Comparison of the Structure and Polypeptide Composition of Three Double-Stranded Ribonucleic Acid-Containing Viruses (Diplornaviruses): Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus

Comparison

of

the Structure

and Polypeptide

Composition

of

Three

Double-Stranded

Ribonucleic

Acid-Containing

Viruses

(Diplornaviruses):

Cytoplasmic

Polyhedrosis

Virus,

Wound

Tumor

Virus,

and

Reovirus

L. J. LEWANDOWSKI AND B. L. TRAYNOR

DepartmentofMedicalMicrobiology,Stanford UniversitySchooloJMedicinie,Staniford, Californzia 94305anid Departmentof MolecutlarBiology anid Virus Laboratory, UniiversityofCalifornia, Berkeley, California94720

Received for publication 17July1972

Iodinationofreovirus, cytoplasmic polyhedrosisvirus (CPV),and wound tumor

virus(WTV),and theirrespectivesubviralforms,followedby analysisof thelabeled

polypeptides by usingpolyacrylamide gel electrophoresis, has beenused tocompare

theprotein contentsof these three diplornaviruses. Thisapproach, whencombined

with electron microscopy and buoyant density determinations, appears capable of

localizing individual polypeptides in some ofthe viral and subviral forms. CPV

(p = 1.435 g/cm3) seems to resemble reovirus cores (p = 1.440 g/cm3) in both

ultrastructureand polypeptide composition. CPV iscomposedof five polypeptides

with molecular weights of about 151,000, 142,000, 130,000, 67,000, and 33,000.

The polyhedral matrix, which in nature encapsulates the virions, is, in turn,

com-posed mainly oftwo polypeptide species with molecular weights ofabout 30,000

and 20,000, and several minor proteins. The proteins of WTV consist mainly of

four species of polypeptide with molecular weights of about 156,000, 122,000,

63,000, and44,000, and several minor components. These molecular weight

deter-minationsareconsistentwith thehypothesis that,ashasbeensuggested for reovirus,

the viral proteins ofCPV and WTV seemto becoded for by monocistronic mes senger RNA molecules transcribed from distinct segments ofthe double-stranded

RNAviral genomes.

In previous communications it was observed thatthestructureofthenucleic acid of the insect pathogen,

cytoplasmic

polyhedrosis

virus (CPV),

was similar to that ofthe mammalian reovirus,

inthatbothgenomeswerecomposed of10distinct

double-stranded ribonucleic acid (dsRNA)

seg-ments present in equimolar amounts within the respective virions (15, 20). Furthermore, these

viruses, as well as a third virus-containing

seg-mented dsRNA (12), the plant oncogenic virus,

namely, wound tumor virus (WTV), contain

enzymatic activitiesin their superstructures

capa-ble of transcribing their respective dsRNA

genomes into discrete corresponding

single-strandedRNAmessengermolecules (3, 4, 13, 22).

The

common

presence ofthese two properties, a

segmenteddsRNA genomeandavirus-associated transcriptase, suggests that, despite their rather

diverse host systems, these dsRNA viruses (the

nomenclaturediplornaviruseshas

been

suggested

by Verwoerd [31]) share the samebasic mode of

replication.

In the present study we have continued the

structural comparison of reovirus, CPV, and

WTV with special emphasis on the number and

location of specific

polypeptides.

The combined

techniques of electron microscopy and the in

vitro labeling ofproteins with 1251 (19) have been

used for this

purpose.

lodination

studies with

influenza virus have recently been used to suggest

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LEWANDOWSKI AND TRAYNOR

that the rate of such in vitro labeling of viral

polypeptides can be related to the position ofthe

individual polypeptide on the surface of the

virus(26).

MATERIALS AND METHODS

Chemicals. Sodium 125I (250 mCi/ml) in 0.1 M NaOH was obtained from New England Nuclear Corp., Boston, Mass. Chloramine-T and sodium-m-bisulfite were obtained from Eastman Kodak Co., Rochester, N.Y. Sodium dodecyl sulfate (SDS) was from Sigma Chemical Co., St. Louis, Mo. a-Chymo-trypsin was obtained from Worthington Biochemical Corp., Freehold, N.J. Electrophoresis grade acryl-amide, methylene bisacrylamide, ammonium persul-fate, and N,N,N',N'-tetramethylethylene diamine

wereobtained from BioRadLaboratories, Richmond,

Calif. Allotherchemicals werereagentgrade. Preparation ofviruses.ReovirustypeIIIwasgrown

in Lcells and purified by the method of Watanabe

et al. (33). Reovirus cores were prepared following

theprocedureof Smithetal. (25). CPV was grown in Bombyx mori,and bothpolyhedralbodies and virions

were purified as described in an earlier report (13).

WTV-infected tumor root tissue from sweet clover

(Melilatus alba) and virus-infected plant cuttings for vegetativepropagation werekindly providedbyL. M.

Black. Virus was extracted and purified as described by Kimura (3) with threeexceptions. Exposure of the tumor tissue to carbon tetrachloride waseliminated,

virus was concentrated by sedimentationonto a65%

sucrose-D20 cushionin placeof pelleting, and virus

waspurified through twocycles on 30 to60%j>o linear

sucrosegradients.

Equilibrium density centrifugation was performed

using preformed gradients of CsCl instandard buffer

and ranging in densities from 1.453 to 1.25 g/cm3.

Sedimentation was at 36,000 rev/min in an SW50.1 rotorfor 9 hrat4 C.Fractionswerecollected and

re-fractive indicesweredeterminedon aZeiss refractrom-eter. Buoyant density values were calculated accord-ing to theformula, pCsCl = 10.860nd25C - 13.4974

(32).

Electron microscopy. Samplesfornegative staining

weredialyzed against 0.01 Mammonium acetate, pH

7.0, mixed with an equal volume of 2%/o

phospho-tungstic acid (PTA) (pH 7.0), and sprayed with a

nebulizerontoaplatinum,7-hole Siemensgridwhich

hadacarbon-coveredcollodionfilm. Forshadowing,

the samples were sprayed on collodium-covered,

200-meshcoppergrids, air dried, and uranium shad-owed. Forthin-sectioning, samples werefixed in 2%7 glutaraldehyde, postfixed in osmium, embedded in epoxy, and sectioned with the LKB ultratome. Sec-tionswere poststained, first with 20% uranyl acetate inmethanol for20min, followedbylead citrate for5

min, and examined inaSiemens Elmiskop Ielectron microscope.

Dissociation of virus. Samples were dissociated eitherpriorto,orafter,iodination.Intheformercase,

CPV and WTV samples in buffer were dissociated with

17%

SDS at 100 C for 2 min, whereas reovirus

was dissociated with

I1,%c

SDSat 100 Cfor 2min, or

with 0.2% SDS and 6 M urea at 45 C for 15min. For dissociation of iodinated CPV and WTV, samples were exposed to 1 % SDS, 6 M urea, 0.03 M dithio-threitol (DTT), 0.1 M tris(hydroxymethyl)amino-methane (Tris), pH 7.5, at 100 C for 2min.lodinated reovirus was dissociated in 0.6% SDS, 6 M

urea,

0.007 M DTT, and 0.007 M phosphate buffer, pH 7.0, at 37 C for 1 hr. Unlabeled CPV used for molecular weight determinations in Fig. 8 was disrupted in 1% SDS, 0.014 M DTT, and 0.006 M phosphate buffer, by heating at 45 Cfor 20min.

