most, 50% ofthematerial in

the Hirt extract is SV40 circles. This suggests

that theefficiency of extractionmustbeatleast 50% and perhaps as much as 70%. In another experiment, nuclei collected after Dounce ho-mogenizationwerereextracted in Triton

extrac-tion buffer, and the extract was analyzed on a

sucrosegradient. No 70Scomplexwasobserved

inthe sucrose gradient, suggesting that

extrac-tion with the newprocedure is at least as effi-cientasTriton extraction.

The extraction of[3H]thymidine-labeled NP complexes from total cells and from nuclei

pre-pared from Nonidet P-40-lysed cells yields the

same profile in thesucrose gradient. However, in thewhole-cell extractthere is extensive

con-tamination of NP-Iby cytoplasmic material,

es-pecially ribosomes. These contaminations were

mostly eliminated by extracting SV40complexes from the isolated nuclei obtained bylysing in-fected cells with Nonidet P-40. Figure 3shows

thesucrosegradient profileofanuclearextract

labeled with

[3H]thymidine

and

[3H]lysine

from

24to 48hpostinfection and with 32P from36 to

48hpostinfection. Host protein contamination

was greatly reduced (Fig. 3). Complexes ob-tainedfromnuclearextract weretherefore used foranalyzingitsprotein compositions.

Properties of SV40 NP complexes. The physical as well as biochemical properties of SV40 complexes are summarized in Table 1.

Theyare discussedbelow.

(i)Physical properties.Thefollowing phys-ical properties of SV40 complexes isolated in

sucrosegradientwereanalyzed.

(a) Morphology ofSV40complexeswas

ana-lyzed by electronmicroscopy.Different fractions

in the sucrosegradient containingisotonicbuffer

corresponding to NP-I, NP-II, and V were

di-luted in either isotonic TD bufferor0.1MTris,

pH 7.4, and deposited on the electron

micro-scopegridsasdescribed inMaterials and

Meth-ods. Whensampleswerepreparedin TDbuffer

and extensive surface tension during sample

o

x

E

I

Iv,

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E

I?

'0

'E

-J-I?

oo 0 4 0 8 o l2

'0

x~~~~~Foto No.

E

a-0 40 r60 80 100 120

Fraction No.

FIG. 3. Profile ofSV40 complexes in a 5 to 40%O

sucrosegradientlabeled with(a) [3H]thymidineand

(b) [:IH]lysine from 24to 48 hpostinfection and(c) with 12Pfrom36to48 hpostinfection.SV40complexes

were extractedfrom the nuclei of infected cells as

described in thetext. Comparetheprofile of12-h32P

labelingwith thatof12-h['Hithymidine labeling,as

shown inFig. 13e.

preparation was avoided, NP-I was seen as a

"beaded" nucleosomalstructure(Fig.4B). In the

same method ofsample preparation, NP-II

ap-pearedas a morecondensed structure (Fig.4C).

Whenthesampleswerepreparedin 0.1 M Tris

(pH 7.4), NP-I was reproducibly observed to

consist of thin DNA-protein fiber about 80

Athick. Mostof the NP-Icomplex spreadunder

this condition showed a supercoiled structure

(Fig. 4A, dashed arrows) similar to that of the

free SV40 DNA. Occasionallyrelaxed open

cir-clescanbe observed (Fig. 4A,solidarrow).The contourlength oftheseopencirclesisabout 1.5

Am, similar to that of free DNA. In contrast,

NP-II, when spreadunder thesame conditions,

showedareproduciblymore compact structure

(Fig. 4D).Occasionally, thin fiber similartothat

observed in NP-Isamples could beseenaspart

of theNP-IImolecule. These observations

dem-onstrate that NP-I and NP-IIhave a different

conformation, whichcanbeeasilydifferentiated

inthe electronmicroscope.

