Structure and composition of the adenovirus type 2 core.

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JOURNALOFVIROLOGY,Aug.1975,p.366-387 Copyright01975 AmericanSocietyforMicrobiology

Vol. 16,No. 2 Printed inU.S.A.

Structure and

Composition

of the Adenovirus

Type

2 Core

DENNIS T. BROWN, MONIKA WESTPHAL, BYRON T. BURLINGHAM,I UTE WINTERHOFF,

AND WALTER DOERFLER*

InstituteofGenetics, University ofCologne, Cologne,Germany

Received forpublication 1 April 1975

Thestructure and composition ofthe core ofadenovirus type 2 wereanalyzed

by electron microscopy and biochemicaltechniquesafterdifferentialdegradation

ofthe virionbyheat,by pyridine, orby sarcosyltreatment. Innegativelystained

preparationspurified sarcosyl cores revealspherical subunits of21.6-nm

diame-terinthe electronmicroscope.It issuggestedthat these subunitsareorganizedas

anicosahedronwhich has its axesofsymmetrycoincident with thoseofthe viral

capsid. The subunits are connected by the viral DNA molecule. The sarcosyl

cores contain the viral DNA and predominantly the arginine/alanine-rich core

polypeptide VII. When sarcosyl cores arespread onaproteinfilm, tightlycoiled

particles are observed whichgradually unfold giving riseto arosette-like pattern

due to the uncoiling DNA molecule. Completely unf'olded DNA molecules are

circular.Pyridine coresconsist ofthe viral DNA and polypeptides Vand VII. In

negatively stained preparations of pyridine cores the subunit arrangement

apparent inthesarcosylcoresismaskedbyanadditionalshell whichisprobably

formed bypolypeptide V. In freeze-cleaved preparations ofthe adenoviriontwo

fracture planes can'be recognized. One fracture planeprobably passes between

the outer capsid of' the virion and polypeptide V exposing a subviral particle

whichcorrespondstothepyridinecore.The second fractureplane observed could be located between polypeptide Vand the polypeptide VII-DNA complex, thus

uncovering a subviral structure which corresponds to the sarcosyl core. In the

sarcosyl core polypeptide VII is tightly bound to the viral DNA which is

susceptible todigestion with DNase. The restrictionendonuclease EcoRI cleaves

the viral DNA in the sarcosyl cores into the six specific f'ragments. These

fragments can be resolvedon polyacrylamide-agarose gels provided the sarcosyl

cores aretreatedwith pronase afterincubationwith the restriction endonuclease.

When pronasedigestionisomitted,acomplexofthe terminalEcoRIfragments of

adenovirus DNA and protein can be isolated. From this complex the terminal

DNAfragmentscanbe liberatedafter pronasetreatment.The complex described

is presumably responsible for the circularization of the viral DNA inside the

virion. The nature of' the protein(s) involved in circle formation has not

yet been elucidated.

The chemical composition ofthe adenovirus

core has been studied intensively. The core

contains the viral DNA and two different

pro-teins, polypeptidesVand VII(17, 18, 22, 23, 32,

33, 34) accordingtothe nomenclatureofMaizel

et al. (26). Polypeptide VII is the

alanine/argi-nine-rich protein (17, 22, 32, 34, 38) which

contains 18 to

19%.0

of alanine and 21 to 23% of

arginine residues. The core proteins have been

isolated by several methods (17, 18, 22, 33, 34)

and have been characterized as to molecular

weight and number of polypeptide chains per

virion (17, 18). Polypeptides V and VII have

molecular weightsof48,500 and 18,500,

respec-'Present address: Division of Bioldgy, Kansas State

University,Manhattan,Kan. 66506.

tively, and itisestimated thatthere are 180 and

1,070molecules per virion, respectively (17, 18,

32, 33, 34). Polypeptide VII comprises

approxi-mately

14%7r

ofthe viral protein, and it has been

calculated that the arginine residues of this protein are sufficient to cover approximately

60% ofthe phosphate charges of the viral DNA

(32). Laver (22) has suggested that the alanine/

arginine-rich protein molecules are evenly dis-tributed along the DNA molecule.

As yet, the arrangement ofthe core insidethe

adenovirion and the relation between the viral

DNA and thecore proteins have received little

attention. Robinson et al. (36) and Doerfler et

al. (11) have provided evidence that the viral

DNAinthe virionis keptina circular

configu-366

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CORES FROM ADENOVIRUS TYPE 2 367

ration perhaps through an as yet unidentified

"linker protein" (36). Everitt et al. (18) and

Everitt and Philipson (17) have proposed that

the core polypeptide V is associated both with

the viral DNA and the penton bases, whereas

polypeptide VII is tightly bound to the viral

DNA.

Recently, Everittetal. (16) have developeda

refined model of the type 2 adenovirion. This

model is based on results obtained by

en-zymatic iodination of intact and disrupted

vi-rions, by immunoprecipitation of intact virions

with specific antisera, and by chemical

cross-linkage of the structural proteins. These results suggest that polypeptides V, VI, VII, and VIII

are located inside the virion. Polypeptide V is

found in close proximitytothe penton-bases,to

hexons, andto protein IIIa. Polypeptide V can

be chemically cross-linked also to polypeptide

VII, hence some molecules of polypeptide V

mustbe located closetopolypeptide VII.

In the present study, the ultra structure of

adenovirus cores was investigated by electron

microscopy offreeze-etched preparations of

vi-rions in infected cells and by electron

micros-copy of negatively stained cores isolated by

severalprocedures.The data obtainedsupporta

model oficosahedral arrangementofthe

DNA-polypeptideVIIcomplex inside the virion.

Sar-cosyl cores of adenovirus type 2 consist ofthe

viral DNA and polypeptide VII predominantly. In contradistinction, pyridine cores (32, 33)

contain viral DNA andpolypeptides Vand VII.

Acomparison of thecompositionofsarcosyland

pyridine cores therefore suggests that the type

ofassociation of thesetwopolypeptideswiththe

coremust befundamentally different. Sarcosyl

cores are tightly packed, gradually unfold on

storage,andcontain circular viral DNAwhich is

sensitive both to pancreatic DNase and the

EcoRI restriction endonuclease. Evidence is

presented that the terminal EcoRI

endonucle-asefragmentsofadenovirustype2DNA, A and

C,arelinkedbyprotein.Acomplexbetween the

DNA termini andprotein has been isolated.

MATERIALS ANDMETHODS

Media and solutions. Cells were grown in Eagle medium or Eagle medium modified for suspension cultures(15) supplementedwith 10% calfserum.Calf serumwaspurchasedfromFlowLaboratoriesorfrom Grand Island Biological Laboratories. The

composi-tion of phosphate-buffered saline (PBS) was de-scribedpreviously (14); TEis 0.01 M Tris-hydrochlo-ride, pH7.2to7.5, 0.001 M EDTA; CsClsolution A consisted of 15gofCsCl (Merck, Darmstadt)and 10 ml of TE. Buoyant CsCl solution contained ngrams

of CsCl and n/2 ml of 0.02 M Tris-hydrochloride, pH8.0. Forthedeterminationofradioactivity

toluene-methanol- and toluene-based scintillators were used which consisted of 5 g of 2,5-diphenyloxazole (Merck, Darmstadt) and 0.3 g of 2,2'-p-phenylene-bis-(5-phenyloxazole) (Merck, Darmstadt) per liter of a 1:1 toluene methanol mixture or of toluene. Electro-phoresis buffer (TEB buffer) consisted of 0.089 M Tris-hydrochloride, 0.089 M boric acid, 0.0025 M EDTA, 0.01 M propylamine, and 0.5% sarcosyl.