In vitro iodination. The tyrosine residues of the samples under study were iodinated by using chlor-amine-T as the oxidizing agent. The reaction mixture consistedof 50,uliters of virus or subviral component, 35 ,uliters of 0.14 M Tris-hydrochloride, pH 7.5, and 5 /Aliters ofNa'251 in 0.2 M Tris, pH 7.5 (200

,uCi/

sample). Labeling was initiated by addition of 20

,uliters ofchloramine-T (10mg/ml) and was allowed

tocontinue at room temperature for the various times listed in the figure legends. The reaction was termi-nated by addition of 20,liters ofsodium-m-bisulfite (24mg/ml) and 1 ,uliter of 0.01 M potassium iodide. Excess 1251 was removed by overnight dialysis with several changes of buffer (0.01 M Tris-hydrochloride, pH 7.5, 0.001 M ethylenediaminetetraacetic acid [EDTA], 0.1 M NaCl for intact virus, and 0.01 M Tris-hydrochloride, pH 7.5, 0.001 M EDTA, 0.1% SDS for dissociated virus). When polyhedral bodies

wereiodinated, excess 125I was removed by

sedimenta-tionthrough 30% sucrose in an SW65 rotor at 10,000 rev/min for 15 min at 4 C. The pellet was resuspended in 5 ml of buffer (0.1 M Tris-hydrochloride, pH 7.5, 0.001 M EDTA, 0.1 M NaCI) and resedimented in a Sorvallrefrigerated centrifuge at 10,000 rev/min for

10 min. This final pellet of 1251-labeled polyhedral bodieswassolubilized with 1% SDS-8 Murea at100C for 2 minpriortoelectrophoresis.

Polyacrylamide gel elfctrophoresis. Dissociated viral fractions were made 5% in sucrose, 0.005% in

bromophenol blue dye, and analyzed on 5%

poly-acrylamide gels (120 mm inlength) prepared in 0.1 M sodium phosphate buffer, pH 7.2, 10%glycerol, 0.13%

bisacrylamide,0.02 MEDTA,0.1%SDS,0.1%temed

(N, N, N',N'-tetramethylethylenediamine),0.08% per-sulfate. Electrophoresis buffer consisted of 0.1 M sodiumphosphate, pH 7.2, 0.1%SDS, 0.02 M EDTA.

Electrophoresis was usually doneat6mapergelfor

25hr oruntilthedyefrontwas nearthebottomof the

gel.Fordeterminationof1251content,gelswerefrozen

in ethanol-dry ice and fractionated by a mechanical

gel fractionater into 1-mm slices. The gel fractions

weredried and countedinatoluene basescintillation fluid (4 g of2,5-diphenyloxazole and 50mg of

1,4-bis-2-(5-phenyloxazolyl)benzene per liter of toluene)

in an Intertechnique liquid scintillation counter at

full window. Molecularweightdetermination of

poly-peptideswasdonefollowingthegeneral procedureof

Weber and Osborn (35). Polypeptide staining was performed with Coomassie brilliant blue (0.25%)

in amixtureof 50%methanol-10%glacialacetic acid for 3 hr at roomtemperature. Gels were allowed to remain in destaining solution (7.5% glacial acetic

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acid-5%methanol) for1 hratroomtemperatureprior

to electrophoreticdestaining inthesamesolution.

RESULTS

Reovirus:

efficacy

ofiodination as a means of localizing virus polypeptides in relation to virus surface. Studies with reovirus type III

(25)

have shownthat reoviral

polypeptides

canbe isolated from three sources: whole

virions,

viral

"top

component"

(RNA-free

viral

shells),

and sub-viral core particles obtained

by

enzymatic

re-moval

of

the outercoat

polypeptides.

This same

report has shownthat reovirus

polypeptides

are

distributed in three

classes,

X

(containing

two

components, X1 and

X2),

,u

(containing

two

com-ponents,

1,u

and

A2),

and o-

(containing

three

components,

o,,

O2,and

a3).

Becauseit is

already

knownwhichofthereovirustypeIII

polypeptides

composethe externalcoatand which the internal

core proteins

(25,

16), this system seemed to

provide an excellent control to ourgeneral

pro-posal

to examine the

position

of

diplornavirus

polypeptides

by

using

the chloramine-T iodina-tion

technique.

Equilibrium sedimentation and electron

micros-copy of reovirus. When a crude

preparation

of reovirus typeIII was sedimented to

equilibrium

in preformed

gradients of

CsCl,

the

sample

separated

into

three

major

areas of

light-scatter-ingparticles, adense

major

band,

two less dense

satellite bands, and a third series of even less

dense minor bands. These three areas were

col-lected and repurified on separate gradients of

CsCl. Based on buoyant density determinations

of 1.38, 1.36, and 1.30 g/cm3, the respective bands seen in Fig. 1 were arbitrarily designated

V, S, andTtorepresent virions, satellite virions,

and top component. Representative samples of

these three preparations were then examined in the electron microscope. Figure 2 shows that,

although both populations Sand V are approxi-mately 75 nm in diameter and morphologically similar icosahedrons, the satellite virions (p =

1.36 g/cm3, Fig. 2a) have uniformly allowed little penetration of stain, whereas the major

virion fraction (p = 1.38 g/cm3, Fig. 2b) shows

heavypenetration by PTA. Top component

ma-terial (p = 1.30 g/cm3, Fig. 2c) showed empty

intactshells which werecompletely permeableto PTA and composed primarily of an inner shell membrane and a relatively intact outer capsid

structure.Upon storage at 4 C, these shells tended to degrade into intact shell membranes and

random aggregates of capsomeres (Fig. 2d),

while both populations S and V appeared to

remainintact.

Iodination and gel electrophoresis ofthe

poly-peptides of reovirus and its subviral particles. Samples of these three populations were then

dissociated with urea-SDS, labeled with 1251,

FIG. 1. Sedimentation ofreovirus subfractions on apreformedgradient ofCsCl centrifugedfor 9 hr in an

SW50.1 rotor at36,000 revlmin. V, Virion (p = 1.38g/cm3;S, satellite virion (p = 1.36g/cm3); T,top com-ponent (p = 1.30g/'cm3).

