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0022-538X/80/12-0787/09$02.00/0 Vol.

Structural

Relationships Between the Surface Antigens of

Ground

Squirrel

Hepatitis Virus and Human Hepatitis B

Virus

WOLFRAM H.GERLICH,tMARK A. FEITELSON, PATRICIAL. MARION, AND WILLIAM S. ROBINSON*

Division of Infectious Diseases, Department of Medicine, Stanford University, Stanford, California 94305

Several physical, chemical, andserologicalproperties of surface antigen

parti-cles from ground squirrel hepatitis virus(GSHsAg) and human hepatitis B virus

(HBsAg) were compared. GSHsAg and HBsAg particles were purified from

positiveserabygel chromatographyandisopycnic centrifugation. Both antigens

consisted mainly of sphericalparticles with an average diameter ofapproximately

20 nm and a buoyant density in CsCl of approximately 1.19 g/ml. Their UV

absorptionspectraindicatedthe presence of more tryptophane than tyrosine and

theabsence of detectable nucleicacid.GSHsAgwas found tocontain two major

polypeptides of approximately 23,000 and 27,000 daltons, with electrophoretic

migration rates distinctly faster than those of the two major polypeptides of

HBsAg particles. After radiolabelingof purified antigen preparations with

Bolton-Hunterreagent,thetwomajorpolypeptidesofGSHsAgshowedalmostidentical

tryptic peptidemaps. The tryptic peptide map ofthe major polypeptide from

GSHsAgcontained 13of37spotsalsopresentinthemap ofthe majorHBsAg

polypeptide, and13of27spotsinthemapofthe major HBsAg polypeptide were

alsopresentin themapofthe majorGSHsAg polypeptide.This suggests

consid-erablesequencehomologybetween the major surface antigen polypeptidesof the

twoviruses.However,therewasonlyaweakserologicalcross-reactivity between

antigens of thetwoviruses.Usingananti-HBs-containingserumwitharelatively

strongcross-reactivity,GSHsAgwasfoundtoconsist ofatleasttwoantigenically

differentsubspecies.Themorestrongly cross-reactingformhadaslightlyhigher

buoyant densitythanthe otherantigenicform.

Whenhepatitis B virus(HBV)wasfirst

char-acterizeditwasfoundtohave severalinteresting

differences from viruses of therecognizedvirus

groups.Itsultrastructure andantigenicstructure

were considered unique (reviewed in 20). Its

small circular DNA hadan unusual structure,

includingthepresenceofalarge single-stranded regionof differentlengthindifferent molecules

whichwasrepairedbyavirion DNA

polymer-ase.Itcausedfrequent persistent infection with

completeviral forms (the42-nmDaneparticle) andincompleteviral forms(22-nmsphericaland filamentousparticles)continuouslyintheblood

inhighconcentrations,andpersistentinfection

wasassociated with chronichepatitisand

hepa-tocellular carcinoma. Both thecompleteandthe

incomplete viralforms inbloodappear to

con-tain on their surfaces a virus-specific antigen

now designated hepatitis B surface antigen

(HBsAg). HBsAg is a complex antigen. The

tPermanent address: Department of Medical Microbiol-ogy, HygieneInstitute, UniversityofG6ttingen,Gottingen,

FederalRepublicofGermany.

HBsAg'sfrom allseraappeartocontaina

group-specificdeterminanta aswellasdoryandw or r type-specific determinants (1, 15). Antigenic

variation of some of these determinants and

additional determinants suchas xand q have

also been described(4, 5).Severalpolypeptides

isolated frompurifiedpreparationsof22-nmand

filamentous HBsAg particles have been shown

tocontaintheHBsAgspecificities (7,10, 12,22).

Recently it has become apparent, however,

that HBV is not a unique virus. Viruses with

similarultrastructural, antigenic,molecular,and biologicalfeatures have been found in eastern

woodchucks (25), Beechey ground

squirrels

in

northem California(16),anddomestic ducksin

China (J. Summers, personal communication).

Thesevirusesprovideimportantanimal models

of HBVinfectioninhumans,and it is ofinterest

to determine how closely related they are.