Cells and virus. KB cells(CCL17fromthe Ameri-can Type Culture Collection) were grown in

mono-layer cultures in Eagle medium (15) or in Eagle medium for suspension cultures both supplemented with 10% (vol/vol) calf serum. Human adenovirus type 2(Ad2) was propagated in KB cells in suspension cultures andwaspurified aspublishedpreviously (10, 11, 13). The production of Ad2 radioactivelylabeled with [6-_Hlthymidine, [14C formate-sodiumor a mix-ture of 3H-labeled amino acids described elsewhere (10, 11, 13). Allradioactivelylabeled compounds were purchased from Amersham-Buchler, Braunschweig, Germany.

Viral DNA. Adenovirus DNAwasextracted from CsCl-purified virionsbymethodsreported previously (5, 11, 13).

Infection of cells for electron microscopy. KB cells growinginmonolayercultureson 100-mm

diam-eter plastic dishes were infected with CsCl-purified

Ad2 at a multiplicity of 50 to 100 PFU/cell as describedpreviously (3).

Preparation of sarcosyl cores from adenovirions. (i) Equilibrium sedimentation in CsCl density gradients. Between 5 and 10optical density unitsat

260nm ofCsCl-purified Ad2("4C-labeled)in 0.3ml of buoyant CsCl solution were mixed with0.07mlof 10%

sarcosyl (N-lauroylsarkosin sodium salt)in waterand

0.63ml of TEandincubatedatambient temperature

for 30 min. In some experiments 5jigof 3H-labeled Ad2 DNA was added as density reference.

Subse-quently, this mixturewaslayeredontop of 3.5to4.0

ml ofCsCl solution A inanitrocellulose tube of the SW56 rotor and centrifuged to equilibrium in an

L2-65B Beckman ultracentrifuge at 37,000 rpm and

4C for at least 60 h. After centrifugation, 5-drop

fractions were collected. Refractive indices of every tenth fractionweredeterminedin aZeiss

refractome-ter.Radioactivitywasmeasuredin 5- to

10-Mgl

aliquots

ofeach fraction.

(ii) Gel filtration on Sepharose 2B columns.

Sarcosyl cores were also obtained by adjusting the

CsClpurifiedAd2preparationto0.5%sarcosylinTE

and passing this mixtureover aSepharose2Bcolumn (1 cm indiameter by 10 to 15cm in length)

equili-brated with thesamebuffer. The columnwas devel-opedat 4 C with0.5%sarcosylinTEat aflowrateof2 ml/h.

Preparation of pyridine cores. Pyridine cores wereprepared accordingtotheprocedureofPrageet al. (33, 34). Ad2 ("IC-labeled), which had been

purified byequilibrium centrifugationinCsCldensity

gradients, wasdialyzed against0.005MTris, pH 8.1,

and disrupted in 10% pyridine and the cores were

subsequently isolatedbyzone sedimentationon10to

25% sucrosedensitygradientsin 0.002M

Tris-hydro-chloride, pH 7.5, and0.0002 MEDTA. Thesamples were centrifuged in the SW41 rotor of the Spinco

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368 BROWN ET AL.

L2-65B ultracentrifuge at30,000 rpm for 125min at 4 C.

Disintegration of adenovirus particles byheat. CsCl-purified preparations of Ad2weredialyzed into PBS or 0.02 M Tris-hydrochloride, pH 8.1, and were heated at 56 C for 5 min (39). These virions were examined in the electron microscope directly, or after rebanding in CsCl density gradients. In some cases, the polypeptide composition of the rebanded virus preparations was also analyzed by electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gels.

Enzymes and reaction conditions.DNase, electro-phoretically purified, wasbought from the

Worthing-tonBiochemicalCorp., andwasusedwithout further pretreatment. Adenovirus cores inavolume of0.1ml wereincubatedin 0.01MTris-hydrochloride,pH 7.5, and 0.005 M MgCl2 with 1 ug ofDNase per ml at

ambient temperature for 5, 10,or20min.Insomeof the experiments, DNase digestion was performed in

0.01MTris-hydrochloride, pH7.2,0.01MCaCl2, and

0.01 MMnCl2 (35). After DNase treatment, someof the samples were incubated with500ug of pronase per mland 1%SDS at 37 C for30minandsubsequently were extracted with 0.5 ml of redistilled phenol saturated with TE. The phenol and aqueousphases

were separated by centrifugation, and the aqueous phase was dialyzed against one-tenth strength elec-trophoresis buffer and analyzedby electrophoresison

polyacrylamide (1.5%)-agarose (0.8%) gels as de-scribed below.

Pronase B was obtained from CalBiochem, dis-solved in 0.01 M Tris-hydrochloride, pH 7.5. at a

concentration of5 mg/ml, and preincubated at37C for2h.Each lot of Pronase B purchased wasassayed afterautodigestion for the absence of endo-or

exonu-clease activity by incubating 3H-labeled Ad2 DNA with 250 Mg of preincubated pronase in 0.01 M

Tris-hydrochloride, pH 7.2, 0.1 MNaCl, and 0.002 M

MgCl2for 60 min at 37C. Subsequently, the size of

the DNA was determinedonneutral sucrosedensity gradients (6).

Restriction endonuclease RI from Escherichia coli (EcoRI). The purification of the restriction endonuclease EcoRI has been described previously (11;R. N. Yoshimori, Ph. D. thesis, Univ. of Califor-nia, San Francisco, 1971). For the experiments de-scribedinthis report, the DEAE-cellulose fraction of the endonucleasewasdialyzed against0.01M

potas-sium phosphate, pH 7.0, 0.35 M NaCl, 0.001 M EDTA, and 0.007 M

$l-mercaptoethanol

and was chromatographed on a phosphocellulose (Whatman, P-11) column (1 by 10 cm) previously equilibrated with 0.01 M potassium phosphate, pH 7.0, 0.35 M NaCl, 0.001 M EDTA, 0.007 M f,-mercaptoethanol, and 10% glycerol. Thephosphocellulose column was developedwithagradientofNaCl from 0.35 to 0.70 M

in the same buffer. The EcoRI endonuclease was elutedatapproximately0.5M NaCl. Theenzyme was storedinthe same buffer it was eluted in, and proved stable at 0 to 4 C for several months. The reaction conditionsforthisenzyme as employed in this labora-tory werereported elsewhere (11).

Sarcosylcoresof Ad2tobedigested with the EcoRI

restriction endonuclease weredialyzed into TE and treated, in atotal volume of 100to 150 ul of 0.05 M Tris-hydrochloride (pH 7.5) and 0.01 M MgCl2, with

10 to 30 Ml of the phosphocellulose fraction of the enzymeat37Cfor 60 min.Subsequently, 5Ml ofa0.2 MEDTAsolution, and insomeofthe experiments 10

Mul

each ofa Pronase solution (5mg/ml) and a 10%

SDS solution, was added and incubation was contin-uedfor 30 minat37C. In other experiments,Pronase addition was omitted (for details see legend to Fig. 13).The mixture was then extracted with phenol (TE saturated), the aqueous and phenol phases were

separated by centrifugation, and the aqueous phase wasdialyzed against one-tenth strength electrophore-sis buffer and analyzedby electrophoresis on polya-crylamide-agarose gels asdescribed below.

Gelelectrophoresis. (i) Analysis of polypeptides in SDS-polyacrylamide gels. The procedures of Maizel (25) were used with minor modifications. Samples of sarcosyl or pyridine cores or intact adeno-virions were dialyzed against 0.01 M ammonium-acetate, lyophilized in a Virtis lyophilizer,

resus-pended in 0.1 ml of sample buffer (0.0625 M Tris-hydrochloride, pH 6.8, 2% SDS, 1% mercaptoethanol, and 10% glycerol), and heated to 100 C for 2 min. Subsequently,5Ml of a 0.25%solution of bromophenol blue was added, and the samples were analyzed on 13%polyacrylamide gels. Cylindrical gels of 14 cm in length were used. The composition of the gels and of theelectrode buffer was described previously (13, 37). Electrophoresis was performed for 5.5 to 7 h at ambient temperature and 100 V. The gels were then fixed in 20% trichloroacetic acid, stained in 0.25% Coomassie blue in 20% methanol and 7% acetic acid for periods ranging from 30 min to 16 h and were destained in 20% methanol and 7% acetic acid for several days. In some of the experiments in which

radioactively labeled proteins were analyzed, the gels

were cut with a slicer and each slice (usually 0.8 to 1.0 mm thick) was processed as described below. For someexperiments slab gels were used of identical composition (21) andelectrophoresis was performed in

anapparatusasdescribedby Studier (41).