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LEWANDOWSKI AND TRAYNOR

FIG. 2. Electron micrographs ofreovirussatellite (a),reovirus virion (b), top component (c), topcomponent stored at4C(d),andreovirus core(e). Particlesshown in a-d were isolatedfrom bands ofCsClequilibrium density gradients as shown in Fig. 1,dialyzedversus0.01Mammoniumacetate, pH 7.0,negatively stained with 1% PTA

atpH7.0,andsprayedwith anebulizeronto a7-hole, platinumSiemensgridwhich had acarbon-coated collodion

film. Particles shown in(e)weretreatedin0.01 Mammoniumacetate,pH 7.0, with 100 ,ug ofa-chymotrypsin/ml

at37Cfor60minprior tonegative staining.a-d, X160,000;e, X260,000.

and

subjected

to

polyacrylamide gel

electrophore-sis. Figure 3a shows that the three

major

classes of viral

polypeptides, X,

A,

and

o,

and theminor

peptide component VIII aresusceptibleto

iodina-tion. Thedistribution of 1251 label is 21, 38, 36, and 4%,

respectively,

and agrees

favorably

with

the values of24, 40, 32, and4% obtained when

virus is labeled in vivo with either 14C or 3H

amino acids (25). This distribution pattern

re-mainsconstant within thosetime periods tested,

from 1 to 15 min. Though not diagrammed, no

major differences could be detected in the

poly-peptide compositions of virion, satellite, or top

components. This

finding, together

with the

inability to detect any gross differences in total

nucleic acid content betweenthetwopopulations

Vand S (Lewandowski and Traynor, unpublished observations),suggeststhat the

increased

buoyant density and susceptibility toPTA uptake of the majority population may be due to loosening

ratherthanremovalofsurfacepolypeptides.Such

arealignment would explain why populations of particles withthe same

RNA-protein

ratio exhibit differential uptake and

binding

ofsmall molecules likeCsCl(21),and PTA.

Previous results suggested to us a potential relationship between reovirus core particles and purified CPV. Reovirus cores and CPV possess

similar segmented dsRNA genomes of

approxi-mately 15 x 106 daltons (14), similar buoyant

densities inCsCl, 1.435 4 0.005 g/cm3 (25, 15), and similar transcriptase activities demonstrable

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Froctions (mm)

3-0

(c)

6.0 _ 1

to~~~~~~~~~~~~~~~~~~~~~~~I

3) 40 50 60 70 80 90 100 110

Froctions (rr,rr)

in vitro (4, 13, 22, 24). Accordingly, intact

reo-virus was digested with

chymotrypsin

and sedimented to

equilibrium

inCsCl. Two

nucleo-protein core components resulted, a major one

at thedensity of1.44g/cm3

(Fig. 2e)

andaminor

satellite component at thedensity of 1.42g/cm3.

Littledifference couldbeobservedbetweenthese

two populations by electron

microscopy.

The most striking physical feature of reoviruscores is

the presence of regularly

spaced,

chimney-like

spike projections (Fig. 2e; reference 18) on the 12 fivefold vertices of the icosahedron. The two core populations were disrupted with urea-SDS, labeledwith 1251, andanalyzed for their

polypep-tidecontent by

polyacrylamide

gel

electrophore-sis. The superimposed patterns are presented in

Fig. 3b. In agreement with the literature data

(16, 18), reovirus core consists mainly ofthe X polypeptides, and polypeptide 02, and a minor

amount of ul. Satellite core, however, has

addi-tional labeled material migrating between the ,u

and a- peaks, and as

low-molecular-weight

pep-tide. Satellite core apparently represents an

incompletestageofremovalofthediscrete

degra-dation products X, Y, and Z (37), and small

peptides of the major coat polypeptides IA and

03 thereby

causing

the core particles to exhibit

a decreased buoyant

density.

Localization ofreovirus polypeptides. We next

investigated whether one could use the 1251 in vitro labeling procedure to

successfully

dis-criminate between surface and internal polypep-tides in the intact virion by the rate of labeling of the tyrosines of each class of polypeptide.

Intact reovirus type III was treated with 1251

reagent for 60 sec; the subsequently obtained

electropherograms of the dissociated proteins

are shown in Fig. 3c. Two striking features are

observed: first, the X core polypeptides contain

very little label, about

7%

of total compared

withthe 21

¼o

of totalwhen

dissociated

viruswas

labeled with iodine (Fig. 3a), and second, the

external coatpolypeptides ofthe ,u and

a-

classes are

appreciably

labeled, and, in particular,

o-has a disproportionate amount of label

(58%-)

compared to the amount observed

(36%)

when

dissociated virus is iodinated. Thus, limiting the

[image:5.493.44.233.51.570.2]

iodination time appears to discriminate between

FIG. 3. a, Polyacrylamide gel electrophoresis of reoviruisdissociatedwith6 -ifurea-0.2%'SDSat45C for15 mini priortoiodinatioln. Two huundred,lAiters of sample wasiodiniatedJor 15 miniat room temperature

asdescribedin Materialsacid Methods. The iodinated

sample was analyzed oln a 5c% polyacrylamide gel.

Migration wasfor6 hrat 3 mAper tube, anid 1-mm

sliceswere exami,iedfortheirradioactivecontent. Di-rection ofmigration isfrom le,ft to riglht. b, Reovirus

coreparticle (A) aiid satellite core (-), dissociated

with5 mi irea-0.2%,G SDSat45 C for45 miii prior to

iodination. lodination was 5miii atroom temperature.

Tlhe iodiniatedsamples wereplacedoii 4%

polyacryla-midegels aiid run,fbr 6hrat3mApertube.c,Reovirus iodinated prior to dissociationi with 6 -i urea-0.2%

SDSat100Cfbr2 min.Onte huintdred ,uliters of'sample

wasiodinated for60sec asdescribedin Materials alid Methods. Electrophoresis was oii 5% polyacrylamide

gels for27hrat7 mApertutbe.

0

c~j

0

re)

c'

50 60

Froctions (mm)

0

LI'

2

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LEWANDOWSKI AND TRAYNOR

polypeptidesin the undenatured virion. Not only

is thetechnique capable of accurately confirming

which classes of viral peptides comprise the

ex-terior coat, but also which particular class (in

this case

a)

is most accessible to iodination and

presumably, therefore, most exterior. These in

vitro dataare in good agreement with the recent

data

showing that,

in

vivo,

U is the first

protein

toberemoved during the uncoating process (23)

followed

by

g2.

Whenintact reovirus cores were

iodinatedprior to SDS dissociation (diagram not

shown)core polypeptideo inparticular showed a

lower percentage of total label relative to the X

components than was seen when cores were

dis-sociated prior to labeling (Fig. 3b). Thus, in the

intact reovirus core, the tyrosines of the X poly-peptides appear to be more available for

iodina-tion than are those of the ,u and a- core

poly-peptides.

CPV: electronmicroscopyofpolyhedral bodies.

CPV, perhapsthe mostwidespreadviral pathogen

of insects (1), derives itsname from light

micros-copy observations

W*'

that infection is associated

/. ' 4'

..

....s

T.w..,. _..

t1

b.]ili&

with the formation of unique cytoplasmic

struc-tureswhich are designated "polyhedral inclusion

bodies." Inasmuch as polyhedral bodies of

cer-tainshapes(icosahedral, hexahedral, and

pyrami-dal [10]) can becloned to give riseto polyhedra

of thatparticular shapeinsubsequent infections, polyhedral shapesappear tobecontrolled by the

strain of viruswhichthepolyhedracontain.