Al-though the surfaceantigensof woodchuck

hep-atitis virus (29) and ground

squirrel

hepatitis

virus(GSHsAg) (16)clearlycross-reactin

sero-logicaltestswith HBsAg, itis not known how

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788 GERLICH ET AL.

similarthe surface antigens of these virusesare. Here we describephysical, chemical, and sero-logical properties of GSHsAg and compare it with HBsAg. The results clarify the degree of similarity of thesetwoantigens and indicate that the respective viruses differ serologically more thando HBV of differentHBsAg subtypes.

MATERLALS AND METHODS Isolation of 20-nm HBsAg and GSHsAg parti-cles.HBsAg (subtypeadw) concentrations inpatient

serahighin HBV-specific DNApolymerase activity (16) andGSHsAgingroundsquirrelserahigh in virion DNA polymerase activity (16) were determined by electroimmunodiffusion (11), using sheep antiserum (S-2/4) to HBsAg (anti-HBs). One-half to 2 ml of serumwithhighconcentrations ofHBsAgorGSHsAg

waschromatographedover aBio-Gel A-5M (Bio-Rad) column in TNEbuffer (0.01MTris-hydrochloride[pH 7.4],0.1 M NaCl, 5mMEDTA) as described in the legendtoFig.2.Fractionsstrongly positive for surface antigen in electroimmunodiffusion were pooled and concentratedapproximately fivefold with Ficoll (Phar-macia). Each 2.5 nil of concentrated material was made upto adensity of 1.30g/ml by addition of solid CsCl andlayeredover1.3mlofaCsClsolution with adensityof1.35g/mlinatube for theSpinco SW40

rotor. Two-milliliter volumes of CsCl solutions with densities of 1.27, 1.24, and1.18g/mlwereconsecutively layeredoverthesample, followedby0.8ml of 0.05 M phosphate buffer, pH 7.5. The surface antigen was centrifugedtoequilibriuminanSW40rotor at34,000 rpmfor40hat 10°C.Antigenwasagain located by electroimmunodiffusion, and positive fractions were pooledandstored inCsClat4°C.

Since theconcentration ofHBsAg inthe human serawas,onthe average, 10-fold less than concentra-tionsofGSHsAgin theground squirrelsera,HBsAg inserum wasconcentrated and partiallypurified by polyethyleneglycol6000precipitation in some exper-ixnentsbefore Bio-Gel A-5M chromatography.Briefly, 200ml ofHBsAg-positive serum was made up to 6.8% (vol/vol) with polyethylene glycol (from a 30% [wt/ wt]stock) and centrifuged for5minat5,000 rpmand 4°C.Theresultingsupernatantwasmade up to 12.5% with additional polyethylene glycoland centrifuged again. The 12.5% polyethylene glycolpellet was dis-solvedin4mlof TNE.

Radiolabeling and chemical modification of HBsAgandGSHsAg.Forradioinmmunoassay, pun-fiedpreparations of HBsAg and GSHsAg were radio-labeledby oxidative iodination, using Iodogen (Pierce ChemicalCo.; 8). Free 125Iwasremoved by chroma-tographyin Sepharose6B-Clwith 1%bovineserum albumin.Specific activities up to 10,uCi/,ugofprotein wereobtained. Forpeptide mapping, both HBsAg and GSHsAg preparations were dialyzed against 0.01 M sodium boratebuffer (pH 8.5)-0.1% sodium dodecyl-sulfate (SDS), lyophilized, andlabeled with Bolton-Hunter reagent (3) as described by Feitelson et al. (submittedforpublication). Briefly,0.2to 0.5

Ag

of p-hydroxyphenylpropionic acid N-hydroxysuccinimide

ester (Calbiochem) dissolved in 1 to2

pl

ofdioxane wasadded to50

pl

of 0.5Msodiumphosphate buffer,

pH 7.5, andrapidlyiodinatedbyadditionof0.5mCi of

125I

and100,ugofchloramine-T. The labeledester was

immediately extracted into 100 1p of benzene, the aqueousphasewas removed, and theorganic phase

wastransferredtothelyophilized protein sample. La-beling andextraction of theester werecarriedoutin less than 40 s to prevent excessive hydrolysis in aqueousmedia. The benzenewasdriven offbyagentle streamofair,the vialwascooledto0°C,and5

A1

of 0.1Msodium boratebuffer, pH 8.5,at0°Cwasadded toeachsample.Afterincubationat0°Cfor1 h,the sampleswerestoredat4°Covernight.Thenextday 200,ugofp-hydroxyphenylpropionic acid N-hydroxy-succinimideester(indioxane)wasaddedtoeach

sam-ple,and the reaction mixtureswereincubatedat0°C for3h.ThepHwasthenadjustedto9in eachmixture, and the sampleswerereductively methylated as de-scribedpreviously(18), with minor modifications(M. A.Feitelson and F. 0.Wettstein, submitted for pub-lication).