(ii) Analysis of DNA fragments in 1.5%

poly-acrylamide-0.8% agarose gels. The composition of

the gels and of the TEB electrophoresis buffer was described earlier (13). DNA samples (0.1 to 0.2 ml) in one-tenthstrength ofelectrophoresis buffer were lay-ered on cylindrical gels, and electrophoresis was

carriedout at40Vatambienttemperature for 8.5 to

12 h. In most experiments the EcoRI fragments of 14C-labeledAd2DNAwere added as molecular weight markers. Immediately after electrophoresis, gels were

cut into 0.8- to 1.0-mm slices and the slices were processed for counting. Individual gel slices were placed intoplastic tubes or directly into scintillation vials and extracted with 0.2 to 0.5 ml of 0.1% SDS with shaking at 37 C for 18 h. The extracts were adsorbed to GF/83 glass-fiber filters, dried at 80 C,

andcounted in5 ml ofthetoluene-based scintillator

inaPackardTriCarb liquid scintillation spectrome-ter, model 3385 or 3330. In someexperiments portions J. VIROL.

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CORES FROM ADENOVIRUSTYPE 2 369 ofthe extractswere counted directly in 5 ml ofthe

toluene/methanol-based scintillator.

Electron microscopy. Monolayers of KB cells

infectedwithAd2weretransferredtoanicebath and

fixed with ice-cold 2% glutaraldehyde in PBS. After fixationonicefor 60min, the cellswerescraped from

theplates, washed with cold PBS, and storedat4C

until prepared forelectronmicroscopy.

Infected cells were prepared for thin sectioning after glutaraldehydefixation as previouslydescribed (3). Cellswerepostfixed for1hwitha1%solution of osmiumtetroxide in Millonig phosphate buffer (27).

The post-fixed cellswereembeddedinepon812by the

procedure of Luft (24). Silverto silver-gold sections werecuton aReichartOM U-2 ultra microtome.

Freeze etching of glutaraldehyde-fixed virus-infected cells was carried out by the procedure

de-scribed by Brown et al. (4). Some specimens were fractured and etchedafterwashingindistilledwater priortofreezing.

Negative stainingwascarriedoutby the procedure ofAnderson (2). Phosphotungstic acid was prepared

asa2%solutionindistilledwaterandwasadjustedto pH7.2by additionof NaOH.

Preparation of purified viral coresforthe electron microscope wascarriedout byamodification ofthe aqueousspreading procedure of Davisetal. (9).The spread monolayers of DNA and protein were trans-ferred to carbon-coated grids and were rotary shad-owedatan angle of 100 with platinum carbon.

Specimens were photographed in a Siemens 101

electron microscope utilizing either bright or dark fieldoptics. The magnificationofthe instrumentwas calibrated withacarbongratingreplica having2,160 linespermm (Ernest Fullam Co.,no. 1002).

RESULTS

Morphology of Ad2. It is generally accepted

that theadenovirions havea mean diameter of

about 80 nm (20, 43) and that the adenovirus

capsid is composed of hexons which are 7 to 8

nmindiameter andpentonswhichare8.0nmin

diameter. Morerecentstudieshave revealedthe

hexon to be a rod-like structure composed of

three proteins withanoverall diameter of9nm

and a lengthof 11 nm and the penton base to

representasphere of 8-nm diameter (30, 45; G.

Wadell, Ph.D. thesis, Karolinska Institutet,

Stockholm, 1970). We reinvestigated the

di-mensions of the type 2 adenovirion for the

purpose of relating the observations on the

internalmorphology of theparticletothe

struc-ture oftheoutersurface. We measured the size

of adenovirus hexons and pentons in both the

intact virion (Fig. la) and as free structures

after release from the virion by heating (Fig.

lb). We also determined the center-to-center

spacing ofthe hexons onthe face of the intact

virionandingroupsof hexons releasedfromthe

triangular facesofthe virus icosahedronduring

heating (Fig. lb). The dimensions of the various

structures are compiled in Table 1. The

pre-dicted size of the virion obtained by measuring

the center-to-center spacing of the hexons was

86.16 nm using the equations E = dT½ and D

= E/0.618 (40), where E is the edge of one

equi-lateral triangular face, D the diameter of the icosahedron, d the center-to-center spacing of

the hexons, and Tthe triangular number of the

icosahedron (for adenovirus T = 25). The

mea-sured value for the particle was 97.5 nm. This

suggeststhat in negatively stained preparations

the virions are distorted by compression as

described by Anderson (1) and by Moody (28), such that the observed cross-sectional diameter isincreasedby 13%. Byutilizing theequation V

= 2.1817 E3, where V is the volume of an

icosahedron and E the edge of one of the

equilateral triangular faces, we foundthe total volume of the virion to be 3.29 x 105 nm3.

Morphology

of thecoreof Ad2.

Subtracting

the thicknessofthe hexon-penton shellfromthe complete virion left a subcapsomere space of

volume1.35 x 105 nm3andaninternal diameter

of 64.15 nm. The measured diameter of cores

seen in thin-sectioned intact virions was 63.9

nm. The smaller size probably results from

shrinkage during the embedding process (29).

The volumeofthe space attributed to the core was therefore 41% of the total volume of the

virion.

The 63.9-nm electron dense core seen in

ultra-thin sections ofthe intact virion (Fig. 2) revealednoinformationregarding the

organiza-tionofthecomponents of the core. Weinitially

attemptedto study the interiorofthe adenovi-rionby freeze-cleaving ofpurifiedvirionsandof

virions attached to the surface of cells. Such

TABLE 1. Measured and calculated dimensionsof Ad

2and its structural components

Averagesize

Structure Technique in nm in nm Hexondiameter Negative stain 9.1

Hexonlength Freezeetching 12.0 Hexonlength Thinsectioning 10.5

Penton diameter Negativestain 8.3

Center-to-centerspac- Negativestain 10.7

ingofhexons

Widthof virion Negativestain 97.5

Widthof virion Thin section 85.6

Widthof virion Freezeetching 87.0

Widthof virion Calculated 86.16 Volume of virion Calculated 3.29x10'nm'

Diameter of core Thin section 63.9 Diameter of core Freezeetching 60.0

Diameter of core Calculated 64.1

Volume of core Calculated 1.35x10'nm9 VOL.16,1975

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4 V4

FIG. 1. Negatively stained preparations of(a) intact type 2 adenovirions and (b) virus capsomeres released from the virion by heat treatment. The intact virions (a) show the classicalarrangement of six- and fivefold organized capsomeres. It is possible on a number of the virions to measure accurately the center-to-center spacing of the capsomeres and the diameters of the subunits. The capsomere in the disrupted viral capsid are also organized in sixfold arrays (b). The hexons have the same cross-sectional diameter as those in the intact virion and in addition have a recognizable substructure. Pentons with fibers can be seen in the background

(arrow). Magnification, x226,400.

370

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* .. ' S .

4n '

*rr i

* 9. * .'.! ; '

**-''

.s

-0'' *-.".

,*

S.,-_

* B-w@- S.;r'>

.* . t!. * *

*0

-. '. ,

* -S.

S.. * x*x,

'

*S .