Thin-section electron microscopy was

per-formed on hexahedral polyhedral bodies

pro-ducedinsilkworm (Bombyxmori) larvae. Figure

4a showsthe gross structure of nativevirions as theyexistintheocclusion bodypriorto dissocia-tion of the polyhedral matrix. At the edges of

these polyhedra one can observe (arrows, Fig.

4a and 4b) native virions as they are partially

embedded orjust being released from thehighly

crystallized polyhedral protein matrix. The

ex-truding virion appears to have adensecore area

surrounded by an outer shell membrane which

has discernible spikes but no thick outer

capsid

structureanalogoustothereoviruscoat.

Equilibrium sedimentationand electron

micros-./

FIC;. 4.

Tlhin-sectioni

electronmicrograph ofCPVpolyhedralbodiesandvirus. Polyhedralbodieswereprepared

asdescribedin MaterialsandMethodsandfixedin2%glutaraldehyde,postfixedinosmium,embedded inepoxy,

and sectioned with the LKB ultratome. Sections werestained with20%0 utranylacetate in methanolfor20min,

poststainedinleadcitratejor5mini,andexaminedin aSiemensElmiskopIelectronmicroscope.a, X84,000; b,

X390,000.

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copyofpurified CPV. To observe the fine

struc-ture of CPV,

purified polyhedral bodies

were

exposed to pH

conditions known

to

solubilize

the polyhedral matrix without destroying the

infectivity of the released virions (13). Density

gradient centrifugation of such concentrated

ex-tracts onpreformed

gradients

of CsCl

produced

three detectable light-scattering components at

equilibrium. A typical light-scattering pattern (Fig. 5) shows the

major

virus band

(V)

atp =

1.435 g/cm3, a minor diffuse shoulder area

(S)

at1.425g/cm3,andavery

sharp

minortop

band

(T) at p = 1.30

g/cm3.

Previous work

in our

laboratory (15) has shown that the

major band

(V) iscomposed of

virions

containing

the entire

genome complement

of

10 dsRNA segments

in

equimolaramounts,whereas the diffuse shoulder

area contains

satellite virions

(S) which

are de-ficient insomeof thesmallerRNAsegments.By analogy to

reovirus,

the RNA-free material equilibratingat p = 1.30 g/cm3 was designated

Tfortopcomponent.

When

analyzed by

electron

microscope,

parti-clesfrom themajor virus bandwerebynomeans

homogeneous with respect to their response to PTA

staining.

This

variation

in

uptake

of

stain

was at times so

striking

thatwe selected several

micrographs

(Fig.

6) which

highlight

different structuralfeatures of CPV. Virionsappear to be

icosahedrons

with an average diameter

of

ap-proximately 65

nm,

a

capsomeric-like

surface

structure,thesuggestion ofanearly six-sided

out-line with 20 capsomeres often visible

along

the circumference, and distinct

pyramidal spikes

on

thecorners of the

polygon

(Fig.

6aand

6b).

These pyramidal spikes which seem to originate deep within the central core area of the virus are

particularly

prominent

in those

particles

which

are

heavily internally

stained and are

distinct

from the

chimney-like

spikes

seen with reovirus

cores

(Fig. 2e). Though six

spikes

are

readily

discernible,

one could

predict,

basedon the

ob-servedstructureofthe

virion,

atotal of either 10,

or, more

probably,

12

(9). Frequently,

virions (Fig. 6c) seem to be extruding long, tail-like

structures. Due totheir6to 7nm diameter, itis

unlikely

thatthesetailsaresingle RNA duplexes which would measure

approximately

2 nm.

More likely, these structures represent duplex

RNA bundles or

nucleoprotein

structures

some-what like thoseof

eucaryotic

chromosomefibers

(6).Whenvirus samples aregiven minimum

elec-tron beam exposure (36), one can observe the

capsomeric-like surface structures but not the

spikestructuresingreaterdetail (Fig. 6d). Ingeneral,nodifference was observed between

the structure of virions banding at 1.435

g/cm'

and particles in the less dense, diffuse satellite

FIG. 5. Equilibrium sedimentation of CPV on a preformed gradientofCsCl (p = 1.370 to1.453g/cm3)

for 9 hr at 36,000

rev/min

in an SW50.1 rotor. V,

Virion(p = 1.435g/cm3);S,satellitevirion(p = 1.425

g/cm3); T, top component (p = 1.30g/cm3).

zone, except thatsatellitevirionsmorefrequently

show tail-likestructures protruding from virions.

Exposure of CPV to chymotrypsin under the

sameconditionsthatproduce reovirus cores (25)

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LEWANDOWSKI AND TRAYNOR

FIG. 6. Electront micrographs showinig various morphological aspects of CPV isolated from CsCI

denisity

gradientsasshown inFig. 5. a, Virions withnegativestaininig;b,shadowed virioni;c, viriontextrudingnucleic acid

containing tails; d,negatively stainied virionis givenminiimum electron beamexposure;antde,niegativelystainedtop

component.a, X215,000; b, X160,000; c, X95,000;danide, X320,000.

from whole reovirus showed no gross effect on

either CPV ultrastructure or buoyant density.

Electron microscopeexamination ofthematerial

banding atadensity of 1.30 g/cm3shows empty

virions completely permeable to PITA (Fig. 6e) and, as was true of the RNA-free uppermost

reovirion band, aggregates of partially

dissoci-ated viral shells.

Polypeptide composition of CPV and its

sub-viral forms.The

polypeptide

componentsofCPV

were investigated by the same techniques

em-ployed in model experiments with reovirus. Samples of the three viral components, virions, satellite area, and top component, were first

dissociated with urea-SDS, labeledwith

1251,

and

subjectedtoanalysison

polyacrylamide

gels.The

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[image:8.493.62.450.79.520.2]

results are seenin theelectropherograms of

Fig.

7.

Disrupted

whole virions

(Fig. 7a)

were found

tocontain five distinct iodine-labeled

polypeptides

and about 10% of total 1251 migrating as small peptide material in front of the dye marker (insert). The proportionof total radioactivity in

each of the viral

polypeptides

obtained after iodination of dissociated whole virus was: I,

36%; II, 16%; III,

34%;

IV, 2%; and V, 10%,

respectively.

When dissociated satellite and dissociated top

componentof CPVweresubsequently labeled in

vitro and analyzed on parallel

polyacrylamide

gels, the

polypeptide

patterns shown in

Fig.

7b

and7c wereobserved. Attentionmight be directed

to the occurrence of

polypeptide

breakdown as

indicated by the presence of

high

background label

migrating

between components III and V

(Fig. 7b). This breakdown phenomenon is also

apparentin the analysis ofUdissociated top

com-ponent

(Fig. 7c).

Becausethe size

distribution of

6

0-C E

>:e

0

n.-.