Alternatively,somepreparationswereradiolabeled byoxidativeiodination,usingchloramine-T(13). All samplespreparedforanalysis bySDS-polyacrylamide gelelectrophoresis (PAGE) werereduced in 10%

2-mercaptoethanoland2Murea at100°Cfor3minand alkylated with approximately two chemical equiva-lents of iodoacetamide. The labeled proteins were

separated from the other reaction components by chromatographyonBio-GelP-10 in0.05MNH4HCO3 and0.1%SDS, lyophilized,andredissolved ina solu-tionwith final concentrations of2 M urea, 3%SDS, and 0.001%bromophenolblue forSDS-PAGE.

SDS-PAGE and peptide mapping. Discontin-uous SDS-PAGE was carried out with 1-mm-thick slabgelscontaining5%stacking gels (with N,N-meth-ylenebisacrylamide cross-linker) and 12.5% running gels (withN,N'-diallyltartardiamide cross-linker).The buffer system and other conditions usedwereidentical tothoseofLaemmli (14).Electrophoresiswasusually carriedoutovernight (16 h)at aconstant currentof 10mAandstoppedwhen thedyereached the bottom of therunninggel.Gels with non-radiolabeledproteins

were stained with 2% Coomassie blue in 10% acetic acid and 25%isopropanol overnight, destained in the samemixture without Coomassieblue,and dried un-dervacuum.Forradiolabeledproteins,thegelswere fixedovernightin10% acetic acid and 25%isopropanol, dried undervacuum for 2 h, and autoradiographed using an intensifying screen and Kodak XR-5 film. Individualradiolabeled bandswerecutfrom thegel, dried underhigh vacuumovernight, and then rehy-drated with0.5ml ofasolutioncontaining250Agof trypsin (Calbiochem) and0.05MNH4HCO3atroom temperatureovernight. The supematantswere lyoph-ilized,and eachwasredigestedwith 100,ug oftrypsin in20

pl

of0.01Msodiumboratebuffer, pH 8.5. Two-dimensionalpeptide mappingwascarriedout on cel-lulose thin-layer plates (20 by 20 cm; Brinkmann) under a layer of n-heptane as described elsewhere (Feitelsonetal., submitted for publication). Electro-phoresiswascarriedoutinthe first dimension for 2.25 hat14°C,using 8% acetic acidand2% formicacid, pH 2,asthe running buffer. After electrophoresis, thin-layer chromatographywascarriedoutin n-butanol-water-ethyl acetate-acidic acid-pyridine (120:60:20:4: J. VIROL.

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1, vol/vol), using thetop phase of themixture only. Thin-layer plateswerethen exposedtoKodak XR-5 film, usinganintensifyingscreen(26).

RESULTS

Antigenic heterogeneity of GSHsAg. GSHsAg was detected in the sera of infected

ground squirrels by its cross-reaction with anti-bodiesagainst HBsAg. For its qualitative deter-mination, a commercially available test kit for HBsAg (Ausria II, Abbott Laboratories) was

used,asdescribedearlier(16). Positivesera were

assayed quantitatively by electroimmunodiffu-sion, using asheep antiserun (S-2/4) directed

moststrongly against the group-specifica

deter-minant of HBsAg. Most other antisera against HBsAg tested didnotshow sufficient

cross-reac-tivitytopermitquantitation of GSHsAg. In

con-trast toHBsAg, which producedonestrongline with this antiserum (Fig. la, well 1), GSHsAg always producedtwoweakerprecipitationlines

of different intensities (Fig. la,wells3-8). This

result suggests antigenic heterogeneity in both GSHsAg and the cross-reacting antibodies. Al-though the relative amounts of both GSHsAg components were similar in different positive

ground squirrelsera, thefinding thatone

com-ponentformedastrongerprecipitationarccloser

totheoriginthanthe othersuggests that there

was more cross-reacting antibody directed

against the former subpopulation than against the latter.Heterogeneity in buoyant densitywas

also demonstratedbyCsCldensity gradient

cen-trifugation (Fig. lb).