-S....^:;

*:

,,-.tJ

k **;2'; a

**

*

V

|ebla. * / q . ~.J

r:

've:5<~~'t'v

;i

FIG. 2. Ultrathin sections ofintranuclearadenovirusparacrystallinestructures. (a) Lowmagnification of

cellat 36hpostinfection.Smallcrystallinestructuresare visible in the nucleus. (b) High magnification ofa

paracrystallineintranuclear inclusion. The cross-sectioned virions haveadistinctelectron densecenterandan

electrontran-sparent layer ofcapsomeres.Magnification: (a) x10,600; (b) x80,000. 371

'U

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372 BROWNETAL.

experiments never produced fracture planes

whichpassedthrough the interior of thevirions,

but rather always exposed the outer surface of the particles (see ref. 3; Fig. 3). We interpret

this resultto indicate differences in the

hydra-tion oftheinterior ofthe isolatedvirion relative

to the surrounding medium creating a resist-ance to thedeveloping fractureplane. Altering

the concentrations of cryoprotective

(glycerol)

didnotproduce anychangeinthe imageofthe

virion afterfreeze-cleaving. In a further attempt

toproduce fracture planes within the virionwe

have freeze-cleaved intact KB cells late after

infectionwithAd 2. At late times after infection

(35 to 40 hpostinfection), the nucleiofinfected

cells containlocalized areasof veryhigh

concen-FIG. 3. A paracrystalline inclusion of the type shown in Fig. 2 after freeze-etching in 30% glycerol and

shadowing withplatinum carbon. Some of the virions are cross-fractured producing arelativelyflat structure in

which theouterring ofcapsomeres ispoorlyresolved(A).Othervirions have a more distinctly resolved outer ring

ofcapsomeres(doublearrow B). The internal corestructure (C) is resolved in some virions as either a smooth

sphericalstructure (C left) orcontaining a knob-like substructure (C right). Occasionally, virions have been

cleaved such that the inner content is removed showing the inner smooth surface of the capsid (D).

Magnification: x162,000.

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CORES FROM ADENOVIRUS TYPE 2

373 rsa~~~~~V

FIG. 4. Anareaof a largeintranuclearparacrystalline inclusion afterfreeze-etching in distilledwaterand shadowing with platinum carbon. The hexagonal arrangement of the virions ispreserved inthefreeze-etched

paracrystals (large doublewhite-black arrows). Many of the virionsare brokenshowinginternal substructure

(singlewhite-edged arrow). Magnification: x140,000.

trations of virions

(paracrystals) (Fig.

2a,

b).

within the nucleus could be cross-fractured.

It was hoped that the virions in the cell

Cleavage

planes

passed through

theouter

layer

nucleus would be similar in their cleaving ofcapsomeres and

exposed

inavaried manner

characteristics tothe surroundingnucleoplasm the internal structure of the virions. In some

allowing fracture planes to pass through the instancesthe virions were cut sothatasmooth

outer layer of capsomeres and exposing the surface was

produced

which

barely

revealed the

interior of the virion. The

paracrystals

were outerringof capsomeres

(Fig. 3).

Such

particles

readily revealed by freeze-cleaving (Fig. 3 and were fractured close to the center and had a

4), andin contrast toextracellularvirus, virions cross-sectional diameter

slightly

larger

than

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374 BROWN ETAL.

that found in thin-sectioned virions (Fig. 2b).

The difference in the size of the freeze-etched

virions compared to thin-sectioned structures

can probably be accounted for by shrinkage

occurring during dehydration and embedding

for thin sections. The diameters of the

freeze-etched virions of 87 nm agrees with the size of

the virion predicted in Table 1.

In other virus particles the fracture plane

passed through the outer layer of capsomeres

and then over the internal contents revealing

the inside of the virion as a smoothsphereoras

composed of closely packed spheres with a

diameter of 9to 10 nm(Figs. 3and 4). In virions

fractured in this fashion the thickness of the

outer hexon layer was 10.5 to 12 nm. Some

virions were broken such that the core was

removed and the inner surface of the capsids

was exposed. This inner surfacewas smooth in

appearance(Fig. 3).

Isolation of sarcosyl cores from purified

virions. When CsCl-purified preparations of

[I4C

]formate-labeledAd2 weretreated with

sar-cosyl and subsequently centrifuged to

equilib-rium in CsCl density gradients as described

above, the bulkofthe "4C-label inAd2 banded in a density position

approximately

0.2 g x cm-3lighter than thatofthe3H-labeled marker DNA which was purified from Ad2 virions

by

the SDS-Pronase procedure (13; Fig. 5a). The

"4C-label

ontopofthegradientwasduetoviral

protein. The apparent buoyant density of Ad2

DNA in this experiment was higher than the

value of p = 1.716 g x cm-3 reported

previ-ously(10), perhapsduetothe presence ofsmall

amounts of sarcosyl. For the analysis of the

sarcosyl cores (seebelow)fractions27to29 were

used. It will be shown by electron microscopy

(see Fig. 8) that these fractions contain viral

cores.

The data presented in Fig. 5b and c reveal

that sarcosyl cores can be readily separated

fromthebulkofthe soluble viral proteinsby gel

filtration on Sepharose 2B. When [3H

Ithymi-dine-labeled Ad2 wastreated with sarcosyl, the

cores eluted in a single sharp peak (Fig. 5b),

whereas Ad2 labeled with a mixture of

3H-labeled aminoacids yieldedtwopeaksof

[image:9.503.281.447.73.526.2]

radio-activity upon treatment with sarcosyl and gel

FIG. 5. Isolation of sarcosylcores. (a) Equilibrium sedimentation in CsCl density gradient. Approxi-mately1.8optical densityunits at260nmof [14C]for-mate-labeled Ad2 in buoyant CsCl solution were

treated withsarcosyl andcentrifuged to equilibrium in a CsCl density gradient together with 3H-labeled Ad2DNA (0.3ug)asdescribed. The refractive indexes

2000 1,9

1.7

1500

16.

1.5

1000- '

500

-3Hcpm 10 20 30 40b

-0-b

6000-

5000-

4000-

3000-

2000-1000,

3Hcpm 10 20 30

00

C

-0-

750-

500-

250-Frcaction

Number

were measured ineverytenthfractionandthe densi-tieswere calculated asdescribedelsewhere (44). The

3Hand 'IC activities were determined in 5- to J0-Mul

portions ofeachfraction as described. (b) Gel

filtra-tion on Sepharose 2B column. Approximately 2.3

optical density units at 260 nm of

[3H]thymidine-labeled Ad2 in buoyant CsCl solution were treated withsarcosylin TE andpassed overaSepharose2B columnasdescribed. The bar indicates theposition of

the dinitrophenyl-alanine dye which was used as a

marker.(c)Inasimilarexperiment2.0optical density units at 260 nm ofAd2 labeled with a mixture of

3H-labeled amino acids were used under conditions

identical,tothose described in (b).

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CORES FROM ADENOVIRUS TYPE2 375

filtration (Fig. 5c). The minor peak represents

the sarcosyl cores, the major peak the viral

proteins (Fig. 5c). In this experiment, about

3.9% of the 3H-amino acid label was recovered

with the sarcosyl cores. This finding indicates

that only part of polypeptide VII is present in the sarcosyl core, since polypeptide VII

represents 14% of the viral protein (32). A part

ofpolypeptide VII wasapparently releasedand

could be found in the main peak (Fig. 5c). At

thepresent

time,

the mechanismofthisrelease

is notunderstood.

Sarcosylcores prepared by gelfiltration were

indistinguishable in the electron microscope

from sarcosyl cores obtained after equilibrium

centrifugation.

Athird way of preparing Ad2 cores wasby the

pyridine method (33) which yielded structures

different from sarcosylcores asjudged by

elec-tron microscopy and the polypeptide

composi-tion (see below).

Electron microscopy ofpurifiedcores. Sub-viral particles

produced

by heattreatment (39),

pyridine treatment (33), or by treatment with

sarcosyl were examined in the electron

micro-scope. It was necessary to utilize a variety of

preparative procedures for the analysis of each

type ofsubviral particle, as aprocedure which

produced goodresultswith one type ofsubviral

a

structure frequently produced no information when applied to another.