20

50 60 70 Froctions (mm)

specificbreakdownproducts of the

larger

species

might well overlap several

of

thenative

species

in such a multicomponent system, it is

generally

rather difficult to relate breakdown products to

a particular native component. The

exception,

however, seems to be breakdown

product

IL

which, because of its size, can only be derived

from component I. The release of thedsRNA of

CPV, resulting first in a satellite area deficient inaportion ofthe RNA (15) and subsequently in

the fraction

called

top component

which,

by virtue of its low buoyant

density,

is

apparently

devoid ofanyof theRNA genome, seems

there-fore to be accompanied by the breakdown

particularly of the largest

polypeptide

species,

component I.

Localization of CPV polypeptides. The rate at

which thepolypeptidesofintact CPV virions are

iodinatedwasdetermined in the same manner as

with reovirus. The electrophoretic pattern of

labeled polypeptides obtained when

CsCl-puri-Froctions(mm)

FIG. 7. Electropherograms ofiodinated polypeptides of CPV virion (V), satellitevirion (S),andtopcomponent

(T) isolatedfrompreformnedgradients ofCsCl(Fig. 5).Samplesin a to c were dialyzedagainst 0.1 M

Tris-hydro-chloride(pH7.5)-0.001 M EDTA-0.1mNaCl,dissociated with 1% SDS, andiodinatedfor 60 sec asdescribed in

Materials and Methods.Thesampleindwasiodinatedfor 60 sec prior todissociation. Electrophoresis was on 5%

polyacrylamide gelsfor 25 hr at 6 mA per tube. The insert in a was included to demonstrate thepresence of small

peptide material whichmigrates ahead of the dye marker.

on November 10, 2019 by guest

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LEWANDOWSKI AND TRAYNOR

fiedwhole viruswasiodinatedfor a briefperiod

prior to dissociation is shown in Fig. 7d.

Com-ponent III has a higher portion (56%), whereas

components II, IV, and V have lower portions (5%, 1%, and 2%, respectively) oftotal label

than when whole virus is dissociated prior to

iodination

(Fig. 7a). Component I

(34%)

is slightly lower. ComponentsI and III may

there-forerepresent outer,and componentsII, IV,and

V, inner polypeptides of the CPV particle. It

must be understood, however, that the specific

location ofthe tyrosineresiduesin theindividual

viral

polypeptides isnot known. Thus, the

possi-bility

that tyrosine groups of the surface poly-peptidesareburied or those ofinternal

polypep-tidesare exposed cannot yetbeeliminated inany

interpretation

by using this technique of 1251

labeling

ofintact virusor subviral particles. Molecular weight determinations of CPV

polypeptides. Molecular

weights

of the poly-peptides from CPV were determined by

com-parison of their electrophoretic mobilitieson SDS

gels with those of proteins of known molecular

weights (35); polypeptides usedasstandardswere

tektin a' and a (220,000 and 200,000); myosin (200,000); Escherichia coli RNApolymerase sub-units

,3'

and ( (163,000 and 155,000); gamma

globulin H2L2 (150,000); ,B-galactosidase (130,000); bovine serum albumin (67,000);

gamma globulin H chain

(50,000);

ovalbumin

(45,000); gamma

globulin

Lchain

(22,000);

and

tobacco

mosaic virus

(TMV)

coat

protein

(17,500).

Figure 8a illustrates the comparative migrations of CPV and markers on 5%

poly-acrylamide gels. When plotted as in

Fig.

8b,

these data show that, in the gel system

used,

migration

islinear within the220,000 to

130,000

range and again within the 110,000 to

18,000

molecular

weight

ranges. The molecular

weights

of CPV polypeptides I through V, determined

from the plots of

Fig.

8b,

are

151,000,

142,000,

130,000, 67,000, and 33,000,

respectively.

Break-down product 1L seems to have resulted from

the

cleavage

ofa

fragment(s)

of

5,000

to

6,000

molecular weight from

polypeptide

I.

An incidental

point

arising

from these

de-terminations of molecular weights concerns the

Xi

and

X2 polypeptides

of reovirus type III.

Literature values place these two

species

at

155,000 and 140,000, a difference of

15,000

(25). Wehave, however,

consistently

observed a

difference ofonly

approximately

6,000

(Fig.

8a).

Recently ithas beenshown that

electrophoretic

mobilitiesin SDSgelscanbeaffectednot

only

by

changes in molecular sizebut also

by

changes

in charge and molecular conformation

(29)

caused bythechemicalmodification of

polypeptides.

To

determine whetherthe iodination

procedure

alters

theapparentmolecular weights of the viral

poly-peptides, iodinated

CPV polypeptides were

sub-jected

to co-electrophoresis in the same gelwith unlabeled carrier polypeptides (figure notshown). Examination of theindividual slices for both dye stain and 1251 content showed that the iodinated

polypeptides migrated,

ontheaverage, 5% more

slowly

than nontreated proteins.

Consequently,

whenever molecular weight values were deter-mined in subsequent studies by using

iodinated

proteins versus non-iodinated markers, a 5% correction factorwasapplied.

Once themolecular weights of the CPV

poly-peptideweredetermined, it was of interest tosee

if,

as appears to be the case for reovirus (37),

a clear correspondence exists between the size

of the dsRNA segments and thesize ofthe

dis-crete viral polypeptides. The genome of CPV

consists of 10 discrete dsRNA segmentsranging

in size from the largest, estimated at 2.7 X 106 daltons, to the smallest, at 0.37 X 106 daltons (14). It would appear (Table 1) that viral poly-peptides I through V could be coded for by dsRNA segments I, II,III, VI, andVIII, respec-tively.Thisleaves, however, five genome segments

either without a corresponding viral structural

protein or coding for proteins which might be

presentin the virus inamounts sufficiently small

to be undetectable by either dye retention or

1251I labeling.

Analysis of the polyhedral proteins of CPV.

We have already noted that the shape of the polyhedral body seems to be determined by the

strainof virus which itcontains. Itseemed

possi-ble, therefore, that the highly organized poly-hedral matrix (Fig. 4b) is composed of proteins which are also direct gene products ofthe viral

genome. To investigate this possibility,

poly-hedral bodieswerepurifiedon astep-gradient of

sucrose (13) andiodinated. The bulk ofthe

poly-hedral body appears, by microscopy, to be

ma-terial other than

virions-proper

(Fig. 4a). Hence

it seemed reasonableto assume that the pattern

of in vitrolabeling would reflect the highcontent

of the polyhedral

polypeptide(s).

lodinated polyhedral bodies were separated from free,

nonreacted1251byseveralcyclesofsedimentation

through step-gradients of sucrose and were

solubilized with urea-SDS. The labeled protein

was then analyzed by

polyacrylamide

gel elec-trophoresis in the presence of unlabeled viral

markerpolypeptides. Theresults are seenin Fig.

9.

The labelingpattern would suggest that poly-hedral protein consists

primarily

of two major polypeptides with approximate molecular weights of29,500 and 19,500. There are,

however,

minor

amounts of labeled

protein

in discrete

positions,

1062 _J. VIROL.

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M- e0mom

-H2L2EliliS-I

-H

-2L2

_-J4_ -I __ft

-M

-x

-(3- gal

.*4W -M-

_.