Size of GSHsAg. GSHsAg-containing ground squirrelserumwaschromatographedon a columnof 4% agarose beads (Sepharose 4B). Totalproteinwasdetectedbyabsorbanceat 280

nmandGSHsAgwasdetectedby

electroimmu-nodiffusion. GSHsAgwaseluted inasingle 280-nmpeak characteristic ofserummacroglobulins

(Fig. 2). Incontrast, HBsAg preparations have been shown to elute with immunoglobulin M andbeta-lipoprotein, but slightly before the

a-macroglobulin peak (11). If the Stokes radius of humana-macroglobulin is 8.9 nm, and the

di-ameterandcomposition of this moleculearethe same in both species, GSHsAg would have an

averagediameter of 18to20nm.Larger

filamen-tousandspherical particles, detectedearlierby

electronmicroscopy (16), were apparently

bro-ken up oradsorbed tothe column, because all

fractionsbefore theantigen peakwerenegative even by the sensitive enzyme-linked

immuno-sorbents assay for GSHsAg (data not shown). Further, there was no detectable DNA

polym-coN ._E

E

sm

3

nc

30

20

10 '

FIG. 2. Sepharose 4B chromatography of ground squirrelserumcontaining GSHsAg. Serum (0.8 ml) wasloadedon an80-cm columncontaining135mlof Sepharose4B in 0.02 MTris-hydrochloride(pH 7.4)-0.13 M NaCI andeluted at4 ml/h with the same buffer. The solid line is a tracing of the 280-nm absorbanceof the eluate passing through a

3-mm-light path cuvette. Fractions were assayed for GSHsAg by electroimmunodiffusion asdescribedin thelegendtoFig. 1, and the lengths ofthe long (0) and short (0) precipitin arcs were measured. Bar shows thefractions pooled for further purification of GSHsAg.

a

1

1 2 3 4 5 6 7 8 15 16 17 18 19 20

FIG. 1. Electroimmunodiffusion of GSHsAgandHBsAg with sheep anti-HBs. Ten microliters ofeach

HBsAg-containing sampleor a1:10 dilutionofeachGSHsAg-containing samplewaselectrophoresedat20 mAfor8 h inaslab(25 by75by1.5mm) of1%agarose(Bio-Rad) containing0.034 M sodiumbarbital(pH 8.4) and 20%(vol/vol)antiserum. Thegelwasthenwashed, dried,andstained with 0.2% Coomassie blue in 7.5% acetic acid. (a) Well 1, humanserum with 35pgof HBsAg (adw)perml; well2, normalground squirrel serum;wells3 to8,AusriaII-positive ground squirrelsera.(b) Wells 15through 20, fractions15through 20, respectively, from the CsCl density gradient described in the legend to Fig. 3a after centrifugation to

equilibrium forpurification of GSHsAg.

20F 30F

40k

50h

70-80 Vt

40 50 60 70 80 90 100 110 120 30 140

36,

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790 GERLICH ET AL.

erase activity in any of the fractions, even

though the starting materialwasvery strongly

positive.

Density ofpurifiedGSHsAg. GSHsAg

par-tiallypurifiedfrom0.8ml ofserumbygel

chro-matographywas adjustedto asolution density

of1.3g/mlwithCsCl andcentrifugedto

equilib-rium inaCsCldensity gradient. Antigenic

activ-itywasdetectedbyelectroimmunodiffusionina

prominent280-nmabsorption peakat adensity

of 1.183 g/ml (Fig. 3a). The low base line of

absorption around thispeak indicated that the

a

1.0 I 1.3

0.5 1.

o 0~~~~~~~~~~~~~~~~~~~~~~~e

0sL0

o

11.0

A

0.5 1.

2 6 10 14 18 22 26

[image:4.496.66.255.208.509.2]

Fraction Number

FIG. 3.

