JOURNAL OF VIROLOGY, Feb. 1971, p. 241-249 Vol. 7, No. 2Copyright ( 1971 American Society for Microbiology Printed in U.S.A.

Structural Proteins of Rabies VirusFRANTISEK SOKOL, DANIEL STANCEK, AND HILARY KOPROWSKI

The Wistar Institute of Anatomy and Biology, The World Health Organization International ReferenceCenter for Rabies at The Wistar Institute, anid The Children's Hospital of Philadelphia,

Philadelphia, Pennsylvania 19104

Received for publication 23 November 1970

Purified rabies virions, unlabeled or labeled with radioactive amino acids orD-glucosamine, were dissociated into their polypeptides by treatment with sodiumdodecyl sulfate in a reducing environment and fractionated by electrophoresis insodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights ofindividual polypeptides were estimated by comparison of their rate of migrationwith that of protein markers of known molecular weight. Purified viral nucleocapsidand a mixture of envelope components, isolated from virions disrupted by sodiumdeoxycholate, were analyzed by the same procedure. The number of molecules pervirion of each polypeptide was estimated from the proportions of the separatedcomponents, the known molecular weight of the viral ribonucleic acid, and thechemical composition of the nucleocapsid. The protein moiety of the nucleocapsidparticle was estimated to consist of 1,713 molecules of a major polypeptide (molecu-lar weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecularweight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein com-ponent (molecular weight, 80,000 daltons), the viral envelope contains 789 and1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000and 25,000 daltons).

The structural complexity of rabies virus (9)indicates that it is composed of several distinctiveproteins. It was reported recently that treatmentwith sodium deoxycholate (DOC) causes disrup-tion of the protein-lipid envelope of rabies virionsand release of the helical nucleocapsid, a nucleo-protein containing the viral ribonucleic acid(RNA; reference 22). The disrupted virions werefractionated into nucleocapsid and a mixture ofcoat components on the basis of markedly differ-ent rates of sedimentation and buoyant density.Several properties of purified nucleocapsid prep-arations have been described, but properties ofthe individual coat components remain largelyunknown.

In the present study, purified rabies virions ortheir components, dissociated by treatment withsodium dodecyl sulfate (SDS) and 2-mercapto-ethanol (2-ME), were analyzed by electrophoresisin polyacrylamide gel to determine the number,the molecular weight, and the structural identityof the polypeptides of rabies virions. Of the fourmajor polypeptides of different molecular weights,the second largest in molecular size is associatedwith the viral RNA in the nucleocapsid, whereasthe remaining three form the viral envelope. Theenvelope polypeptide with the largest molecularweight is a glycoprotein.

MATERIALS AND METHODSVirus. The 1210/A clone of the high-egg-passage

(HEP) Flury strain (21) and the W clone of the ERAstrain (14) of rabies virus, isolated by T. J. Wiktor,were used in all experiments.

Tissue culture. BHK/13S cells were propagatedin roller cultures. The 2-liter bottles were seeded with4 X 107 cells in 100 ml of BHK cell growth medium(17) supplemented with 10% fetal calf serum andwere incubated at 37 C. The cell cultures becameconfluent in about 3 days and contained, at the timeof infection, approximately 2.4 X 108 cells per bottle.BHK-21 cells were grown in 1-liter Blake bottles asdescribed previously (21).

Infection of the cells and labeling of the virus.Cultures of BHK/13S or BHK-21 cells were infectedwith 1 plaque-forming unit of virus per cell, asdescribed previously (21). After 1 hr of adsorptionat 35 C, the inoculum was removed and the cellswere refed with 50 ml of Eagle's minimum essentialmedium supplemented with 0.2% bovine serumalbumin (BSA). For labeling of the virus, the mediumcontained 2.5 ,Ci of a mixture of 16 3H-amino acidsper ml (average specific activity, 17.6 Ci/mmole;Schwarz/Mann mixture no. 3130-08; Schwarz Bio-Research, Orangeburg, N.Y.), 0.5 MACi of a mixtureof 13 14C-amino acids per ml (average specific ac-tivity, 250 mCi/mmole; Schwarz/Mann mixture no.3122-08, Schwarz BioResearch), or 1 ,uCi of 3H-D-glucosamine per ml (specific activity, 850 mCi/mmole; New England Nuclear Corp., Boston, Mass.).

241

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

SOKOL, STANCEK, AND KOPROWSKI

Also for protein labeling, the concentration of thecold amino acids in the medium was reduced to one-fifth of the regular level. The cultures were incubatedat 33 C. Extracellular virus was harvested approxi-mately 72 hr after infection.

Purification of the virus and isolation of viral com-ponents. The virus purification scheme includedprecipitation by zinc acetate, desalting on a Sepha-dex column, treatment with ribonuclease and deoxy-ribonuclease, pelleting by high-speed centrifugation,and banding of the virus by centrifugation in asucrose density gradient (21). Procedures for thefractionation by velocity centrifugation in a sucrosedensity gradient of DOC-disrupted virions into nu-cleocapsid and a mixture of coat components, aswell as the purification of the nucleocapsid by iso-pycnic centrifugation in CsCl solution, have beenpreviously described (22).