-SA-

41

-IV-OA

- -4

41 -QA-

_.J

., V

-L

I'l-T_

-TM V

20

40

60

80

10

Distance

Migrated

(mm)

FIG. 8. Molecularweightdeterminations oj CPVpolypeptidesbasedonelectrophoreticmigrationofassorted

markerspecies.a,Samplesweresolubilizedwith1%SDSandsubjectedtoelectrophoresison5%

polyacrylamide-0.1% SDSgelsat8 mApergel for17.5hr. Abbreviations: Ta' andTa, tektin a' anda;M, myosin;B' andB, E. coli RNApolymerase subunits B' and B; H2L2, gamma globulin H2L2; f8-gal, j3-galactosidase; SA, bovine

serum albumin; H, heavychain ofgammaglobulin; OA, ovalbumin; L, light chain ofgamma globulin; TMV,

TMVcoatprotein; I-V, CPVpolypeptides;X, IA, a, reoviruspolypeptides. Migration isfromtop to bottom, b,

Plotofmolecularweightversus distancemigrated.

1063 _4-w -Ta'

_swE -M(Ta)

-/3-9al

---crC a

40

10

0 >X

4

II

0

b

To'

/3;

m

9L

----_

-H2L

I I

3-gal

'III

F~~~

OA

K

I I I I I I I

)O

120

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[image:11.493.97.386.55.602.2]

[image:12.493.265.459.75.238.2]

LEWANDOWSKI AND TRAYNOR

TABLE 1. Correspondence of'geniome segmenit size of cytoplasmic polyhedrosis virus to the size

of viral antd polyhedral polypeptides

CPV genome com-ponent no. I III IV v VI VII VIII Ix x Molecular weight Observed dsRNA segment' (X 106) 2.70 2.44d 2.15 1.85 1.14 0.83 0.62 0.54 0.37 Estimated ssRNAb transcript (X 106) 1.35 .22d 1.07 0.92 0.57 0.41 0.31 0.27 0.18 Estimated gene productc 150,000 136,000d 119,500 102,800 64,000 46,000 34,000 30,000 20,500 Polv-peptide observed in the virion 151,000 142,000 130,000 67,000 33,000 Poly-peptide observed in poly-hedral matrix 122,000e lOG,00(' 48,OO0e 29, 500 19,500

aCalculated from Lewandowski and Leppla (14) byusing reovirusRNAsegmentsasmolecularweightimiarkers.

bssRNA,Single-strandedRNA.

cEstimated molecular weightof gene pr-oduct = mol wt

ssRNA message/[mol wt nucleotide/(3 nucleotides'aa)] X mol wtaa. Mol wt niucleotide, 3 X 102; inol wt aa, 102;aa,

aminoacid.

dAverage valuesforcomponents anid 111 which migrate

closely.

eMinorpeaksindicatedbyarrowsinFig.9.

a fl

I lI12

CPVI: m I

ICe r!~~~~~~~~~~

1~~~~

Cr--05-1 ft _

A(" dr) r,n ar) '7n O -- ,A ^IA

JIJF4u bs60 70 80 9C

Fractions (mm)

FIG. 9. Electropherogram of polypeptides from

iodinated,dissociated polyhedralbodiesofCPV.

Poly-hedral bodieswereputrifiedonzsucrosegradientts as

de-scribedinMaterialsanldMethods,

iodinated.for

15miii, anidsoltibilized with

1%Z7O

SDS-8 Ml uireaat 100 C for 2

mini. This labeled sample was mixed with unlabeled

marker CPV polypeptides anid sutblected to electro-phoresis oni 5c polyacrylamide-0.JO SDS gels at 7 mApergel,fbr21.5 hr. Migrationi isfrom lefttoright.

Arrowsindicate thepositionsofthreeminorpolypeptide species which, inadditionztothetwomajorpeaks,

corre-spontdinsizetoexpected viralgenieproduicts (seeTable 1).

someof which overlapthemarkerviral structural

polypeptides and several of which do not.

Com-parison with unlabeled CPV structural protein

markers and application of the 5 correction

factor associated with the iodination procedure

provide the molecular weight estimates seen in

the lastcolumn of Table1;themolecularweights

of the two major and three of the minor

poly-hedral polypeptides suggest that they might be

direct gene products of the five remaining

cis-tronsofthegenomeof CPV. As thepolypeptides

of the polyhedral matrix are being laid down around individual virusparticlesinthecytoplasm

ofthehostcell (27), it ispossiblethat othergene

products which, although perhaps not part of

the final virion superstructure but which were

functioning during virus replicationand

matura-tion, might easilybecome entrappedinthe

grow-ing polyhedral structure. This may explain the

presenceofthe minorpeaksinFig. 9.

WTV: Equilibrium sedimentafion and electron

microscopyof WTV.Arelationship between

reo-virusand WTV hasbeenproposedbasedon

elec-tron microscopy data showing somewhat similar

external morphologies andonthecommon

pres-enceofdsRNA as the geneticmaterial (2, 8, 28,

30).When sucrose-purifiedWTVwassedimented

to equilibrium in preformed gradients of CsCl,

thepreparation separated into threemajor classes

of light-scattering particles: two lower bands

which made up the bulk of the preparation, a

diffuse middle area, and an upper area of

obvi-ouslyless dense material. Immediate

resedimenta-tion of these three areasresulted in the fractions

seeninFig. 10.Thetwolower bands had buoyant

densities of 1.445 and 1.425 g/cm3, whereas the

middle diffuse area and the upper bands equili-brated at 1.39 to 1.41 and 1.30 g/cm3,

respec-tively. Dueprimarilyto previousreportsthat the

buoyant density of WTV ranges from 1.395 to

1.410 (D. R. Black, Ph.D. thesis, Univ. of

Cali-fornia, Berkeley, Calif., 1970; reference 7), the

middle diffuse area was arbitrarily designated V

(for virion). Becausethebuoyantdensities of the

lower bands approximately resembled those of

intact CPV and reovirus cores, these fractions

weretentatively designatedC(for cores),whereas

the top band was designated T (for top

compo-nent). The presence of upper bands (T) in the

centergradientwhich receivedonlythelower ma-terial(C) suggestedthatTcould ariseas a

break-downproductofC.

Electron microscopy of the original

sucrose-purified preparation and of the three fractions

resulting from CsCl resedimentation is shown

in Fig. 11. Figure lla shows a typical

sucrose-1064

J. VIROL.

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[image:12.493.62.256.91.278.2]

FIG. 10. Equilibrium sedimentation of wound tumor virion and subviral components. Sucrose-purified WTV

hadpreviously been sedimented to equilibrium in preformed CsCI gradients (p = 1.453 to 1.30g/cm3),anldthree

majorclasses ofliglht-scatterinzgparticlesresulted: two lower bandsequilibratinigatp = 1.445anidp = 1.425g/

cm1, termedfractiontC,amiddlediffusearea with a density from 1.39 to 1.41g/cm3, termnedfractioni V,anidan

upper area equilibratinigat p = 1.30g/cm3, termedfractionz T. Immediate resedimenztationz ofthese threeareas

resultedinthefractionis seent above. Allpreformedgradienlts were 3 ml in volunmewitha2-mlsampleoverlayanzd

wereruniat40,000

revlmiii

Jbr12hrinanSW50.1 rotor.

purified preparation containing mainly intact virions

approximately

66nm in diameter. Figure

1lb was selected to show some disrupting and

apparently empty virions as well as bundles of strands, possibly analogous to those seen with

CPV. The virions are hexagonal, possessing an

innercore area35to40nmin diameter(Fig. 1 c)

surroundedbyan outercapsid membrane. Figure

lid,

e, and f contain V, C, and T components,

respectively. It is apparent that, compared to

reovirus or CPV, prior exposure of WTV to

cesium chloride has a strong degradative effect

when virus is subsequently dialyzed to remove

salts and observed by PTA negative staining.