Isopycnic

centrifugation

of

GSHsAg

(a)or

HBsAg (b)in CsCl.After gelchromatography of0.8 mlofserumwith GSHsAgand2mlofserum with HBsAgasdescribed in thelegendtoFig. 2,thepooled antigen-containing fractions ofeach were

concen-tratedbyosmoticdehydrationwithdryFicollto4ml andadjustedto adensity of1.30g/mlwithCsClfor centrifugation in a CsCl density gradient as de-scribed in thetext.After centrifugation inaSpinco SW40rotorfor40 hat34,0X)rpmand10°C, fractions

were collectedfromthe bottomofthe tube. Absorb-anceat280nm(0)ina cuvettewitha1-cmlightpath

andsolutiondensity(Ax)byrefractometrywere deter-minedforeachfraction. Bars representthelengths oftheprecipitation arcsin the

electroimmunodiffu-sion assayforGSHsAgorHBsAg.

antigen was already highly purified after only

two purification steps. HBsAg from 2 ml of.

serum, purified in parallel by the same

proce-dure(Fig.3b),wasfoundina280-nmpeakat a

density of 1.190 g/ml. Although the starting

volume of humanserum was 2.5times greater

than the volume ofground squirrelserumused,

thedensitygradient profiles showed muchmore

GSHsAg than HBsAg. In another experiment

with serum from a secondpositive animal, an

evenlargeramountofGSHsAgwas extracted,

suggesting that the serum concentration of

GSHsAg in infected ground squirrels was

ap-proximately10times that ofHBsAginthe

high-titer human sera used.

Electroimmuno-diffusionanalysis of the three fractions making

uptheGSHsAg density gradient peak(fractions

17, 18,and19,Fig. 3a) revealedtwoprecipitation

lines in the two fractions of highest density

(fractions17and18, Fig. lb) andonly the faint

j.recipitation line in the antigen-containing

frac-tionof lowestdensity (fraction19,Fig. lb). This

resultsuggested thatGSHsAgwas notonly

an-tigenically heterogeneous but also

heteroge-neousinbuoyant density. Electron microscopy

of the GSHsAg peak fractions revealed both

sphericaland short filamentous forms withmean diameters of 18 to 20 nm. Although the

mor-phologyof theseparticleswassimilartothat of

HBsAgparticles, theapparentaveragediameter

was slightly less than that reported (2, 6) for

sphericalHBsAg particles(20to 22nm).

UV absorption spectrum of purified GSHsAg. The UV absorption spectrum of GSHsAg,like that ofHBsAg (11), had peaksat

280and 290.5nm as well as ashoulder at 275 nm (Fig. 4), which were indicative of a high tryptophane/tyrosineratio. Sincemostproteins have alow

tryptophane/tyrosine

ratio,and the

absorption spectrum of tyrosine obscures the

290.5-nm peak, the UV absorptionspectrum of

thispreparationsuggests arelativelyhigh degree

ofHBsAgpurity. Further, thespectrumshowed

no evidence of thepresence ofnucleic acid. If

the specificabsorbance ofGSHsAg isassumed tobethe same asthatofHBsAg, which is 4.2 at 280 nmfora 1-mg/ml solution, then the serum

from which the GSHsAg was purified in this

experiment would have contained

approxi-mately 300,ug ofantigen protein per ml. Sera

fromotheranimals had between40 and 300% of

that concentration when tested in

electroim-munodiffusion.

Serologicalcross-reactivityof antibodies

against HBsAg and GSHsAg in

radioim-munoassay. Antibodies directed against

HBsAgorGSHsAgdeterminants were detected

in sera bysolid-phase radioimmunoassay (21).

Briefly, dilutionsof test serawere added to U-J. VIROL.

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SURFACE ANTIGENS FROM HEPATITIS VIRUSES 791

0.3-,03

0.2

-240 250 260 270 280 290 300 310 320 Wavelength nm

FIG. 4. UV absorption spectrum of purified GSHsAg. Fraction 19 from the CsCI density gradient shown in Fig. 3a was measured in a Zeiss PM2 spectrophotometer at 0.1-mm slit width.

shaped wells of microtiter plates coated with

GSHsAg or HBsAg, and antibody bindingwas

detected by subsequent binding of

"2'I-labeled

antigenof thesametypeusedto coatthewells.

Mouse and ground squirrel antisera against

GSHsAg and guinea pig and sheep antisera

against HBsAgweretitratedby thisassay,using

purified GSHsAgorHBsAgasthereagent(Fig.

5). All antiserafrom four mice immunizedwith

GSHsAg bound labeled GSHsAg tosaturation

(75% of theradioactivityadded)atlowdilutions,

and 50% was boundwhen antiserum dilutions

between 1:2,000 and 1:10,000 were used. With

HBsAgasreagent, twoof the four antiserawere

negative(<0.3%binding), and the othertwo sera

bound 0.5 and 2% of the added

"2'I-labeled

HBsAg at a dilution of 1:10 (Fig. 5a and b).