Protein determination. Protein was assayed asdescribed by Lowry et al. (16) by using BSA asstandard.

Standard procedure for the preparation and elec-trophoresis of polypeptides. Proteins from dilutedsolutions were precipitated at 0 C with 10% trichloro-acetic acid. The precipitate collected by low-speedcentrifugation was washed at 0 C with 2 ml of 2%trichloroacetic acid and then with the same volumeof ice-cold acetone. The sediment was then dried in astream of warm air and dissolved in 0.01 M phosphatebuffer (pH 7.2) containing 3.33% (w/v) SDS and0.67%7o (v/v) 2-ME. If necessary, the pH of the solu-tion was adjusted to 7.2 by addition of a few micro-liters of 1 N NH40H. In all instances, more than 94%Oof the original amino acid or glucosamine label wasrecovered in the dissolved precipitate. Sufficientlyconcentrated samples were directly adjusted to 3.33%in SDS and dialyzed at room temperature overnightagainst the SDS- and 2-ME-containing phosphatebuffer. The polypeptide samples were stored at 4 C.Sucrose was then added to make a final concentrationof 15%. Samples not exceeding 0.2 ml in volume andcontaining 20 to 400 ,ug of polypeptides were heatedat 100 C for 1 min (18) and analyzed.

Nine volumes of a solution containing 7.5% acryl-amide, 0.27% N,N'-methylenebisacrylamide, 0.1%SDS, and 0.05% N,N,N',N'-tetramethylethylene-diamine in 0.1 M sodium phosphate buffer (pH 7.2),were mixed with 1 volume of 1%/ ammonium per-sulfate and polymerized to form gels 20 cm in lengthand 0.6 cm in thickness. The gels were prerun in theelectrophoresis buffer (0.1 M phosphate buffer, pH7.2, containing 0.1% SDS) for 4 hr at 2 ma per gel.The sample was then placed on the top of the geland overlaid with the electrophoresis buffer. Elec-trophoresis was carried out at 2 ma per gel for 20min and then at 5 ma per gel for 11.2 to 16.5 hr.Some gels were stained for 2 hr at 20 C with 1%Amido Black B-10 in 7% acetic acid and then de-stained in several changes of 7% acetic acid. All gelswere cut into 1-mm slices, unless otherwise stated,and solubilized in 0.5 ml of 15% hydrogen peroxideat 90 C for 30 min. The radioactivity of the fractionswas determined, after the addition of 10 ml of Aquasol

(New England Nuclear), in a Packard liquid scintil-lation spectrometer.

Protein standards for determination of molecularweight. The following proteins, purchased from MannResearch Laboratories, New York, N.Y., were usedas markers for molecular weight determination:human gamma globulin (160,000 daltons), BSA(67,000 daltons), ovalbumin (45,000 daltons), whalesperm myoglobin (17,800 daltons), and horse heartcytochrome c (12,400 daltons).

RESULTS

Polypeptides of rabies virions. When purifiedrabies virions, labeled with radioactive aminoacids, were disrupted by treatment with SDS and2-ME and analyzed by electrophoresis in poly-acrylamide gel, four major peaks of radioactivitywere repeatedly observed (Fig. la). Similarly, theoptical density tracing of virus polypeptides, frac-tionated by the same procedure and stained byAmido Black, showed four main bands of ab-sorbance (Fig. lb). The peaks of the stainablebands coincided with those of the radioactivebands (Fig. la, inset). The radioactivity or theabsorbance recovered in the four main bandsrepresented 85 to 90% of the total radioactivity orabsorbance in the gel. A variable amount (5 to12% of the total) of polypeptides of a molecularsize larger than the slowest of the four major com-ponents was detected in each preparation of dis-assembled virus. For reasons which will be givenlater, we believe that most of these componentsrepresent aggregates (dimers) of the major poly-peptides. Peaks of radioactivity observed in elec-trophoretograms of dissociated cellular proteins.obtained from cell-free fractions of mock-infectedcultures and subjected to the same sequence offractionation procedures used for the purificationof the virus did not coincide with the peaks ofviral polypeptide bands (Fig. 2). Therefore, con-taminating cellular proteins would be easily de-tected in electrophoretograms of insufficientlypurified preparations of dissociated virions. Al-though the material not contained in the fourlarge bands probably represents either aggregatesof the major viral polypeptides or cellular contam-inants, it is possible that minor structural proteinsof the virus were not detected by the fractionationprocedure used.

Other methods of releasing rabies virus poly-peptides, such as treatment with SDS and 2-MEat 37 C with or without previous precipitation ofthe virions by trichloroacetic acid, replacement of2-ME with dithiothreitol, additional treatment ofSDS- and 2-ME-dissociated virions with 6 Mguanidine or 10 M urea, were also tried. In allcases, the electrophoretic patterns obtained were

242 J. VIROL.

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

RABIES VIRUS PROTEINS

I..-.;trOD

.0~~

;s 500 -

1000 -

500

tz

..