Electron micrographs of structures produced

by heating adenovirions to 56C for 5 min are

shown in Fig. 6. Best preparative results were

obtained by negative staining of these

struc-tures with neutral phosphotungstic acid. The

subviral structures observed were similar to

those described by Russell et al. (39). Free

hexons, groups of hexons, pentons, and phous structures were seen (Fig. 6a). The

amor-phous structures were larger than the intact

virions from which they were derived and had

anaverage diameterof 167nm, suggesting that

they represent particles which have lost their

structural integrity and have relaxed into a

larger volume. The subviral particles produced by this procedurewerefoundtocontain most of

the viral structural proteins (see below). Some

substructure was recognized in the amorphous spheres (Fig. 6b). Sphericalstructures about20

nm indiameter with acenter-to-centerspacing

ofabout30nm canbedistinguished.Itwasnot

possible to determine if these substructures

reflect aninternalorganization ofthevirion or a

topological alteration oftheviral surface.

Particles produced by treatment with

pyri-dine produced similar results whether stained

positively with uranyl acetate or negatively

b

FIG. 6. Negativelystainedpreparation ofAd2after heatingat56 Cfor5min.(a)Lowmagnificationshowing

viralcapsidcomponentsandlarge amorphouscores(arrows). (b)Higher magnificationofasubviralstructure showingasubunit-likeorganization.Inthebackground can beseenfree capsomeres. Negativelystained with

phosphotungsticacid.Magnification: (a) x80,OOO; (b) x,200,000.

VOL.16,1975

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376 BROWN ET AL.

stained with

phosphotungstic

acid(Fig. 7).The

pyridine core structure was best preserved by dilutingthe preparationin 0.37%formaldehyde

inPBS aftergradientpurification.The pyridine

cores were tightly packed and had a mean

diameter of 66 nm (Fig. 7a). Occasionally, partially decomposed structures were observed

which seemed to contain avariable number of

smallerspheres approximately22 nm in

diame-ter (Fig. 7b). The internal spherical structures

seen in the pyridine cores had a haloof

associ-ated material which appeared to beofuniform

thickness. It is possible that the

sphere-associated material holds the 22-nm

substruc-tures together in the tightly packed structures

(Fig. 7a) and coats the entire core preventing

thevisualizationofsubstructureinthemajority

ofparticles observed.

Sarcosyl cores produced different images in

the electron microscope depending upon the

preparative procedure employed. The sarcosyl

cores were rather fragile when examined by

negative or positive staining, but could be preserved by treatment with formaldehyde as

described above. Positively stained sarcosyl

cores (Fig. 8b) revealed electron dense

struc-tures with asomewhat variable diameter which

measured 66 to 67 nm in the best preserved

structures. The structures tended toaggregate,

aN "I..L

a~~~~~~~~~~~~~~~~4

and

frequently

fibrous

material, possibly

DNA,

radiated from the

centrally

located electron dense structures. The size variation described above

appeared

toresultfrom thereduction in

size of the

centrally

located dense

body

asmore

DNA wasreleased fromthe

particle.

Whenthe

particles

were

negatively

stained

(Fig. 8a),

it

was no

longer

possible

to

identify

the DNA

fibersortofindsuch welldefined electron dense

centers as seen in

positively

stained

prepara-tions. The

negatively

stained structures,

rather,

appeared

as a collectionofsmallersphereswith a constant diameter of 21.6 nm. Discrete

clus-ters of

eight

to ten 21.6-nm diameter

spheres

wereobserved

(Fig.

8c).

A

comparison

ofthe

morphology

of

positively

and

negatively

stained

sarcosyl

particles

sug-gests that the structures of 67 nm diameter

described above are

composed

of a number of

spheres

with a diameter of 21.6 nm. These

spheres appeared

very electron denseand were not

distinguished

in positivelystained

prepara-tions. In

negatively

stained preparations the

differential penetration of staininto the 67-nm

particle

and the partial exclusion of stainfrom theinteriorofthe 21.6-nmsubstructures

facili-tatesvisualizationoftheinternalmorphologyof thesarcosyl

particles.

We attempted to verify that the fibrous

I. '-. 9 4 '

b

FIG. 7. Viralcores releasedfrom adenovirions bytreatment withpyridine. (a) Typical pyridine cores. The structures arehydrophobicandadhere to one another. The surface is smooth and bulges as though there were

spherical structures under the surface. (b) An unusual pyridine core. The particle appears to have opened somewhatrevealing thesphericalstructures within. The spheres are coated with some material which may have

been on the outer surface of the core (arrow). Negatively stained with uranyl acetate. Magnification: (a)

x370,000; (b) x300,OOO.

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CORES FROM ADENOVIRUS TYPE 2 377

'.

_.re*

tE'';;-

_' ;-;

.8w;417

s ' * g _ wW

'' *'.'s,. i;'''

,&, :

., o.'.

#.'.''.'..-;o

' s.

FIG. 8. Cores released from Ad2 after treatment with sarcosyl. (a) Cores negatively stained with uranyl

acetate.Onecannotdetect the freeDNAor awell-defined central dense body. Rather the cores are made up ofa

numberof smaller spherical structures. Unlike the pyridine cores these sphereshave nohalo of material around them. Thecoresubunitssometimes appeartobe heldtogetherby fibrousmaterial(arrow). (b)Asarcosyl core

positively stained with uranyl acetate. The arrowpoints toDNAfibers protrudingfrom the electron dense

core. (c) High magnification ofa negatively stained sarcosyl core, showing 9orpossibly 10 subunits.

Mag-nification:(a) x116,000;(b) x156,000;(c)x420,000.

VOL16,1975

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I :, 4'.. 1-Y

.4- ,*

1.

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(13)

378 BROWN ET AL.

structures seen radiating from the positively stained sarcosyl cores was DNA

by

spreading sarcosyl cores on a protein

monolayer.

To our

surprise, freshly prepared particles did not

spread into the random DNA structure as

expected, butratherappearedas

tightly

packed particlesofapproximately thesizedescribedfor

thepositivelystained particles (Fig. 9a). Occa-sionally, these particles had small protrusions

ontheirsurface suggestingthe association with these structures of free DNA (Fig. 9a). If the particleswereallowedtostandfor afewdaysat 4 C, the amount of free DNA

protruding

in-creased, whereas the size of the central core

decreasedconcomitantly (Fig. 9b). Free endsof DNAwererarelyseen. Asthe DNAwasreleased

fromthe densecenter oftheparticle,itformeda

rosetteofclosed loops around the central

struc-ture. The

loops

of DNA were

frequently

highly

twisted

along

their

length,

but

opened

attheir distal ends like hair pins

(Fig.

9b, c).

With increasing time ofincubation at4

C,

the cores

seemed torelease

progressively

moreDNAinto

long random structures, some of which

rep-resented one genome

equivalent

of DNA (10

to 11 ,um in

length).

Even in these highly

disorganized arrays free ends of DNAwereonly rarelyseen, and theDNA wassimply organized

inlarger loops which radiatedasbeforefromthe

surface of asmaller core structure (Fig. 9d).

Ifthe preparationswere allowedtostandfor a

period ofabout 1 week, thecore-like structures

were only rarely seen. Instead randomly

ar-ranged circles of oneadenovirusgenomelength similar to those described

by

Robinson et al.

(36) wereobserved (Fig. 10).Open moleculesof

one andtwovirallengthscould also be foundin

these preparations. Treatment ofthe purified sarcosyl cores with pronase at any stage after

preparation converted the dense bodies into

linear moleculesofone genome length. Polypeptide composition of sarcosyl cores

from Ad2. Since the

buoyant

densityofsarcosyl

cores was found to be

considerably

lower than

that of Ad2 DNA (Fig. 5a), it was likely that

sarcosyl cores consisted of DNA andprotein. It

is not known whether sarcosyl can bind to the

cores and alter their

buoyant

density.