Using HBsAg,antibodycouldbe detected in the

two anti-HBs-containing sera diluted up to 1:

10,000. The sheep antiserum (S-2/4) boundup

to48%ofGSHsAgwhenused undiluted and 28%

at a 1:10 dilution (Fig. 5c), and the guinea pig

antiserum boundonly5.6% ofGSHsAgata1:10

dilution (Fig. 5d). It should be noted that the

sheep antiserum S-2/4 was directed mainly

against thea determinant ofHBsAgbecauseit

wasprepared by boostingwithan HBsAg

sub-type different from that used for the primary

inoculation. Six monoclonal antibodies to

HBsAg (agiftof LizJones)werealsotested with

GSHsAg. Onlyonebound 0.5% of theGSHsAg

atdilutions upto 1:100, whereasthe otherfive

antibodies showednodetectablebindinginthis

sensitiveassay.

SDS-PAGE analysis of GSHsAg- and

HBsAg-associated proteins. Purified

prepa-rationsofHBsAgandGSHsAgeachcontaining

50 ,ug ofprotein were reduced, alkylated, and

analyzed bySDS-PAGE,andtheproteinbands

wererevealedbyCoomassie bluestaining. The

HBsAg preparation yieldedtwomajor

polypep-tides with apparent sizes of 25,000 and 29,000

daltons (Fig. 6a, lane 2), similar to the findings

inprevious studies (7, 10, 12, 17, 22). Two major

polypeptides were also found in the GSHsAg

preparation (Fig. 6a, lane 1), and although their

apparent sizes were similar, they consistently

migrated fasterduringelectrophoresis(apparent

sizesof23,000and 27,000 daltons) than the

cor-responding majorHBsAgpolypeptides. Surface

antigen preparations radiolabeled by

chlora-mine-T (Fig. 6b)orBolton-Hunterreagent (data

notshown) revealedthe same two major

com-ponentsby autoradiography.

Several minor components migrating more

slowly in SDS-PAGE than the major

polypep-tides werealso foundin surface antigen

prepa-rationsfrom bothviruses when Coomassie blue

staining or either method of radiolabeling was

usedtodetectpolypeptides.Minor polypeptide

components have been repeatedly found in

HBsAg preparations previously (7, 10, 12, 17,

22).

Table1summarzesthe findings fromseveral

SDS-PAGE

experiments

in whichpolypeptides

weredetected by Coomassie blue stainingorby

autoradiography ofpreparations radioiodinated

by the iodogen procedure. None of the minor

Coomassie blue staining polypeptides with ap-parentmolecularweightsgreaterthan30,000in

GSHsAg preparations comigrated with any of

those from HBsAg. In contrast, the major and

-, -2 -3 -4 -, -2 -3 -4 0 -, -2 -3 -4 -5 -, -2 -3 -4 '0Log Dilution

FIG. 5. Cross-reactivity of antisera against GSHsAg andHBsAginsolid-phase radioimmunoas-say. A50-,lI portion ofmouseantiseraagainst puri-fied GSHsAg (a and b), sheep antiserum against HBsAgdeterminant a (c),orguinea pigantiserum againstHBsAgsubtypeadw(d), eachdiluted in 1% bovineserumalbumin (BSA), wasincubatedin mi-crotiterwells precoated with 10 ngofpurifiedHBsAg (0)orGSHsAg(0)for15hat4°C.Aftertwo wash-ings withBSA, 40,000 cpmof'2I-labeledHBsAg(2.5

,uCi/pg)or8,000cpmof"LI-labeledGSHsAg(0.5liCi/

pg) in50

j1

ofBSAwereincubatedfor24hat4°C. After threewashingsthe wellswerecounted. Approx-imately 75%ofeitherradiolabeledantigen wasthe maximumamountbound. Thebackgroundwith

nor-malhumanseruminsteadofantiserumwasbetween 0.2and 0.4%(datanotshown).

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792 GERLICH ET AL.

a

FIG. 6. Polypeptides ofGSHsAgan ticle preparations.Purified preparatic (a, lane1) andHBsAg (a, lane2) we) SDS-PAGE and Coomassie blue $tai GSHsAg preparations from ground s

lane1)and 147(b, lane 2)wereradioio iodogen method andanalyzed by SI autoradiography.

TABLE 1. Apparent molecularweig)

polypeptidesseparatedby SDS-PAG.