20 40 60 50 10 120 140START

FRACTION NUMrBER

BIn

zw

- Z0 40 60 80 100 120 140START

DISTANCE (mm)

FIG. 1. Fractioniationt of rabies virus polypeptidesby electrophoresis in polyacrylamide gel. Purifiedrabies virus [higlh-egg-passage (HEP) Flury strain],labeled with a mixture of 3H-ainino acids, was dis-sociated with sodium dodecyl sulfate in a reducinzgenivironmnenit anid electrophoresed in 7.0%c gel Jbr 13.5hr. Thle 3H-aminio acid-labeled polypeptides were

stainied before the gel was sliced (inset, panel a). Theoptical denisity profile of the viral polypeptides (HEPFlury strain) electrophoresed for 11.2 hr was obtainiedby scanniin7g the stained gel in a Gilford spectrophotom-eter at 550 nim (b).

similar to those observed after fractionation ofpolypeptides released from the virions by thestandard procedure, i.e., treatment with SDS and2-ME followed by heating at 100 C for 1 min.The electrophoretic pattern of disassembled

ERA virions (Fig. 3) was similar to that of disso-ciated HEP Flury virus (see also Table 1).

Estimation of molecular weights of rabies virusstructural proteins. In the presence of excess SDS,the differences in the original net charge of variouspolypeptides are suppressed. Thus by electro-phoresis in an SDS-containing polyacrylamidegel, polypeptides are separated largely on the basisof molecular size, the distance migrated beinglinearly related to the logarithm of the molecularweight (20). With proteins of known molecularweight as standards, the molecular weights of thefour major HEP Flury virion polypeptides were

found to be 80,000, 62,000, 40,000 and 25,000daltons, respectively (Fig. 4).

Identification of the nucleocapsid and envelopeproteins of rabies virions. Purified rabies virions,labeled with a mixture of 3H-amino acids, were

disrupted by treatment with DOC and subjectedto velocity centrifugation in a sucrose densitygradient so that the slowly sedimenting envelopecomponents were separated from the 200S nucleo-capsid (22). The nucleocapsid was then furtherpurified by isopycnic centrifugation in a CsClsolution (22), treated with SDS and 2-ME, andanalyzed by electrophoretic separation of thereleased polypeptides (Fig. 5). One major and twominor bands were repeatedly observed. On theaverage, 90.4% of the total radioactivity was asso-

ciated with the polypeptide of intermediate size,having a molecular weight of about 62,000daltons. The molecular weight of the heavy com-

ponent, corresponding to 6.2%, of the total radio-activity, was about 130,000 daltons, indicatingthat it could be a dimer of the major nucleocapsidpolypeptide. The identity of the major nucleo-

capsid polypeptide with the virion polypeptidehaving a molecular weight of 62,000 daltons was

500-

z0

400

w 300

z

2 200

_ 1oo0z

20 40 60 80 100 120START

FRACTION NUMBER

140

FIG. 2. Electrophooretogram of polypeptides de-rived from host componientts which have beeni releasedspontanteously inito the tissue culture fluid from mock-intfected cells. Mock-infected cultures were labeledwith 3H-aminio acid mixture antd inicubated in the same

way as infected cultures. Thze tissue culture fluid(290 ml) was subjected to the same procedures usedfor the purificationi of the virus. The host componenitswere then mixed with ani excess amounit of nionilabeledpurified virus, dissociated by sodium dodecyl sulfatein a reducinig enivironiment, an1d electrophoresed in7.-0% polyacrylamide gel for 12.7 hr. The gel was firststained for determination of the positioni of viral poly-peptides (arrows). It was theni cut inito slices for the

determiniation of radioactivity conitainied in thle frac-lion2s. For comparison, the amounizt of viral polypep-tides used in the experimenzt showil in Fig. la, was

derived from 29 ml of ine ctious tissue culture fluid.

VOL. 7, 1971 243

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

SOKOL, STANCEK, AND KOPROWSKI

confirmed by co-electrophoresis of disassembled'4C-amino acid-labeled virions with dissociated

,0o

20 40 60 80 loo 120 140START

FRACTION NUMBER

160 180

FIG. 3. Electrophooresis of dissociated ERA virionsin polyacrylamide gel. Purified rabies virus, labeledwith 3H-aminio acids, was dissociated with sodiumdodecyl sulfate an2d 2-mercaptoetlhaniol antd electro-phoresed in a 7.0% polyacrylamide gel for 14 hr.Abbreviations: EP, enivelope proteini; BSA, bovinteserum albumnin; NCP, niuicleocapsid proteini; GlP,glycoprotein.

14-

2-i

10-

C.)

z

cm

6

462-

4.00 4.25 4.50 4 75 5.00 5.25

LOG MOLECULAR WEIGHT

FIG. 4. Estimationi of the molecular weights ofrabies virus polypeptides. Polypeptides released frompurified rabies virions (higlh-egg-passage Flutry strain)by treatmentt with sodiumti dodecyl sulfate (SDS)anid 2-mercaptoethlaniol were separated by electro-phoresis in 7.0% polyacrylamide gel for 13.5 hr.Marker proteins of known molecular weight were

treated with SDS and electrophoresed in parallel.The gels were then stainied with Amido Black, and thedistance from the origin of each band was plottedagainist the molecular weights of marker proteinis.The four major componienits wvere designated GlP(glycoprotein), NCP (nlucleocapsid protein), anid EP(enivelope proteini) 2 antd 3.