(b) Buoyant density of SV40 complexes was

analyzedinCsClgradientsafterglutaraldehyde

fixation or in a metrizamide gradient without

fixation. NP-I has adensity of1.45 g/mlinthe

CsCl gradient, whereas NP-II and the virion

have densities of 1.35 g/ml (Fig. 5). Although fixed NP-II and the virion have thesamedensity

in CsCl, unfixed NP-II is disrupted into free protein and DNAcomponentsinaCsCl gradient

whereasthematurevirionisnot.Inmetrizamide

gradientanalysis, NP-I and NP-II have thesame

density, 1.18 to 1.20 g/ml, whereas the mature

virion has a density of 1.26 g/ml (Fig. 5). The

secondcomponent ofdensity1.2g/ml in Fig. 5C

is due to the presence of NP-Il material in a

viruspeakinthe sucrosegradient. Thepresence

ofNP-II material in the virus peak isalso

ob-served by electron microscopy as described

aboveandby analyzing unfixed materialin the

virus peak intheCsCl gradient.

(c) Electrophoresis of SV40 complexes in a

1.0% agarose gel shows that NP-I, NP-II, and

virion have different mobilities (Fig. 6). NP-I and NP-II have broad distribution in the gel. However, the ratio of[3H]lysine to

["4C]thymi-dine label remains approximately constant

throughout the band, indicating that the

breadthofthe band is not theresult of

dissocia-tion of proteins during electrophoresis, but

ratherdue totheinherentheterogeneity of the

samples.

(ii)Biochemical characteristics. TheDNA of SV40 NP complexes is mainly supercoiled

DNA(R90%),asdetermined by electron

micros-copy and agarosegelelectrophoresis. The

num-ber ofsuperhelicalturnsintheDNA ofall three complexesappears to be the same aswhen

an-alyzed by agarosegel electrophoresis (14). The

DNA is arranged in a nucleosome structure in

TABLE 1. Physicalandbiochemicalproperties of SV40 complexes

Property NP-I NP-II Virion Sedimentation coef- 70S 180S 210S

ficient

Density (CsCl; 1.45 1.35 1.35

jg/ml)

Density (metriza- 1.18 1.18 1.26 mide;,ug/ml)

Morphology (elec- Open Condensed Virion tron microscopy)

Replication activity Yes No No invivo

Transcription activ- Yes No No ity in vivo

Replication activity Yes No No invitro

Transcription activ- Yes Variable No ityinvitro

Supercoil density Same Same Same (DNA)

Micrococcal nuclease Nucleosome Nucleosome digestion

HistoneHi Yes Yes No

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'-C

15E

Pli,~~

FIG. 4. Electron micrographs ofSV40 NP complexes.SV40 NP complexes obtained from a sucrose gradient in TDbuffer were diluted into TD buffer (B, C) or 0.1 M Tris, pH 7.4 (A, D). Pallodion-coated grids were touched to adrop of complexsolutionfor 5 s. After excess solution was removed, the grids were shadowed

withplatinum-palladium(80:20). (A, B) NP-I; (C, D) NP-II.

NP-I and NP-II as manifested by micrococcus nuclease digestion (Fig. 7). A resistant core of about 150 nucleotides inlength was observed. This result is similar tothatobtained for

poly-omavirusby Ponderetal. (16).

(a) Preliminary analysis of theprotein

com-ponents of SV40 complexes is given in Fig. 8.

NP-I is composed mainly of the five groupsof histones and material migrating in the same

position as VP-1 protein of SV40 virus in a

sodiumdodecylsulfate-polyacrylamide gel.The majorproteinsin NP-II, however, arethe viral capsidproteins VP-1, VP-2, and VP-3. Histone proteinsarealsopresentbut in loweramounts.

Histones, exceptfor

Hi,

arepresent in the

ma-ture virion. Thepresence ofHiinNP-Iand

NP-II but not in the mature virion has also been

confirmed bythe acid-urea gel electrophoresis technique (15; M. Coca-Prados, G. Vidali, and

M.-T.Hsu,manuscriptinpreparation). Further

characterization of the protein components of

theSV40 NP complexesand their modification

willbereportedelsewhere.

(b) Endogenous RNA and DNA polymerase

activities associated withSV40complexeswere

analyzed byinvivo andinvitroincorporation of RNAorDNAprecursors, Toanalyze the in vivo transcription activities,SV40-infected cellswere pulse-labeledwith[3H]uridinefor1min at37°C.