There-fore, reliableestimates oftheamount ofprotein remaining associatedwithsarcosylcores cannot

be made from the buoyant density. The

poly-peptidecompositionofsarcosylcores was

deter-mined by electrophoresis on

SDS-polyacryl-amide gels using both 3H-amino acid-labeled

(Fig. 11a-c) and unlabeled cores (Fig. 12, slot

no.2). Acomparisonofthepolypeptidepatterns

presented in Fig. llb and c demonstrates that

digestion

of the

sarcosyl particles

with DNase

prior

to

electrophoresis

does not affect the

ap-parent

polypeptide

composition

ofthe

sarcosyl

cores. In the

experiment

shown in

Fig.

12

(slot

no.

2)

the

sarcosyl

cores were

analyzed

after

digestion

with DNase which also

registered

in

the

gel

pattern. The

identity

of

polypeptide

VII was demonstrated

by

comparing

slot no. 2

with slotno. 3which containedDNase

by

itself

(Fig. 12).

The data indicate that it is

predom-inantly

the

alanine/arginine-rich

polypeptide

VIIwhichwasassociatedwith the

sarcosyl

cores.

The

identity

of this

polypeptide

was further documented

by

co-electrophoresis with ["C

1-formate-labeled

polypeptides

fromadenovirions

(Fig.

lla),

and

by

the

high

proportion of

arginine

and alanine in the amino acid

com-position

of

sarcosyl

cores. The sarcosyl cores

contained also a small amount of the hexon

polypeptide (II).

From the data presented in

Fig.

llaand

b,

itcanbe estimated that <3%of

the total hexon

polypeptides

were still

associ-ated with sarcosyl cores. Small amounts of

polypeptides

VIII and IX were also present in the

sarcosyl

cores.Thisfindingisnotsurprising, since polypeptides VIII and IX are associated

with the hexons (16, 17,

18).

Asa

control, pyridine

coreswereprepared

by

the method of

Prage

et al. (33) and were

analyzed by electrophoresis

on

SDS-polyacryl-amide gels (Fig. 12, slot no. 5). It has been

shown previously (17, 18, 32, 33) that

pyridine

cores contain

exclusively

polypeptides V and

VII,

and this

finding

is also documented

by

the results shown in Fig. 12, slot no. 5. This

control supports the conclusion that

sarcosyl

cores arefreeofpolypeptide V, since this

poly-peptide

can

readily

be detected in

electro-phoretograms

of corepreparations.

Whenpurified virionswereheatedto56Cfor 5 min

(39),

repurified by equilibrium

centrifu-gation in CsCl density gradients, and then analyzed by electron microscopy and electro-phoresis on

SDS-polyacrylamide

gels, the

re-sulting

cores had a typical structure (Fig.

6b)

and contained most of the viral polypeptides

(not shown).

The organization oftheviral DNA in

sar-cosyl cores. The sarcosyl cores contain intact

viralDNA. When [3H]thymidine-labeled

sarco-syl

cores were extracted by the

SDS-pronase-phenol method (10, 13), Ad2 DNAwasobtained

which co-sedimented at 31S with "4C-labeled,

intact Ad2 marker DNA in neutral sucrose

density gradients. After incubation of

[3H ]thymidine-labeled sarcosyl cores with

pan-creatic DNase for 5 to 20 min as described

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-

ia~~~~~W

A; '

I i. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

.--V 4b~~~~~~~lw~'r

FIG. 9. Electronmicrographs of sarcosylcoresprepared by spreadingon aprotein monolayeratvarioustimes after isolation. (a) First day of purification: thecores are tightlypackedstructuresfrom whichoccasionallya small amountof DNA protrudes. (b) Second day after isolation:moreDNAprotrudes inaradialfashion from the cores. The loops of DNAare closedstructures. Free endsare not seen. (c) Thirdday after isolation: the centralcorebecomes smaller and the freeDNAloops larger. Theloops remain closed. (d) Fourtofive days after isolation: thecentraldensebody has almost disappearedandthefreeDNAloops represent nearly the complete genomelength. Magnification: (a) x40,OOO; (b) x38,000; (c) x60,OOO; (d) x60,OOO.

379

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380 BROWN ET AL.

FIG. 10. DNA of i mately 1 weekafter is viral genomeunitlend

Fig. 9 is no longer appi

illustrated in Fig. 13a this protein was removed by pronase digestion.

This interpretation was confirmed bv the

datapresented in Fig. 13c. Upon extraction and

Pronasedigestion of the 3H-labeled material on

top ofthe gel showninFig. 13b, the 3H-labeled

- --^ EcoRI A and C fragments were released from

this complex and could be identified by

co-electrophoresis with the appropriate "4C-labeled

-,SsFlar marker fragments (Fig. 13c). Fragments A

and C are located at either terminus of the

Ad2 DNA molecule (31) (insert to Fig. 13a) and

are presumably involved in the circularization

oftheDNAmoleculeinsidethevirion(36).The

major polypeptide found associatedwith

sarco-syl cores waspolypeptide VII. It is not yet clear

whethersomeofthe polypeptide VII chains or, perhaps more likely, another as yet unidentified polypeptide present in minute quantities is

4d2. Sarcosyl core at approxi- responsible for complex formation with

frag-olation. Thegenomeis acircleof ments A and C. This complex is interesting

gth. The central core as seen in because it might stabilize the viral DNA in a

rarent. Magnification: x32,710. circular configuration.

above, all the 3H-label was converted to low-molecular-weight material which migrated with

the bromophenol blue dye front upon electro-phoresis in polyacrylamide-agarose gels.

Whenthe sarcosyl cores were incubated with

EcoRI restriction endonuclease and when the

incubation mixture was subsequently treated with Pronase, the [3H ]thymidine-labeled Ad2

DNA inthe sarcosyl cores was cleaved into the

six specific fragments (31) which

co-electro-phoresed withthe six specific EcoRI fragments from

"4C-labeled

marker Ad2 DNA (Fig. 13a).

These results were obtained independently of

thetime afterisolation ofthe cores and indicate

that, although theviral DNA was closely

associ-ated withpolypeptideVII in the core, the DNA

remained fully susceptible to digestion with

pancreaticDNase and that the six palindromic

sites recognizedbythe restriction endonuclease

EcoRI were accessibleto and canbe cleaved by the enzyme.

Strikingly different results were obtained

when sarcosyl cores were treated with theEcoRI

restriction enzyme and were subsequently not

digested with pronase (Fig. 13b). In this case the amount of the 3H-labeled EcoRI A fragment

was reduced in amount relative to the

l4C-labeled marker DNA, and the 3H-labeledEcoRI

C fragment was missing. Some of the 3H-label

was retained on top of the gel (Fig. 13b); this

could be due to the missing DNA fragments

which are complexed with protein and thus

cannot enterthe gel, whereas in the experiment

DISCUSSION

(i)Morphology ofthe adenoviruscore.The

electron microscopical observations presented

here suggest that the interior of the

adeno-virion is structurally highly organized and not

asimple amorphous electron dense mass.Upon

treating the intact adenovirion with sarcosyl,

spherical structures 66 to 67 nm in diameter

arise. This value is slightly larger than the

size of the core in the intact virion as

meas-ured in electron micrographs of thin sections of freeze-etch preparations. The slightly larger

size of the free core mayresult from distortion

due to compression of the particles which

generally occurs in air-dried preparations.

Al-ternatively, the increase in the size of the

free core may result from an expansion of

the structure once it is freed from the

con-fines of thecapsidshell.

The DNA ofthe 67-nm core ispacked into a

number of smaller spherical structures each

havinga diameterof 21.6 nm. There are8 to 10

such spheresdetectable foreach core, although

this number isprobablytoolow as thosespheres

which arecoveredby others cannot be counted.