3H-amino acid-labeled nucleocapsid. The poly-peptide with the lowest molecular weight, com-prising 3.4% of the total radioactivity, is mostlikely a distinct structural component of rabiesvirus nucleocapsid, since it was present in allnucleocapsid preparations analyzed. Its molecularweight was estimated to be 55,000 daltons.The remaining three major virion polypeptides

were identified as structural parts of the viralenvelope by electrophoresis of 3H-amino acid-labeled envelope components, isolated fromDOC-disrupted virions, with '4C-amino acid-labeled virus (Fig. 6). All preparations of disso-ciated viral envelope were contaminated with asmall amount of nucleocapsid protein. Viral RNAcontained in the nucleocapsid molecules releasedfrom rabies virions by treatment with DOC wasfound to be partially sensitive to the degradingaction of ribonuclease (22). This sensitivity is prob-ably the result of the detachment of some pro-tein subunits from the nucleocapsid particlesduring treatment with DOC. Thus when DOC-treated virions are fractionated by velocity cen-trifugation in a sucrose density gradient, the de-tached nucleocapsid protein is recovered in thefraction of the dissociated viral-envelope.

Glycoprotein component of the viral envelope.Several enveloped viruses, such as myxovirus(5-7, 15, 25), arbovirus (24, 26), herpevirus (1,13, 23), vesicular stomatitis virus (VSV; references2, 12), poxvirus (8), and RNA-containing tumor(4) virus, have been shown to contain carbohy-drates covalently bound to one or several of theenvelope proteins. To determine whether carbo -

hydrates are incorporated into rabies virions, andif so, whether they are covalently bound to someof the polypeptides, we labeled the virus with3H-glucosamine and fractionated the labeled com-ponents of dissociated virus by electrophoresis inpolyacrylamide gel. Radioactive glucosamine was

a ZV), ° 3-

Z u

O trt) M 2'

, X

0 1-

1-- 20 40 60 RO 100 120 140START FRACTION NUMBER

200

2_

- 150 o

0-U ,>

o0z

50

FIG. 5. Fractionzation of polypeptides of rabiesviruts nucleocapsid by electrophoresis in polyacryl-amnide gel. 3H-amino acid-labeled purified nucleocapsidwas mixed with '4C-amino acid-labeled pur ified virions(highi-egg-passage Flury strain), treated by sodiumndodecyl sulfate int a reducinzg eniviroinmenit, alid electro-phoresed in a 7.0%-, polyacrylamide gel for 15 hr.

I.0

1-

(E) I (f)J I 1 4 A

*IL - 4 1 0

,--4-

ONRIPMONO-1

u

w

(20

0 zi

>co4

0

NCP

2:aEm9

p.-

244 J. VIROL.

z0I.-

nQ

crw0-

Z

2irwa.

co

zD0

CY

.D

160 180i

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

RABIES VIRUS PROTEINS

20 40 60 80 100 12(START FRACTION NUMBER

FIG. 6. Fractionation of polypeptides of the rabiesvirus envelope by electrophoresis in polyacrylamidegel. 3H-amino acid-labeled envelope components(highl-egg-passage Flury strain) were mixed with14C-amino acid-labeled purified virions, treated bysodium dodecyl sulfate and 2-mercaptoethanol andelectrophoresed in a 7.0%7 polyacrylamide gel *for15 hr.

selected for labeling because its specificity as alabel is very high, 85 to 97% of the radioactivityincorporated into the glycopeptide components ofvarious viruses still being present in the originalchemical form or in the form of N-acetylgluco-samine (2, 3, 26). Moreover, the glycopeptides ofSindbis virus contain more glucosamine than anyother sugar (3, 26).When partially purified rabies virus, grown in

tissue cultures labeled with 3H-glucosamine dur-ing infection, was centrifuged in a sucrose densitygradient, the peak of the virus band coincidedwith that of the radioactivity (Fig. 7). Two com-

ponents were seen after the purified 3H-gluco-samine-labeled virus was dissociated by treatmentwith SDS and 2-ME and fractionated by electro-phoresis in polyacrylamide gel (Fig. 8). The majorcomponent, comprising 73 to 79%O of the totalradioactivity contained in the gel, had the samemolecular size as the largest of the four majorvirion polypeptides, i.e., a molecular weight ofabout 80,000 daltons. The minor component cor-

responding to 5 to 13% of the total radioactivityhad a molecular weight of about 160,000 daltonsand probably represented the dimer of the viralglycoprotein.