(Aseparatebiochemicalanalysis of 1-min

pulse-labeled RNA showed thattheyrepresent

grow-ing RNA chains [5].) SV40complexeswerethen extracted fromtotalcells andseparated ina 5 to

20% sucrose gradient. Labeled RNAs were

ex-tracted from each fraction of the gradient and assayed for SV40-specific RNA by hybridizing

to anSV40 DNA filter. Figure 9 shows that

1-minlabeledSV40 RNA is associated withNP-I

and notwith NP-Il or virion. The endogenous

RNApolymeraseactivities werealsoassayedin

vitro for the incorporation of

[3H]UTP

or

a-[32P]UTP

into acid-precipitable material. NP-I

canbereproducibly foundtobe active in

tran-scription (Fig. 10). NP-II, on the other hand,

gave low but variable activities. At present we

donotknow thecauseof suchvariabilityinthe

activity of NP-II. Thespecificityof the in vitro

transcriptionofNP-Iwasassayed by hybridizing

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CSCI Metrizomide taining 40 or 20% formamide, elongating RNA

a a' could beseen associated with SV40 DNA. The

-Ao A 6

frequency

of

observing

such structuresis about

l 50, 1% of the total SV40 DNA scored on the grid.

2- l 14513 , This

frequency

remained

approximately

the A 140 1.2

- 135

140

[l2 ;-1

O-

same even when the

sample

was diluted to the

1 v-o / \ 1 35 t' - 2 extent that no more than twoSV40DNAs could

beseenin thesame

grid

areaina300-mesh

grid.

I b b ^ This

suggests

that the structures observed are

° 8- 1\ 0.8 °

unlikely

tobe the result of the

superimposition

6

E- -- 06 E of free RNA and DNA on the

grid.

Treatment

CL6 w'1.-50 1.3 I' 0,6 o

-Q

S>>°}45 [12

1 l , _-°' Q of nuclei with RNase before

spreading

elimi-4-

Itw-

140 11 OA-0 natedtheseSV40 DNA-associated chains,

dem-WI 2 1.35 0.2 I

onstrating

that these chainsareRNAinnature.

The extended RNA chain in 70% formamide

c c' 20 spreading (Fig.

11A,

B, and C) could be seen to

15- 1 A

collapse

into

secondary

structures in 50%

form-s50o 3 A 115 amide

(Fig.

liD, arrow), a

typical

behavior of 10o 145 12 / -e 1 single-strandedRNA. An RNA chain almost as

+-

. 140 11

long

as the

SV40

genome

length

could be

ob-5- 135 10 05 served

(Fig.

liB).

Rarely,

replicating

molecules

. with RNA chain could also be observed (Fig.

0 10 20 30 40 10 20 30 40 llC).

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Sedimentation

FIG. 5. Analysis of density of SV40 complexes. a b c d SV40 complexes were labeled with [3H]thymidine

from 24 to 48 hpostinfection, as in Fig. 2c. Fractions corresponding to complexes I and II and virion (see Fig. 2c) wereanalyzed ina44% (wt/vol) metrizamide gradient in 50 mM Tris, pH 7.4 (a', b', c') or in a preformed CsCl gradient after fixation of SV40 com-plexes with glutaraldehyde (a, b, c). Solid curves representradioactivity. Dashed curves represent den-sities.(a,a') NP-I; (b, b') NP-II; (c, c') virion.

labeledproduct to SV40 DNAfilters.About 50%

of the label became hybridized to SV40 DNA

filters after 24 h of incubation at 65°C and

treatmentof thefilters extensively withRNases.

Underthesameconditions, labeled

complemen-tary RNA transcribed from SV40 supercoiled

DNA by Escherichia coli polymerase

hybrid-ized tothe extentof60%. Thus, most, ifnotall,

ofthe RNAtranscribedinvitrorepresents viral

sequences. Since almost nohost DNA was

ob-served in the DNA extracted fromNP-I complex

when analyzed in the electron microscope, we

believe thatthe lower efficiency ofhybridization

of RNA transcribed from NP-I as compared to

complementary RNA is probably not due to

transcription of host DNA.However, the nature

of in vitrotranscripts as to its sizes andlocations

on theSV40genome has not been characterized.