Whereas both theVand Cfractionsseemtohave

retained aninternalcore structure, both are now

relativelypermeable to PTA anddisplay little of the highly organized capsidstructure seen inthe

preparation not exposed to CsCl. It is not yet

clear whetherthelossofcapsid material occurred

duringCsCI sedimentation or during the

subse-quent dialysis or PTA staining steps. Top

com-ponent (Fig. 1

If)

is composed mainly of empty

shells, completely permeable to PTA and devoid

of much of itscapsid structure and none of the

virus-free strands seen in the V and C

prepara-tions. Exposure ofWTV to chymotrypsin failed

to produce a particle with spike structures

ana-logous to thoseonreoviruscores orCPV.

Polypeptide composition of WTV and its

sub-viral forms. Samples of the sucrose preparation

and fractions C, V, and T from CsCI werethen

dissociated with SDS, labeled with 1251, and

ana-lyzedon polyacrylamide gels. The resulting

elec-tropherograms appear in the upper

portions

of

thefour panels of

Fig.

12. Since the -16 x 106 daltons of viral genome comprise 22% of total

virionmass (12), we cancalculatethattotal

pro-tein mass is about60 x 106 daltons per virion.

With the sucrose gradient-purified mixture, the

1251 content of this protein mass is distributed

between two major polypeptide peaks (II and

IV), two small peiaks(IandIII), andseveralother

peaks not much greater than the background

(arrows, Fig. 12a). With the V fraction from

CsCl,the numberandheightof thesebackground

peaks were considerably increased, but again

peaks It and IV predominated (Fig. 12c).

De-spitethe appearanceinFig. lIeofalossofsome

of theoutercapsomerestructure, thepolypeptide

content offraction

ClI

(p = 1.425 g/cm3) shown

in Fig. 12b is very similar to that of

sucrose-purifiedWTV seeninFig. 12a.Thoughnotshown,

thepolypeptide pattern offractionCI (p = 1.445

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[image:13.493.106.388.71.309.2]

FIG. 1 1. Electron micrographsof WTV,preparedandstained as in Fig. 2. a andb, WTV purifiedon sucrose density gradientsbythe Kimuramodification as described in Materials and Methods;c, WTV partiallydisrupting

torevealan innercore-like area.Rods of potato virus X were added toenhance drop spreading.

d-f;

Viral

com-poieietsfromCsCIdensity gradienitcentrifuigationt (Fig. 10);d, componeiit V;e,

componentt

C;

f;

componentt T.

a-f; X160,000.

1066

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[image:14.493.61.448.46.649.2]

I CPv IH1 & 7

3t ~~~~~~~(a)

- 2- I E

E2

o r 4

- 1!

'

.p

'3

2k

c

c

[image:15.493.107.395.67.421.2]

Frcctions(mm)

FIG. 12. Electropherogram ofiodinated WTVpolypeptides. Upperportions of each part show viralfractionls

dissociatedwith1% SDSfor2minat 100Cpriortoiodinationfor10min.Lowerportionsofeachpanelshow

thesameviralfractions iodiniatedintactfor30secpriortodissociation. A,Sucrose-purifiedWTV. Unlabeled CPV

wasaddedtothissampleto serve asmarker,and thestainledpatternisseen atthe topofthefigure.Arrowsintdicate

the migration positions which wouldcorrespondtothose ofexpectedviral geneproducts (see Table2).b, WTV

"C,," bandisolatedfrom CsCIat theequilibriumpositionofp = 1.425g/cm3. c, WTV "V"areaisolatedfrom

CsCIattheequilibriumpositionofp = 1.410to1.390g/cm3.d, WTV"T"band isolatedfromCsCIatthe

equili-briumposition ofp = 1.30g/cm3.

g/cm3) was also much like that of fraction

CII.

This suggests that the apparent loss of

capso-meres in all the subfractions of

CsCl-exposed

WTV

(Fig. 12b-d)

maynothave occurred

during

sedimentation itselfbut rather as a result of

di-alysis against

low salt buffer or PTA

staining

priorto

microscopy

ofthe

CsCI-exposed

sample. Analysis of the polypeptide constituency of

WTV top component (T) is seen in the upper

portion of

Fig.

12d. In this case, no distinct

peaks arediscernibleatpositionsIandIII. Since

the bulk of the top component appears to be

composed of empty shells, the polypeptide

pat-ternin

Fig.

12dwould suggestthatcomponentsII

and IV

probably comprise

the membrane and

capsid polypeptides of these nucleic acid-free shells.

Location and molecular weight determinations

of WTVpolypeptides. Toinvestigate the locations ofthe viral polypeptides, samples ofthe sucrose

and CsClfractions ofWTV werelabeledprior to

dissociation and analyzed in parallel. Thelower

halves of Fig. 12a-d show that, inall cases, the

polypeptidemost susceptible to iodination in the

intact structures was species II. This tentatively

suggests thatpolypeptide II comprises the outer

capsomeric layer of WTV and its CsCl

subfrac-tions. Of the non-SDS-treated samples, the

tyro-sineresidues of polypeptide IV were available for

iodination only in top component (T), and then

(b) 3_

E

-2

44L

.0 l~

--~I

U-I

xcL\

i3-1

0 0 %

Au

-.41

.e. -?'if T

.

1, In

EZ

I I

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[image:16.493.52.244.104.293.2]

LEWANDOWSKI AND TRAYNOR

TABLE 2. Corresponidelice ofgeniome se

oJ wolutnd tumor viruis to the size oj

associatedpolypeptides W\TV genome component no.

II

11 III IV-V VI-VII VIII IX X-XI XII XIII Molecular weight Ob-served dsRNA seg-menta (X 106) 2.80 2.30 2.08 1.72 1.08 1.01 0.77 0.56 0.54 0.33 Estimated ssRNAb transcript 1.40 1.15 1.04 0.86 0.54 0.50 0.38 0.28 0.27 0.16 Estimated gene product" 155,000 128,000 116,000 96,000 60,000 55,500 42,000 31,000 30 ,000 14,500

,,Calculated from Lewandowski ar

(14) by using reovirusRNAsegmentsas

weight markers.

ssRNA,Single-strandedRNA.

See footnote b in Table 1.

only partially (Fig. 12d). The interpr

the data obtained with WTV seems t

cult,since the virus isunstabletoCsCl.