In almost all preparations, the band, cor-responding to the viral glycoprotein obtained afterelectrophoretic separation of the components ofdissociated virus, was broader than that of themajor nucleocapsid polypeptide (e.g., Fig. 1),indicating that the glycoprotein component was

heterogeneous in molecular size. As with VSVglycoprotein (2, 12), in preparations of decom-posed rabies virions, which were labeled with3H-carbohydrate and 14C-amino acids, the ratio ofcarbohydrate radioactivity to the amino acidradioactivity was significantly different in variousfractions of the glycoprotein band (not shownhere). Occasionally, the glycoprotein was distrib-

0.7- 7'Z0

U.

O 06CY

0.5 S 5 a.

U) 0.4 4TOz

-j 0.3 3 z

FIG. 7.Frationaionopartally urifid,3H0

grdet0A2sup2sooflble iios(.6m)

C)J

5 10 1'5 25 30

BOTTOM VOLUME (ML) TOP

FIG. 7. Fractionation of partially purified, 3H-

glucosaminie-labeled rabies virions (high-egg-passageFlury straini) by cenztrifuigationz in a sucrose density

gradient. A suspension of labeled virions (0.6 ml),partially purified by precipitation with zinc acetate,filtration through Sephadex G-75 column, and pelletingby high-speed centrifugation, was layered over 28.5ml of 10 to 50% (w/w) linear gradient of sucrose itNTE buffer (0.13 m NaCl, 0.05 um Tris-hydrochloride,0.001I ethylenediaminetetraacetate, pH 7.8) andcentrifuged at 4 C and 61,000 X g for 90 min in theSW 25.1 rotor of a Spinco centrifuge. Absorbanceand radioactivity offractions collected from the bottomof tlhe tube were then determined. The absorbing bandlocated approximately in the middle of the tube coin-cides with thtose ofvarious viral activities (20). Symbols:0, optical density at 260 tim; 0, radioactivity of 3H-glucosamine.

400

300

200

100

20 40 60 80too

START

FRACTION NUMBER

120 140

tt

-1000 Z

-500 a.

0

I>

FIG. 8. Electrophoresis in polyacrylamide gel of3H-glucosamine-labeled rabies virus (HEP Flurystrain) dissociated by treatment withl sodium dodecylsulfate and 2-mercaptoethanol. 3H-glucosamine-labeledvirus was mixed with 04C-amino acid-labeled virions,treated with the detergent in a reducing environment,and electrophoresed in a 7.0% polyacrylamide gel for16 hr. Each fraction corresponds to a 1.5-mm gel slice.

I

-.Il 11

i-,-. "N

4d.

VOL. 7, 1971 245

GIt

z0

IL)-ta:LI

a:wQ.

2(rw0.

(f)

zD0C-)

y

laz

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

SOKOL, STANCEK, AND KOPROWSKI

uted in two adjacent bands (Fig. 9). Two obser-vations indicated, however, that the latter type ofextensive heterogeneity represents an artifact: (i)the proportion and the mobility of the separatedglycoprotein "components" were variable indifferent preparations; and (ii) preparations ofdissociated virus which originally showed onlyone broad glycoprotein band became extensivelyheterogeneous in the molecular size of their en-velope glycoprotein after being stored for severalmonths at 4 C. The reason for the lability of theviral glycoprotein is unknown. Precipitation ofthe viral proteins by trichloroacetic acid or heat-ing of the viral polypeptides at 100 C for 1 minwas not responsible for the degradation of theglycoprotein component, since virion prepara-tions not treated with trichloroacetic acid and dis-assembled by SDS and 2-ME at room tempera-ture or 37 C occasionally showed a similar exten-sive heterogeneity.

Relative proportions of rabies virus polypeptides.The relative proportion of the major rabies viruspolypeptides was determined from the amount ofdye bound by these four components, as well asfrom the amount of 3H or 14C radioactivity re-covered from them (Table 1). The differences inthe polypeptide composition of the virions, asdetermined by these three methods, reflect, inaddition to experimental errors, the differences inthe amino acid composition and the specificradioactivities of the two reconstituted proteinhydrolysates used for labeling, as well as thedifference between overall labeling with the dyeand the labeling of specific amino acids withselected radioactive precursors. Moreover, it hasto be assumed that the glycoprotein moiety of theglycoprotein component is not labeled, or only toa negligible extent, with the dye or the radioactiveamino acids. Thus data presented in Table 1 donot include the contribution of the glycopeptideto the relative proportion of the viral glycopro-tein.

Estimates of the molecular weights of the pro-teins of the viral nucleocapsid and envelope, aswell as of the number of molecules of these com-ponents per one complete virion, are given inTable 2. A complete virion is defined here as avirus particle containing one nucleocapsid particleof full length (22). The estimates were derivedfrom the data presented in Fig. 4 and Table 1, aswell as from previous findings that the molecularweight of rabies virus RNA is 4.6 X 101 daltonsand that the viral nucleocapsid is composed of96%o protein and 4% RNA (22). They are alsobased on the assumption that the glycopeptidesdo not affect the mobility of the viral glycoproteinin an SDS-polyacrylamide gel to the extent thatestimation of its molecular weight is unreliable.

ir zwoa--I-

0

z

, Li

a pAJi.