SV40transcriptionintermediates in SV40-in- FIG. 6. Low-salt agarose gel

electrophoresis

of

fected cells could be observed in the electron SV40 complexes. SV40 complexes corresponding to

microscope. When nuclei ofSV40-infected cells fractions 42 (a), 61 (b), 78 (c), and 96

(d)

in Fig. 2c

wereadded to a solution containing either 70% wereelectrophoresed in0.4%agarose in 10 mMTris

(Fig.I1A, B, and C) or 50%(Fig.liD)formamide (pH 7.4)at 50 V for 9 h. The gel is stained with 0.5

andimmediatelyspread ontoahypophasecon- pgofethidiumbromideperml.

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D

C

B

A

FIG. 7. Microccocal nuclease digesti complexes. 32P-labeled SV40 complexes ingtothose inFig.6(approximately1 l digested with500 Uof microccocalnuc for 10 min at 37°C. The reaction was adding EDTA to0.1M and sodium do to0.5%.DNAwasextractedbyphenolan(

andanalyzedin a2.5% agarosegel. (A,

(B, C)complexII; (D)virion.

DNA-synthesizing activities of

plexeswerealsoanalyzed. Pulse-labe for 10 min with [3H]thymidine de that the majorityofDNA-synthesiz is associated withNP-I (Fig. 12).AE

clusionwasobtainedby assayingin

poration of[3H]TTP (datanotshow

Relationship among SV40NPc

Todeterminewhether theremightt

sor-productrelationship amongthe ofSV40complexes,weperformeda I

experiment (see Fig. 13). As descr

ously,shortpulses with[3H]thymid belNP-I.Achase for1h with100ml\ thymidineresulted in theappearanc

the NP-II region.This result suggests that

NP-II isderivedfrom NP-I. Since NP-IL and virions

are not very well separated in the 5 to 20%

sucrose gradient, we further characterized the

complex in the NP-II-virion region by a CsCl

gradient. Mature virions band at a density of

_ 766 1.34

g/ml,

whereas SV40

complexes

are

disso-1~ii

70 ciated into DNA and proteincomponents,which

_I

0 band at densities of 1.7 and 1.2 g/ml,

respec-100 tively. Such an analysis was carried out using

SV40 complexes pulse-labeled with

[3H]thymi-dine fordifferent lengths oftime,with the end

point oflabeling always at 48 h postinfection.

5 25 The

complexes

extractedwerefirst

separated

in

-446 H2b

H4 H2a H3 VP3 H1 VP2 VP1

150~~~~5

0

L)

3

-o ~ b

ion of SV40 >1 2-

correspond-to 5Ag)were 1 leaseperml

stopped by r?

decyl sulfate CD

dchloroform a

ComplexI E

-015-

A

SV40

com-lingin vivo

monstrated .ing activity

similar

con-vitro

incor-rn).

omplexes.

:e a

precur-three forms

pulse-chase

ibed

previ-ine only

la-dunlabeled

eoflabelin

Fraction No

FIG. 8. Sodiumdodecylsulfate-polyacrylamidegel

(14%o) electrophoresis analysis of proteins in SV40 chromatin. (a) Complex I; (b) complex II; (c) virion. Proteins were labeled with['Hilysinefrom 24 to 48 hpostinfection. Gelwasstained with0.2%oCoomassie brilliant blue(inserts), slicedinto 1-mm slices, and counted (solidcurves).

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620

1000

S

E

I 500 400 300 200 100

A

\1

A4

F'

lIt

i

It 'II I'

4

-

I\

I

<d

600 500 400 300 N

200 c

E

100 Li

I 1:

Pellet 10 20 24

Fraction-FIG. 9. Analysis ofnascentRNA associated with SV40complexes. SV40-infected CV-1 cells(5 x 10) were pulse-labeled for 1 min with [3H]uridine (1 mCi/mlof medium). SV40 complexeswereextracted from cells by Douncehomogenization of cells in hy-potonic solution andanalyzedina 5 to20%osucrose gradient. RNAs from each fraction were extracted withphenol andchloroformandhybridizedtoSV40 DNAfilters (1

pwg/filter).