Given the cross-sectional diameter ofthe

sub-structures it is possible to calculate their

vol-ume, assuming they are spherical, by the

for-mula V = 4.189 r3 (40), where Vis the volume

and r the radius of the sphere. The volume of

thisstructurewould be5.2 x 103nm3,and 25of

these spheres would be required to occupy the

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CORES FROM ADENOVIRUSTYPE 2 381

It.sSIDsX,

j}HcpVIm300

X:O,

se '*~

CV U

2000 02 04 0608 1OR,

01 In IVv VI 1 O , \s0V

.~~~~~~~~~~~~~J

'

_

10 0 30 0 50 60 20 80 90 10 110

b

V0 VUIx

0 0 30 4 5 60

s ~~~~~~~~~d

nmIv v VI vn v'll Ix

L

X=

7080o YU 1010l 10 20 30 40 Fraction Number

050 60 70 80 90 100 110

FIG. 11. Analysis of polypeptide composition of sarcosyl cores. (a) Sarcosyl cores were prepared from 3H-labeled amino acid Ad2 by gel filtrationon Sepharose 2B, dialyzed against 0.01 Mammonium-acetate, lyophilized, and resuspended in 0.1 ml of sample buffer. A small amount of "4C-labeled Ad2 marker virus wasadded and the samplewaselectrophoresedona13%polyacrylamide gel(21)for7.5hat100V,asdescribed.

The Romannumeralsdesignate the viral polypeptides. In the insert the logarithmsofthemolecular weightsof eachof the viralpolypeptides areplottedversusthe

Rf

values. (b and c) Sarcosylcores werepreparedfrom

3H-labeled amino acid Ad2 by equilibrium centrifugationinCsCIdensity gradients andweredialyzed against 0.01MNH4-acetate buffer. One portion (b)wasincubated with50 ,ugofDNase under the conditionsdescribed andthereactionwasstoppedbytheadditionofEDTA.Anothersample(c)wastreatedidenticallyexceptthat DNase wasnotadded tothe reaction mixture.After lyophilization and resolubilization in samplebuffer, the samples wereanalyzed by electrophoresis on13%polyacrylamide gelsat100Vfor 7.3 h. (d) Asacontrol the polypeptides of "4C-labeledAd2wereanalyzed byelectrophoresison13%polyacrylamide gels.

entire internal volume (1.35 x 105 nm3) ofthe

virionwhichwehave allottedtothe-core (Table

1). By actualcountthe number of spheres in the

core is lower, as would be expected, since the

21.6-nm-diameter spherescontainonlythe viral

DNAandprotein VII.

It is possible to estimate more precisely the

number of spheres making up the core of the

virion by calculating the volume occupied by

the viralDNA and proteinVII. Ifoneassumes

the viral DNAtoapproximateacylinder2.0nm

wide by 109.4 nm long (12), its volumecan be

calculatedby the equation V = r2 irh(40) tobe

3.4 x 104 nm3 (where V is the volume, r the

radius, and h the height of the cylinder).Everitt

et al. (18) have shown that each virion contains

1,070copies of protein VIIwhichhasa

molecu-larweightof18,500. Assumingthatthisprotein

isglobular, similarto serum albumin, it would

have a volume of about 40nm3 (42). The total

volume of protein VII in the core would

there-fore be approximately 4.3 x 104 nm3, and the

totalvolume of theDNA and protein VII would

be about 7.7 x 104 nm3.Thus, 14ofthe 21.6-nm

diameter spheres would be required to contain

all of the DNA and protein VII.Thisnumber is

probably toohighasthe above calculation does

not take into accountthe fact thatsome ofthe

DNA must run between the 21.6-nm diameter

spheres connecting them together.

Another consideration in estimating the

num-ber ofDNA-proteinVII spheres which the core

contains is that of the symmetry relationship

between the icosahedral outer surface of the

virionand the innercore.Ifthecoreiscomposed

of subunits, as is implied here, and is to be

packed in an orderly manner into the T = 25

icosahedron, that is the adenovirus capsid, a

structuralcompatibilitymustexistbetween the

morphology of the core and thecapsid. This is

particularly desirable if the core isto be

pack-aged into the capsid by a mechanism of

self-assembly. The conditions ofcompatibility are

best met if the core, like the capsid, has

1500-

100-500

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382 BROWNET AL.

1

23 dil

[image:17.503.97.230.75.412.2]

4

5

FIG. 12. Analysis ofAd2andsubviral particlesby

electrophoresison13%polyacrylamide slab gels. Slab gel electrophoresis was performed at 40 Vfor 12 h under conditions as described. All samples were lyophilizedandredissolved insample buffer priorto electrophoresis. The individual slots contained the

following samples: (1) Ad2; (2) sarcosyl particles preparedandtreated withDNaseasdescribed inthe

legendtoFig. llb; (3) DNase; (4) Ad2; (5) pyridine corespreparedaccordingtothemethods described by

Prageetal. (33)andby Everittetal. (18).

icosahedralgeometry (20, 43). Percore, 8to 10

spheres of 21.6 nm diameter were observed in

electronmicrographs, and about14sphereswere

expected by the above calculations. We predict

that in reality each core contains 12 of these

21.6-nm-diameter substructures. This number

takesintoaccountthepossibility that becauseof

superposition ofthe 21.6-nm-diameter spheres

within thecoretheycannotall be distinguished

in electron micrographs, and thatnotall ofthe

viralDNAactuallycontributesitsvolumetothe

spherical structures. The number 12 would

imply that about 19%ofthe viral DNA isnotin

the 21.6-nm-diameter

spheres,

but may in fact link the 12 spheres to each other within the

core. Proposing that the core contains 12 of

the spheres has the added

advantage

that it allows an exact

symmetrical

relationship

be-tween the viral capsidP = 1, T = 25, and the

core P= 1, T= 1,using the notation of

Caspar

and Klug (8). The 12coresubunits would be so

oriented that the 5, 3, 2 axes of symmetry of

the inner core coincide with the same axes of

symmetryoftheouter

icosahedral

capsid.

That 12 coresubunits isnotan

unreasonable

estimate for the

number

of

21.6-nm-diameter

spheres in the virus core is also

suggested

by

examination of an icosahedron

containing

12

structural units. When such a structure is

viewedfrom one direction, aswould be thecase

inthe electron microscope, a maximum of nine

subunits can be

distinguished.

If the

particle

isslightly distorted, amaximum of 9 to 10

sub-unitswould still beseen,the remainingsubunits

being obscured.

Utilizing the above calculations the

DNA-protein VII complex would account for about

57% of the total volume allotted tothe core of

theintact virion

(Table

1).Everittetal. (16,

18)

have suggested that the interior of the virus

directly below the outer

layer

of subunits

con-tains, in addition to protein VII and the viral

DNA viral proteins V, VI, and VIII. Everitt et

al. (18) have estimated the numberofcopiesof

each ofthe proteins per virion with the

excep-tion of protein VIII. Using the

procedure

de-scribed abovewecalculated the total volumeof

theseother proteins, exceptVIII, tobe 4 x 104 nm3. When this volume is combined with that calculated for the DNAandprotein VIIcore, it

amounts to a total ofabout 1.2 x 105 nm3 and

accounts for allbut 11%oftheinternal volume

of the virion which should be occupied by

protein VIII andwater.

(ii)

The protein components of the viral

core. Selective proceduresforthe stepwise

deg-radation of the adenovirion proved useful to

elucidate its structure (16, 18,

22, 33,

39). We

have used heat (56

C) (39),

pyridine (33),

and sarcosyl(11)treatmenttodisrupt the virion and

get access to the core. Each of these agents

produces a specific type of subviral particle.

Althoughsubviralparticles preparedbyheating

purifiedvirions to 56C (39), by pyridine

(33),

or

by sarcosyltreatment have some similarities in

appearance in electron micrographs of

nega-tively stained preparations (Fig. 6, 7, and 8),

theyarestrikingly different in theirpolypeptide

composition(Fig. 12). Virions heated to 56 C for

5min andreisolatedbyequilibrium

centrifuga-J. VIROL.