*48

I- 20START

z0I-

LL

a:

a.

z

0

a- 20START

40 60 80 100 120 140

FRACTION NUMBER

FRACTION NUMBER

FIG. 9. Heterogeneity in molecular size of theglycoprotein component of rabies virus. (a) 3H-aminoacid-labeled virions (ERA strain), disassembled bythe standard procedure, were electrophoresed in 7.0%polyacrylamide gel for 15.5 hr. (b) 3H-glucosamine-labeled virions (high-egg-passage Flury strain) weredissociated by the standard procedure and electro-phoresed in 7.0% polyacrylamide gel for 16 hr. Theposition of the major nucleocapsid polypeptide isindicated (arrow). Each fractionz corresponds to a1.5-mm gel slice. The occasional distribution of theviral glycoprotein in two partially overlapping bandscould also be observed after stainiing of the gel withAmido Black (not showni here).

DISCUSSIONThe results of the present study have shown

that the protein moiety of rabies virus is composedof at least four major and one minor polypeptideof different molecular sizes. The viral RNA isassociated with one of the major and with theminor polypeptide to form the nucleocapsid of thevirion. The remaining major polypeptides arecontained in the viral envelope. One of them, thelargest in size, was shown to be a glycoprotein. Arelatively small amount of components of muchhigher molecular weight (from 100,000 to 160,000daltons) than that of the envelope glycoprotein(80,000 daltons) was detected in all preparationsof disassembled virions. Insufficiently purifiedvirus preparations were shown to contain con-taminating cellular proteins composed of poly-peptides of such molecular size. On the other

.246 J . VIROL .

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

RABIES VIRUS PROTEINS

TABLE 1. Relative proportions of major rabies virus polypeptidesa

No. of Per cent in component (average values)Method Virus strain determina-

tions GIP NCP EP2 EP3

Optical density at 550 nm ERA 2 48.0 32.0 9.5 10.5after staining with Amido HEP Flury 2 49.0 31.0 7.5 12.5Black

Labeling with 3H-amino acid ERA 2 47.0 34.0 8.5 10.5mixture HEP Flury 6 43.5 32.5 10.5 13.5

Labeling with 14C-amino acid HEP Flury 5 43.0 34.5 10.0 12.5mixture

a Abbreviations: G1P, glycoprotein; NCP, nucleocapsid protein; EP2 and EP3, envelope proteins;HEP, high egg passage.

TABLE 2. Protein composition of complete rabiesvirusa

PolypepteMol No. of moleculesPolypeptide Mol wt per virion

GIP 80,000 1,783NCP 62,000 1,713NCP-MC 55,000 76EP2 40,000 789EP3 25,000 1,661

a Abbreviations as in footnote a, Table 1.b Average proportions of the four major poly-

peptides of HEP Flury virus used for the calcu-lation (from Table 1): 44.3%G GlP, 33.0% NCP,9.8% EP2, 12.9% EP3.

c NCP-M, minor nucleocapsid protein; thenumber of NCP-M molecules per virion was cal-culated by using a weight ratio of NCP/NCP-M =26.6.

hand, with sufficiently purified virion prepara-tions, they might represent aggregates (dimers) ofthe four major viral polypeptides because (i) theproportion of these in different preparations ofdisassembled virus varied widely; and (ii) prep-arations of disassembled viral nucleocapsid or of3H-glucosamine-labeled virus contained minorcomponents of approximately twice as highmolecular weight as the nucleocapsid polypeptideand the viral glycoprotein, respectively. A similarconclusion is suggested from the comparison ofthe size of the viral RNA and the total molecularweight of the virion polypeptides. The single-stranded viral RNA having a molecular weight of4.6 x 106 daltons (22) can code for distinct poly-peptides with a total molecular weight no greaterthan 4.6 x 105 daltons. The molecular weights ofthe five structural polypeptides totals 2.6 X 10daltons. If the viral genome would direct thesynthesis of two additional polypeptides, eachwith molecular weight larger than 100,000daltons, the viral RNA could not code for non-structural proteins.

Preparations of purified HEP Flury virus con-tain a small proportion of truncated virus parti-cles (21). It was assumed, in interpreting theresults of the present study, that their proteincomposition was identical with that of completevirions of full length. This assumption is sup-ported by the finding that purified B and T virionsof VSV do not exhibit significant differences intheir protein composition (11, 27). Purified prep-arations of ERA strain of rabies virus containonly a negligible amount of truncated virions (K.Hummeler, F. Sokol, and H. F. Clark, un-published data).VSV, which could be structurally and morpho-

logically compared to rabies virus, contains threemajor polypeptides (11, 27, 28). The nucleocapsidpolypeptide and the glycoprotein (2, 12) of VSVhave molecular weights similar to those of cor-responding components of rabies virus (11, 27).On the other hand, it is uncertain which of theremaining two envelope polypeptides of rabiesvirus (EP2 and EP3) is comparable in molecularsize to the second major envelope protein of VSV.Three laboratories reported appreciably differentmolecular weights for the latter VSV component(2, 11, 27). With sufficient time for electrophoreticseparation, a fourth minor structural polypeptidewas detected in preparations of dissociated VSV(19), the molecular weight of which is comparableto the EP2 polypeptide of rabies virus. Althoughthe exact biological functions of the individualenvelope proteins of rabies virus are unknown, itwas found that the mixture of the three envelopeproteins induces virus-neutralizing antibodieswhen injected into animals (T. J. Wiktor and F.Sokol, unpublished data). Since a glycoproteinisolated from cell- and virus-free lysates of VSV(12) and the glycoprotein components of Roussarcoma virus (4) were shown to bind virus-neu-tralizing antibodies, it can be assumed that theglycoprotein of rabies virus might exhibit a simi-lar activity.