GSHsAg and HBsAg preparc GSHsAg

Animalno. 83

Animalno. Coomassie 125i 147(1251)

blue

75 75

70 70

43 42 42

39 39 39

36 37

34 34 35

27a 27a 27a

23a 23a 23a

20 20

aMajorcomponents.

most of the minor '25I-labeled polypeptides of

) GSHsAg preparationsfromtheseraoftwo

dif-ferent ground squirrels (no. 83 and 147)

radio-2 labeled

by

the

iodogen procedure appeared

to

be identical(Table 1). Therewas,however,

var-iabilityin the intensity andposition oftwo of

theminorcomponentsmigrating in the

34,000-to37,000-molecular-weight range(Table 1, Fig.

6b) in the two GSHsAg preparations.

Radiola-_ 5 beling of bothHBsAg and GSHsAg by the

chlor-7e anmine-T, iodogen, and Bolton-Hunter

proce-dures revealed several autoradiographic bands

42. inthe 14,000- to20,000-molecular-weight range

39 which were not detected on gels stained with

37 Coomassie blue (Fig. 6, Table 1).

35 Typtic peptide mapping of the major

polypeptides ofGSHsAg and HBsAg. The

relationship between the two major

polypep-tides ofGSHsAgwas examinedbytryptic

'25I-labeled peptide mappingof each afterlabeling

2 antigen preparations with Bolton-Hunter

re-agentandrecovering the appropriate

"25I-labeled

2 C polypeptide from a polyacrylamide gel after

SDS-PAGE. Figure 7a shows the map of

'25I-labeled peptides -detected by autoradiography

for the 23,000-dalton polypeptide (P-23) of

4

HBsAg

par-

GSHsAg.

The

peptide

mapof the

27,000-dalton

)nsof GSHsAg polypeptide (P-27) of GSHsAgwasverysimilar

reanalyzed by but not identical.Figure 7b shows acomposite

ning. Purified diagramof thepeptidemapsofthetwoGSHsAg

quirrels 83(b, polypeptides. The spot at position 1 was

de-dinatedby the tected in the mapof 27 and not in that of

P-)S-PAGE and 23. The spot at position 2 was very heavily labeled in P-23 and barely detectable in P-27.

itS _Xl-3)

of

Thus,

the two major polypeptides ofGSHsAg

ts (xlO.).of are closely related in their primarysequences.

tonfprified

Figure 7c shows the

12II-labeled

peptide map of

the 25,000-dalton polypeptide (P-25) derived

fromHBsAg, andadiagram of thismapis shown

HBsAg in Fig. 7d. The tryptic

"2'1-labeled

peptides in

(Coomassie themaps of the major polypeptides of the two

blue)

viruses

werecomparedin

autoradiograms

of the

two mapped separately and by digesting and

72 mappingamixture ofthetwo onasingle

thin-layer plate. Thirteen

125I-labeled

peptidesinthe

63 map ofGSHsAg P-23 were found to be identical 56 to spots in the map of HBsAg P-25, and the

54 remainder ofthe spots weredifferentin the two

maps. Specifically, the shaded spots (13 of 37 spots) in the diagram of the peptide map of

GSHsAg P-23 (Fig. 7b) are identical to spots

present in the map of HBsAg P-25. The

un-31 shaded spots areunique toGSHsAgP-23. The

29a shadedspots (13of 27 spots) in thediagram of

the peptide map ofHBsAg P-25 (Fig. 7d) are

25" those also found in the peptide map, of GSHsAg

P-23. The unshaded spots were unique to HBsAg P-25. It can be concluded that, although

themajorpolypeptides of GSHsAg and HBsAg

J. VIROL.

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a

b

.a

a

0.

c

d

I

2:

_ v

-rA

-FIG. 7. Tryptic'LI-labeledpeptidemaps of the majorpolypeptidesofHBsAgandGSHsAgradioiodinated by theBolton-Hunter method. (a) "LI-labeledpeptidemap of the23,000-daltonpolypeptide(P-23)ofGSHsAg. (b)Diagram of the compositemapof the samepolypeptideand the27,000-dalton polypeptide ofGSHsAg. The darkened spots are identical to spots in the map of the 25,000-daltonpolypeptide (P-25) ofHBsAg. (c) 1251. labeledpeptide map ofHBsAgP-25. (d)Diagram of the map of the same polypeptide, with the darkened spots correspondingtothose identical to spots in the map ofGSHsAgP-23.

possessdifferent mobilitiesonSDS-PAGE,they

share a considerable amount of structural

ho-mology.