Symbols: (0) total

incorpo-ration;(0)hybridizedcounts.

a 5 to 20% sucrose gradient into two general

regions, I andII (seeFig. 13a-e). Complexesin

region II which include both NP-II and SV40 virionswerefurtheranalyzedin a 1.2 to1.6-g/ml linear CsClgradient witha 1.7-g/ml CsCl

cush-iontotrapthe freeDNA(Fig. 13f-g). In the 30-min label, most of the label appears in NP-I

(Fig. 13a),whereasduring the12-hlabelingmost

oflabel appears inthematurevirion and NP-II (Fig. 13e and j). These results and the pulse-chaseexperimentsuggest thefollowing

biosyn-theticpathway for SV40complexes:

' NP-I->NP-II-- virion.

Appearance of label in the NP-IIregionis rather

rapid (within30minof thepulse-labeling time,

see Fig. 2), whereas the SV40 virion is labeled

only after 1h ofpulse-labeling. Sincethe time

requiredforoneround ofSV40 DNAreplication

is about 10 to 15 min, this result implies that

shortlyafter thecompletionof DNAreplication

NP-Iis converted intoNP-II.

Additional experiments have shown that a

similar kinetic relationship could be

demon-strated as early as 16 to 17 h after infection,

shortlyafter theonsetof viralDNAreplication

(datanotshown). However,the amountof

NP-II and virion is muchreduced relative toNP-I

ascomparedwiththatat 48hpostinfection.

DISCUSSION

Using a new isolation procedure, we have demonstrated that thereare atleast three forms ofintracellular SV40NP complexes. That they

are unlikely to be artifacts generated during isolation procedures is shown by the various

controls and by the differential labeling ofthe

complexes. In contrast, extraction with Triton

convertsall the intracellularcomplexes,

includ-ing virions,into the60S-70S form (NP-I).Since

wehaveshownthatNP-Irepresents only a small

portion oftheintracellular SV40 NP complexes late during theinfection cycle, the60S-70S SV40 complexisolatedby the Triton method actually is composed mainly of material derived from

NP-Il andmaturevirions,which are not active inbiosynthesis of SV40 RNA and DNA. Thus, webelieve the isolationprocedure is superiorto

the Triton extraction method inpreserving the

structuresofSV40NPcomplexes and for

study-ing theirbiological functions.

E

0

E I. E f

7

o

E

c

0

.-a

0 0

a- I-0

a.

[image:9.505.91.237.79.315.2]

Fraction number

FIG. 10. In vitroincorporation ofa-["P]UTP into

acid-precipitableRNA bySV40complexes.

SV40-in-fected cellswerelabeledwith[3H]thymidine from44

to 48hafter infection.SV40complexes extractedfrom totalcellsby Douncehomogenization ofcells in hy-potonic bufferwereseparated ina 5 to20%osucrose

gradientcontaining50 mM Tris(pH 7.9),50mMKCI, and 0.5 mMdithiothreitol. Fractions collectedfrom

the sucrosegradientwereassayedfortheactivity of

incorporation ofa-[32P]UTP into acid-precipitable

RNA. Symbols: (0)

a-[32P]UTP

incorporation; (0)

[3H]thymidine incorporation. Pelletrepresents

con-taminatingnuclei.

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[image:9.505.275.458.372.555.2]

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, *' ZrXDD , '

FIG.4 11. Electron micrographs of SV40 transcription complexes. Nuclei from SV40-infected cellswere

iAn4 tZ~~~~~~~~~~~~4 f . V

4*~~~~~~~~~~~~~~ ~ ~ ~ ~,~*~ ~ ~

iDicAtemplaenascntRNAchains. Indark fohrmamidealuysprseRnt.chInsmayreprnextendedNconolymeation whereasa

replicatingmolecule. Thetworeplicating forksareindicatedbybrokenarrows.