VOL. 7,1971 247

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

SOKOL, STANCEK, AND KOPROWSKI

In only one of seven preparations of dissociatedrabies virus could the envelope glycoprotein beseparated by electrophoresis in polyacrylamidegel into two adjacent components differing inmolecular weight by 5,000 to 10,000 daltons. Forreasons given above, we believe that this hetero-geneity of the viral glycoprotein is an artifactcaused by an unknown factor during the purifica-tion or dissociation of the virions. Examination ofelectrophoretograms of VSV polypeptides alsorevealed an occasional heterogeneity of the glyco-protein component (11, 27). Moreover, it was

found that the molecular size of VSV-specificglycoprotein isolated from the virus-free fractionof infective tissue culture fluid was smaller andcontained about twice as much glucosamine thanthe glycoprotein component of the virions (12).Most likely a part of the polypeptide chain can

easily be split off the viral glycoprotein. Attemptsto demonstrate the presence of polypeptideshaving a molecular weight of 5,000 to 10,000 dal-tons in preparations of disassembled rabies virionswere, however, unsuccessful. This suggests that a

portion of the polypeptide moiety of the viralglycoprotein, located at one end of the molecule,may have been degraded to amino acids or pep-

tides, which escaped detection.Another type of heterogeneity in the molecular

size of the viral glycoprotein, detected in almostall preparations of dissociated rabies virus, was

manifested as an extensive width of the band cor-

responding to this component in SDS-polyacryl-amide gel. Moreover, the carbohydrate content ofthe glycoprotein was found to be variable. It wasobserved in the VSV glycoprotein band, obtainedafter electrophoretic separation of dissociatedstructural proteins of the virus, that the peak ofglucosamine label did not coincide with that ofthe amino acid label (2, 12). This finding wasinterpreted as suggesting the presence in the bandof a larger polypeptide and a smaller glycoprotein(2). Another interpretation would be the vari-ability of the proportions of protein and glyco-peptide in individual glycoprotein molecules.

There is some evidence that the glycopeptidemoiety of viral glycoproteins may be of host cellorigin (2, 7, 15, 26). If this is so, then viral glyco-proteins represent a complex antigen, composedof both host- and virus-specific components.Several rhabdoviruses can be propagated in BHKcells in amounts sufficient to attempt isolation andimmunological comparison of their envelope gly-coproteins. It would be of interest to -find outwhether they indeed contain a common host-specified antigen.

Infection by rabies virus does not suppress thesynthesis of host-specified macromolecules inBHK cells (F. Sokol, unpublished data). It would

be, therefore, difficult to investigate the dynamicsof synthesis of structural and functional viral pro-teins by electrophoretic fractionation of compo-

nents of infected cells synthesized during theinfectious cycle. Electron microscopic and im-munological studies on rabies virus-infected cellsindicate, however, that this experimental ap-

proach might still be feasible. Relatively soon

after infection, large inclusion bodies, composedmainly of viral nucleocapsid particles, are formedin the cytoplasm of BHK cells (10). On the otherhand, the plasma membrane of infected cells con-

tains virus-specific antigens demonstrable byvarious immunological techniques (29). Thus anattempt to investigate the kinetics of the synthesisof virus-specific proteins by electrophoretic analy-sis of polypeptides, derived from the cytoplasm orthe plasma membrane of infected cells and labeledwith radioactive precursors during the infectiouscycle, is indicated.

ACKNOWLEDGMENTS

We are deeply indebted to the late Scott C. Newlin, a formergraduate student at the Wistar Institute of Anatomy and Biology,for his help in the early stages of this project. We thank Mary K.Sheehan and Doris W. Grella for excellent technical assistance.

This investigation was supported by Public Health Serviceresearch grants RO1-CA 10594 from the National Cancer Institute,ROI-AI 09706 and AI-02405 from the National Institute ofAllergy and Infectious Diseases, and by funds from the WorldHealth Organization.

LITERATURRE CITED

1. Ben-Porat, T., and A. S. Kaplan. 1970. Synthesis of proteinsin cells infected with herpesvirus. V. Viral glycoproteins.Virology 41:265-273.

2. Burge, B. W., and A. S. Huang. 1970. Comparison of mem-brane protein glycopeptides of Sindbis virus and vesicularstomatitis virus. J. Virol. 6:176-182.

3. Burge, B. W., and J. H. Strauss, Jr. 1970. Glycopeptides ofthe membrane glycoprotein of Sindbis virus. J. Mol. Biol.47:449-466.