Molecular weight determinations

labeled polypeptideswere made by inc

labeled CPVinseveral gels. The migral

stained CPV markers is diagrammati

sentedatthetopof Fig. 12a. Basedon

the following values were assigned tc

distinct peaks of WTV polypeptide

156,000; II, 122,000;III, 63,000;and I

These molecular weight estimates are

in Table 2, which shows that a reaso'

respondence exists between the size

polypeptides presentinthevirion and

several of the estimated gene produ

latter values are derived from data

(

suggest that thegenomeofWTV iscoi

13dsRNA segnents whichareelectrop]

separable into 10 distinct classes. One

matethat viral polypeptides Ithrough

be coded for by dsRNA segments,

VI(VII), and IX. The predicted elect

positions of the remaining estimated g

uctshave beendesignated by arrowsir It is not yet possible, however, to de

the minor peaks of polypeptide labi

around thesepositions correspondtoc

geneproducts.

DISCUSSION

When the genome of a

dsRNA-virion isintroducedintoahostcell,th

?gnmit

size tion which the viral RNA contains cannot be

f virus- translated directly by the ribosomes;

transcrip-tion of single-stranded messages is first required. The RNA extracted from dsRNA viruses does

not appear to beinfectious. Transcription of the

input genome therefore does not appear to be

Polypeptide

accomplished by

any enzyme

pre-existing

in the

observed in_{the virion} uninfected cell, but rather by the RNA-dependent_{RNA polymerase introduced along with the} re-spective templates. It would seem to follow that

156 ooo translation of these messenger RNA species

I then provides the remaining virus proteins

re-122,000 quired for successful multiplication of the virion.

Viral genome-specified polypeptides fall into

63,000 two categories, those which compose the

super-structure of the virion and nonstructural

polypep-44,000X° tides which function in virus-specified

biosyn-thesis. In the case of the dsRNA-containing vi-ruses, these two categories appear to overlap.

Whereas each reovirion has a

chymotrypsin-id Leppla

digestible

outer coat structure

composed mainly

molecular of twoproteins,A2and the outermostcomponent

0J3, with CPV this role of protective outer coat

seems tobe borne on a collective basis by the

poly-hedralmatrix, also composed mainly of two pro-teins. The polypeptides most susceptible to

*etation of iodination with the intact viral or subviral forms o be diffi- are,2and 13forintactreovirus,theX components

for reovirus cores, components I and III for

of these CPV, and class II polypeptide for WTV. Unlike

ludingun- intactreovirus, purified WTV andCPV showno

tion of the detectable response to exposure to chymotryp-ically pre- sin.

thisstudy, All attempts in this laboratory to further

de-the four grade either reovirus core or purified CPV into

label: I, smaller enzyme-active particles have succeeded

[V, 44,000. only in eliminating polymerase

activity,

most

recorded probably by dissociating the enzyme-template

nable cor- complex. Whatever treatment,chemicalor

physi-of those cal, is used toseparate RNAfrom the viral

pro-the size of teins appears also to destroy

biological

activity.

cts. These Furthermore, wehave had to date no success in

14) which obtaining template-free viral proteins in any form

mposed

of which,whencombined withexogenousviralRNA

horetically

in the proper substrate-salt milieu (4, 13, 22),

- can esti- initiates transcription. Based on these facts, the

IIV

might grossstructureofreovirus cores, CPV,andWTV

;rophoretic

are

compared

diagrammatically

in the upper

gene prod-

portion of

Fig.

13. Because WTV is rather

un-nFig. 12a. stable toCsCl,we canonly estimate the minimal

termine if structure of

transcription

which would be

ana-iel in and logous to reovirus cores and purified CPV. We

Jirect viral have assigned this role to fraction C from CsCl

(p = 1.425 to 1.445 g/cm') which, despite the

harsh effects of high salt, retains transcriptase

containing activity

(Lewandowski,

unpublished observation).

einforma- One obvious

question

to arise from this

com-1068 J. VIROL.

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WTV tive aggregate equivalent to procaryotic and eu-caryotic host polymerase.

VIRAL AND TRANSCRIPTASE POLYPEPTIDES

E COLITRANSCRIPTASE

WOUND TUMOR VIRUS -+ t

+--BSUBTILIS TRANSCRIPTASE +--t --- t

CYTOPLASMICPOLYHEDROSISVIRUS --RATLIVER 0bTRANSCRIPTASE

20 15 C G5 02

[image:17.493.47.239.57.235.2]

1oltons*I10

FIG. 13. Top, Diagrammatic presentatioli of the

structuresofreoviruscore, CPV,and WTV, represent-inzg what iskntown todateoftheminimal structure of diplornzavirus transcription. Bottom, Comparisoln ofthe

molecular weights ofselectprocaryotic andeucaryotic DNA-dependentRNApolymerase polypeptideswith the viralpolypeptidesassociated with thesuggestedminimal viral RNA-dependenztRNApolymeraseactivities.

parison concernswhich polypeptides among the

total protein mass of the minimal

enzyme-tem-plate complexareresponsiblefor the actual

proc-ess of transcription. Based primarily on the

striking similarity between the polypeptide

com-positionsofE. colideoxyribonucleicacid

(DNA)-dependent RNApolymerase and reovirus cores,

it has beensuggested that the reovirus cores are

made up entirely of transcriptase (11). Similar

comparisons can be made between the

polypep-tides of CPVandWTV,on onehand,and certain

procaryoticandeucaryotic DNA-dependentRNA

polymerases (5, 17, 34), onthe other (lower

por-tion of Fig. 13). The general pattern of these

compositional similarities suggests to us that

these viral and hostenzyme activitiesmay prove

tobeanalogous, perhaps differing chieflyintheir

preference of double-stranded template, either

DNA or RNA. Such a hypothesis raises the

in-teresting possibility of an evolutionary

relation-shipbetweendsRNA-containingviruses andthose

portions of procaryotic and eucaryotic genomes

codingforpolymerase activities. It must be

cau-tioned, however, that in allcases, including

reo-virus, inlieu ofa directreconstitution assay asis

available for the host polymerases, there is no

direct evidence showing which of the viral

poly-peptides designated in Fig. 13 is responsible for

the viral transcriptase activity. One attractive

possibility is, for example, that each of the

capsomeric-like structures seen onthe surface of

reovirus cores, CPV and WTV, in addition to

representing a major entity of virus

superstruc-ture, is also a multicomponent, biologically

ac-ACKNOWLEDGMENTS

WeareindebtedtoC. A.Knight for his cooperation during the

courseof thisresearch,toL.M.Black for thegenerousprovision of woundtumorvirus-inducedroot tumortissue, and toR. C. Williams, K. E. Richards, and J. Toby for their services in the electron microscopy studies.

This investigation was supported by Public Health Service grants AI 00634 from the National Institute of Allergy and In-fectious Diseases and CA 13169-01 from the National Cancer Institute, bycontract 71-2173 within the Special Virus Cancer Program of the National Cancer Institute, and by traininggrant GM 01389 from the National Institute ofGeneral Medical

Sci-ences.

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