The three formsof SV40NPcomplexes differ

in both physical and biochemical properties.

NP-Iis active in thesynthesis of SV40DNA and

RNA. The DNA in NP-I is more sensitive to

DNase Idigestion thanNP-Il (E. Derman and

M.-T. Hsu,unpublishedobservations). Analysis

of both its physicalandbiochemical properties

suggests that NP-I is the active chromatin of

SV40. Because it can be isolated

uncontami-natedby the other forms ofSV40NPcomplexes

andbythecellularchromatinandbecauseof its

simple genetic complexity, webelieve that

NP-I representsanexcellent modelsystemfor

study-ing thestructures and functions of active

chro-matinineucaryotic cells.

Incontrast toreportsby otherworkers,SV40

NP-Icomplex wasobservedasauniform 8-mm

DNA-protein fiber when spread in 0.1 M Tris,

pH 7.4.Thisstructuremaybe derived from the

nucleosomalstructure by unfoldingthe

nucleo-somes as aresult of charge repulsion between

DNA-protein fibers in a low-salt environment.

Variability of chromatin conformation observed

in the electron microscope as a result of the

difference inmethods and buffersused for

pre-paring samplesiswell known (17). In our

expe-rience, not only are salt concentration and pH

importantfactors inspreadingSV40complexes,

but surface tension of the sample is a major

factorcontributingtothevariabilityof structure

observed in the electron microscope. This

vari-abilitynotwithstanding,theSV40NP-Iand NP-IIcomplexes, when spread under identical

con-ditions, showedreproducibly recognizable

differ-ences in morphologyunder the electron micro-scope.

The major protein components present in

complex I are composed of histones anda

pro-teinthatcomigrates with VP1. At presentwedo

not know the function of VP1 in NP-I. The

possibility that it is aggregated with NP-I during

isolation cannot be excluded. The presence of

the histone Hi fraction in SV40 NP complex

extracted by the Triton method has been

re-ported by Varshavsky et al. (20) but not by

others (2, 14). Our results show thatHi is

pres-VOL. 29, 1979

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1000

V

a-9

C

* 0

to

o

vs 0

I o#

._-._-_I

E on

Fraction number

FIG. 12. Pulse-chase of SV40 complexes with

[3H]thymidine.Atotalof1O SV40-infected cellswere

pulse-labeledwith[3H]thymidine (1mCi/5 mlof

me-dium)for10min (0) and chasedwith 100mM

thy-midine for 1 h (0). SV40 complexes in total cell extract wereanalyzedina5to20%osucrosegradient

containing 50 mM Tris, pH 7.9. Pellet represents contaminating cell nucleiasanalyzedwith the

elec-tronmicroscope.

ent in NP-I and NP-II but not in virion. This

implies that during SV40 infection Hi first

be-comesassociatedwith the uncoated virus and is

later removed when SV40 virus is assembled.

Furtheranalysis is necessary tounderstandthe

functional role ofHiinthe virusinfection

proc-ess.

The histones present in NP-I complex are

highlymodifiedbyphosphorylationand

acetyla-tion. In addition, highly phosphorylated and

acetylated non-histone proteins were also

ob-served (Coca-Pradosetal., manuscript in

prep-aration).

Therole of NP-II in theSV40 infection cycle

isstill unknown. It is derived from NP-I within

20min aftercompletion of DNA replication of

NP-I. At present we do not know what is the

control process that determines the conversion

of NP-I toNP-II with the concomitant change

of conformation and inactivation of the

biosyn-thetic activities ofSV40chromatin. Perhaps it

istriggeredbythe addition of viral coatproteins

Fractions

FIG. 13. Labeling kinetics of SV40 DNApresentin SV40 complexes. SV40-infected cells were pulse-la-beledwith[:H]thymidinefor30min (a,f),90min (b, g),3h(c, h),5h(d,i),or 12h (e,j). Theendpointof labelingperiodswasalways at48h after infection. SV40complexes obtained in total cell extracts were analyzedfirst ina 5to20%osucrosegradient contain-ing 50 mM Tris,pH 7.4. Portions ofthe gradient labeled"II"werepooledandlayeredon a 1.42-g/ml

CsClgradient with a 1-ml cushionof1.7g ofCsCl per ml. Thegradientwasspunat33,000 rpmfor20h in a type 65 rotor. Sucrose gradients (a-c); CsCl gradient(f-j).Sedimentation isfrom lefttoright. In theCsClgradientprofile,thevirionbands at the top ofthegradient(f-j,left),whereasfreeDNA molecules sediment to the bottomofthegradient(f-j,right).