4. Duesberg, P. H., G. S. Martin, and P. K. Vogt. 1970. Glyco-protein components of avian and murine RNA tumorviruses. Virology 41:631-646.

5. Harboe, A. 1963. The influenza virus hemagglutination in-hibition by antibody to host material. Acta Pathol. Micro-biol. Scand. 57:317-330.

6. Harboe, A. 1963. The normal allantoic antigen which neu-

tralizes the influenza virus HI-antibody to host material.Acta Pathol. Microbiol. Scand. 57:488-492.

7. Haukenes, G., A. Harboe, and K. Mortensson-Egnund. 1965.A uronic and sialic acid free chick allantoic mucopoly-saccharide sulphate which combines with influenza virusHI-antibody to host material. Acta Pathol. Microbiol.Scand. 64:534-542.

8. Holowczak, J. A. 1970. Glycopeptides of vaccinia virus. L.

Preliminary characterization and hexosamine content.Virology 42:87-99.

9. Hummeler, K., H. Koprowski, and T. J. Wiktor. 1967.Structure and development of rabies virus in tissue culture.J. Virol. 1:152-170.

10. Hummeler, K., N. Tomassini, F. Sokol, E. Kuwert, and H.Koprowski. 1968. Morphology of the nucleoprotein ofrabies virus. J. Virol. 2:1191-1199.

248 J. VIROL.

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

RABIES VIRUS PROTEINS

11. Kang, C. Y., and L. Prevec. 1969. Proteins of vesicularstomatitis virus. I. Polyacrylamide gel analysis of viralantigens. J. Virol. 3:404-413.

12. Kang, C. Y., and L. Prevec. 1970. Proteins of vesicularstomatitis virus. II. Immunological comparisons of viralantigens. J. Virol. 6:20-27.

13. Kaplan, A. S., and T. Ben-Porat. 1970. Synthesis of proteinsin cells infected with herpesvirus. VI. Characterization ofthe proteins of the viral membrane. Proc. Nat. Acad. Sci.U.S.A. 66:799-806.

14. Kuwert, E., T. J. Wiktor, F. Sokol, and H. Koprowski. 1968.Hemagglutination by rabies virus. J. Virol. 2:1381-1392.

15. Laver, W. G., and R. G. Webster. 1966. The structure ofinfluenza virus. Virology 30:104-115.

16. Lowry, D. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

17. Macpherson, I., and M. Stoker. 1962. Polyoma transforma-tion of hamster cell clones-an investigation of geneticfactors affecting cell competence. Virology 16:147-151.

18. Maizel, J. V., Jr., D. 0. White, and M. D. Scharff. 1968. Thepolypeptides of adenovirus. I. Evidence for multiple proteincomponents in the virion and a comparison of types 2, 7A,and 12. Virology 36:115-125.

19. Mudd, J. A., and D. F. Summers. 1970. Protein synthesis invesicular stomatitis virus-infected HeLa cells. Virology42:328-340.

20. Shapiro, A. L., E. Vifiuela, and J. V. Maizel, Jr. 1967. Molecu-lar weight estimation of polypeptide chains by electro-phoresis in SDS-polyacrylamide gels. Biochem. Biophys.Res. Commun. 28:815-820.

21. Sokol, F., E. Kuwert, T. J. Wiktor, K. Hummeler, and H.Koprowski. 1968. Purification of rabies virus grown intissue culture. J. Virol. 2:836-849.

22. Sokol, F., H. D. Schlumberger, T. J. Wiktor, H. Koprowski,and K. Hummeler. 1969. Biochemical and biophysicalstudies on the nucleocapsid and on the RNA of rabies virus.Virology 38:651-665.

23. Spear, P. G., J. M. Keller, and B. Roizman. 1970. Proteinsspecified by herpes simplex virus. II. Viral glycoproteinsassociated with cellular membranes. J. Virol. 5:123-131.

24. Stollar, V. 1969. Studies on the nature of dengue virus. IV.The structural proteins of type 2 dengue virus. Virology39:426-438.

25. Strandli, 0. K., K. Mortensson-Egnund, and A. Harboe. 1964.Purification of the normal allantoic antigen which reactswith influenza virus HI-antibody to host material. ActaPathol. Microbiol. Scand. 60:265-270.

26. Strauss, J. H., Jr., B. W. Burge, and J. E. Darnell. 1970.Carbohydrate content of the membrane protein of Sindbisvirus. J. Mol. Biol. 47:437-448.

27. Wagner, R. R., T. A. Schnaitman, and R. M. Snyder. 1969.Structural proteins of vesicular stomatitis viruses. J. Virol.3:395-403.

28. Wagner, R. R., T. C. Schnaitman, R. M. Snyder, and C. A.Schnaitman. 1969. Protein composition of the structuralcomponents of vesicular stomatitis virus. J. Virol. 3:611-618.

29. Wiktor, T. J., E. Kuwert, and H. Koprowski. 1968. Immunelysis of rabies virus-infected cells. J. Immunol. 101:1271-1282.

VOL. 7, 1971 249

on April 18, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from