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CORES FROMADENOVIRUSTYPE 2

383

10 20 30 40 50 60 70 80 90 100 110 120 130

[image:18.503.105.392.74.479.2]

Fraction Number

FIG. 13. Cleavage of viralDNA insarcosyl cores with the EcoRI restriction endonuclease. Sarcosyl cores were prepared from [3H]thymidine-labeled Ad2, dialyzed against TE, and incubated with EcoRI restriction endonuclease. Allpertinent techniques have been described. Immediatelyprior to analysis of the cleavage

products byelectrophoresis on 1.5%polyacrylamide-0.8%agarosegels, 1.4to2.8ggof "4C-labeledAd2 DNA

previously cleaved withthe EcoRI endonuclease inaseparateincubationwasaddedasamarker. Subsequent

to the incubation with EcoRIendonuclease, themarkerwas extractedfrom the incubation mixtureby

SDS-Pronase treament andphenolization. Beforeelectrophoresis allsamples weredialyzedagainst 1/10 strength

ofTEBelectrophoresis buffer. (a)Incubation ofsarcosylcoreswithEcoRIendonuclease, followed byPronase

treatment.An amount of sarcosyl particles equivalent toapproximately 1.5Ag ofAd2 DNA was incubated under standard conditions with EcoRI endonuclease for 60 minat 37 C. The reaction was stopped by the additionof5M1of0.2MEDTA, 10

Al

ofPronase(5mg/mI),and10

Al

of10%SDS and incubationat37C

con-tinuedfor30min. (b) Omissionof Pronase incubation. Experimentalconditions wereessentially identicalto

thosedescribed under(a), except thatan amountofsarcosylparticles equivalent to0.8Ag of Ad2 DNA was

used and that Pronase was notused in the incubation subsequent tocleavage by EcoRI endonuclease. The

gelslice infraction1waseluted in0.2mlof0.1%SDS andfurther analyzedasdescribed under(c). (c)Pronase

treatment of theDNA-protein complex. The materialremaining on top ofthegel described under(b) and elutedfrom thegelslice(fraction 1) in0.2 ml of0.1% SDS wasincubatedat 37C with 20M'ofPronase(5

mg/ml)and20

Al

of10%SDSfor45min. Thereaction mixturewaschilled,and"4C-labeled Ad2 DNAfragments

(EcoRI)

wereaddedpriortoelectrophoresis. VOL-16,1975

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384 BROWN ET AL.

tion in CsCl density gradients contain most of

theviralpolypeptides (results not shown),

pyri-dine cores (18, 33) contain polypeptides V and

VII (Fig. 12, slot no. 5), and sarcosyl cores

contain predominantly polypeptide VII and

onlyminor amounts ofpolypeptidesVIIIand IX

and hexons (Fig. 11).

Inthe model oftheadenovirion suggestedby

Everitt et al. (16), theviralpolypeptideVis in

close neighborhood to polypeptide VII and the

penton base, and polypeptide VII is tightly

bound to the viral DNA. In the sarcosyl cores

described in thepresent report, polypeptideVII

isthe predominantproteincomponent, whereas

polypeptide Vcan be removedfrom thecore

by

sarcosyl treatment. Obviously, polypeptides V

and VII must be anchored in different ways in

the viral core. The electron microscope data

presented (Fig. 3, 4, 7, 8) are also consistent withthisinterpretation (see belowandFig. 14).

In fact, the arginine/alanine-rich polypeptide

VII can be quantitatively separated from the

viral DNA

by

proteolytic

enzymes, or alkali

treatment, or

by

boiling

in SDS. Our

data,

however, do not

permit

us to draw definite conclusions about the type oflinkage between

n

IV

FeP

V

VI

VIII

Ix

FIG. 14. Model ofthe type 2 adenovirion. The Roman numerals refer to the viralpolypeptides (26). The

locationof the fractureplanes(FP1andFP2)isderived from electronmicroscopedata (seetext and Fig. 3, 4, 7,

and8). The particlesgenerated by cleavage inFP1isprobably similar to pyridine cores, the particle that arises

by cleavageinFP2isprobably similar to sarcosyl cores. The arrangement of the viral DNA molecule as shown in

the model was chosen not to presuppose any definite organization. The loops extending into each of the

substructures in the model in reality represent tightly packed coils of viral DNA (see Fig. 9). The model represents a modification of the scheme proposed by Everitt et al. (16).

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CORES FROM ADENOVIRUS TYPE 2

385

polypeptide VII and the viral DNA. The poly-peptide analysis of sarcosyl cores by electro-phoresis on SDS-polyacrylamide gels gave very

similar results, regardless ofwhether the

sarco-syl cores were pretreated with DNase or were electrophoresed without predigestion of the DNA (Fig. 11).

Our ultrastructural studies revealed that the

sarcosyl core was atightlypacked sphere which

gradually unfoldedupon storage at 4 C(Fig. 9).

It is not understoodwhat typesof interactions,

DNA-proteinorprotein-protein, areresponsible

for the packing of the DNA or whether other

factors such as polyamines may play a role in

the organization ofthe viral DNA in the core.

The gradual unfolding of the sarcosyl core is

perhaps a consequence of the removal of all otherviralpolypeptideswhich serve tostabilize

the core structure. It is conceivable that

poly-peptide V is at least partly responsible for this

stabilization. Differences in ionic strength and

pH valuesintherangebetween 7.2and8.5did

notalterthemorphologyofthe sarcosylcores or.

noticeably affect the unwinding process.

(iii) The viral DNA. The viral DNA in the sarcosyl core can be

completely

digested

with DNase and is susceptible to cleavage by the EcoRI restriction endonuclease. The data

pre-sented in Fig. 13 demonstrate that the termini ofthe linear Ad2 DNA molecule are linked to

each other probably by a protein, since this linkage can be disrupted by treatment with proteolytic enzymes (Fig. 13c). The finding that, uponincubationofsarcosylcoreswiththe EcoRI restriction endonuclease, a complex

be-tween protein and the terminal EcoRI

frag-ments A andC canbe isolatedontop ofthegel

(Fig. 13b) furtherargues forthe involvementof a

polypeptide

inthis

linkage.

Thenatureofthe

polypeptide(s)

in thiscomplex hasnotyetbeen determined.

TheviralDNA inthesarcosyl coreshasbeen showntobea

circular

molecule

(11;

Fig. 10)

and circular adenovirus DNA has also been found

after treatment ofpurifiedvirionswith

guanidi-nium hydrochloride (36). These circular DNA

molecules are converted to a linear form

by

proteolytic enzymes and the linear Ad2 DNA

has a unique melting pattern (12), hence the

circular moleculemustbe

opened

by

proteolysis

at one specific site.

We concludethat the complex between viral

EcoRIDNAfragmentAandC and the

polypep-tide(s)

responsible

for circularization of the

viral DNA has been isolated in theexperiment

described in Fig. 13b. It is conceivable that

polypeptide VII is involved in the

circulariza-tion oftheviral DNA,

however,

an

additional,

morespecialized polypeptide may be present in

minuteamounts and may hithertohaveescaped

detection.

We also consider the possibility that the adenovirus DNA isa covalently closedcircular

molecule and that an activated form of the

virion-associated endonuclease (7) cuts the

cir-cle at the site of thespecificpalindrome dueto

the terminal repetition ofthe adenovirus DNA

(19, 46). This endonuclease could be activated upon disruptionofthestructural integrity ofthe

virion.

(iv) Correlation between morphological

and biochemical findings. The presence of

protein V inthe pyridine cores and itsabsence

inthe sarcosylcorescorrelates withthe presence ofthe haloofmaterialsurrounding the21.6-nm

core subunits in the particles produced by

pyridine, and the absence of this halo in the

particles prepared by sarcosyl treatment.

Pro-tein V seems to coat the protein VII-DNA

complex and togive the pyridinecores a

stain-ing characteristic which generally makes the internal structure non-distinguishable. In the freeze-cleaved virions, twofractureplanes were found inthe interiorofthevirion. One revealed thecoreas asmoothstructure,the other showed

thepresence ofsubunits. It isconceivable that

in the former case the fracture plane passed

between the outercapsid and protein