0

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[image:11.505.73.263.72.352.2] [image:11.505.273.463.79.521.2]

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(e.g.,VP3 whichreplaces Hi histones) which are

presentin large quantity in NP-II. These added

proteins mustbe in arelatively loosestructure

sinceNP-II has the same densityas NP-I in a

metrizamide gradientand its DNA is accessible

to micrococcusnucleasedigestion. Study of the

mechanism ofNP-I toNP-II conversion should yield information as tohow the geneactivity of chromatin is regulatedatthe level of the struc-tureof chromatin.

The buoyant density of NP-II is similar to

thatofNP-Ichromatin in the metrizamide

gra-dient whereas inthe CsClgradient it is similar

to that of SV40 virions. The protein-to-DNA ratio ofNP-II calculated from its protein

com-position predicts thatits buoyantdensity should be similar to that of SV40 virions. The lower than expected density of NP-II in a nonionic metrizamide gradient isprobablydue to

exten-sive hydration of the loosely structured NP-II complex. SV40 chromatin in NP-II is as

acces-sible to micrococcal nuclease digestion as the

NP-I complex, suggesting that the capsid

pro-teins inNP-Ilareprobably only loosely attached

to SV40 chromatin. The "porous" structure of

NP-IIallowswatermoleculestofreelypenetrate

inside the complex, whereas the interior of the

maturevirionislessaccessible. Thismayexplain

why NP-II bands at a lower density than the virioneventhough they haveasimilar protein-to-DNA ratios.

The population of NP-II is rather

heteroge-neous. Itprobably contains different

intermedi-atesbetween the active chromatin (NP-I) and theassembled virus. Furtheranalysis of NP-II, using the electronmicroscopetechniquetostudy

the process of virus assembly, will be reported

(manuscriptinpreparation).

ACKNOWLEDGMENTS

We thankJ. E. Darnell for constant encouragementduring thisinvestigation.We aregratefultoJ.Cozzitorto forexcellent

technical assistance andtoJ.Ford,E.Johnson, M. Wilson, and G. Vidali forhelpfulcriticism and discussions.

Thisinvestigationwassupportedby Public Health Service National Cancer Institute grant CA19073 to M. T. Hsu. R. F.-M.was anEMBOFellow. M.C.-P. isapostdoctoralFellow of theUSA-SpainCultureCooperationProgram.

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infectedand normalHeLacells.Science162:572-574. 2. Bellard, M.,P.Oudet,J. E.Germond,andP. Cham-bon. 1976.Subunitstructureof simian virus 40 mini-chromosome. Eur. J. Biochem.70:543-553.

3. Burgess, R.R., and J. J. Jendrisak.1975. Aprocedure

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4. Cremisi, C., P. F. Pignatti, 0. Croissant, and M. Yaniv. 1976. Chromatin-likestructures in polyoma vi-rusand simian virus 40 lytic cycle. J. Virol. 17:204-211. 5. Ford, J. P.,and M.-T. Hsu. 1978. Transcription pattern of invivo-labeled simian virus 40: equimolar transcrip-tion beyond the mRNA 3' terminus. J. Virol. 28: 795-801.

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9. Hall, M. R., W. Meinke, and D. A. Goldstein. 1973. Nucleoprotein complexes containing replicating simian virus 40 DNA: comparison withpolyoma nucleoprotein complexes. J. Virol. 12:901-908.

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18. Solari, A. J. 1974. The molecular organization of the chromatinfiber, p. 493-531. In H. Busch (ed.), Thecell

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