DISCUSSION

By physical, chemical, and serological

tech-niques the similaritiesanddifferences between

thespherical surface antigen particles fromHBV

andground squirrel hepatitis viruswere

inves-tigated. The most numerous particles bearing

the surfaceantigens of both viruses hadsimilar

sizes andmorphologies, thesamebuoyant

den-sity,unusually high tryptophane/tyrosine ratios,

serologicalcross-reactivity, and a pair of major

structural polypeptides with considerable

ho-mology in primary structure. These common

featuressuggest that the molecular architecture

ofintact GSHsAg particles is probably similar

tothatofHBsAgparticles. Withoneantiserum

(S-2/4) from asheep immunized withHBsAg,

sufficient cross-reactivity was found to detect

andquantitate GSHsAgin

electroimmunodiffu-sionandbyenzymeimmunoassay.Surprisingly,

this antiserumdistinguished between two

anti-genicforms in GSHsAg, although no such het-erogeneityhasbeendescribed forHBsAg.Since

this antiserum was raised against HBsAg, it is

probable that HBsAg itself hasasimilar

heter-ogeneity, which maybe obscured by the

pres-enceofmanycommondeterminantsonpartially

heterogeneousparticles.Theunevendensity

dis-tribution of thestrongercross-reactive

compo-nents suggests that itsspecificity is related to

glycosylationormasking by lipid. That thereare

commondeterminantson HBsAgandGSHsAg

suggestsasimilarconfigurationofthe

polypep-tide chainsatthesurface of theparticles.It will

be of interest to determine whether chemical

reduction ofGSHsAgreduces itsantigenicityas

muchashas beenreportedforHBsAg (27).

Itis of interest thatGSHsAgparticlescontain

two major structural polypeptides similar but

notidenticalin sizetothemajorcomponents of

HBsAg,providingfurtherevidencefora

similar-ityin structure of the surfaceantigen particles

of thetwoviruses.Based upon amino acid

com-position (22, 28), limited

sequencing

data (19),

andsimilarantigenicproperties (12, 22),thetwo

36, 1980

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794 GERLICH ET AL.

major structuralpolypeptides ofHBsAgappear

tohaveverysimilarprimarysequences.Periodic

acid-Schiffstaining (23) andradiolabeling (17)

experiments indicate that the major HBsAg

polypeptide with higher apparent molecular

weight, and not the smaller one, contains

car-bohydrate and this maycompletelyaccountfor

the difference in the mobilities of the two in

SDS-PAGE. Because of the small amounts of

antigenavailable, thepresence ofcarbohydrate

in the GSHsAg polypeptides has notyet been

studied, but the largerof the twomaycontain

carbohydratebyanalogywith theHBsAg

poly-peptides. The peptide maps ofthe two major

polypeptides ofGSHsAgwere almostidentical,

differing in only two spots (Fig. 7b). Although

differences in the amino acid sequence of the

two proteins could account for these small

changes in the peptide map, glycosylation (or

otherpost-translationalmodifications) ata

sin-gle site could also account for the changes. At

present,studiesarebeing directed toward

defin-ing thenatureofthesedifferences.

The molecular weight estimate by

SDS-PAGE for themajornon-glycosylated

polypep-tide ofHBsAg (25,000) isveryclosetothe

the-oretical molecular weight calculated from the

DNA sequence that appears to code for this

polypeptide (9). If the estimates of therelative

sizes of themajorpolypeptides ofGSHsAgand

HBsAg are accurate, an approximately 10%

smaller DNAcodingsequencewould berequired

to specify the 23,000-dalton GSHsAg

polypep-tide thanto specify the 25,000-dalton

non-gly-cosylatedcomponentofHBsAg.

Forpeptide mapping, the majorpolypeptides

from surface antigen particles of both viruses

wereradiolabeled in vitrobytheBolton-Hunter

procedure, which has been showntolabel

pro-teins more uniformlythan oxidative iodination

(Feitelson et al., submitted for publication).

Wheninterpretingstructuralrelationshipsfrom

peptide maps ofproteins labeled with

Bolton-Hunter reagentanddigested with trypsin,it is

importanttonotethat each-spotdoes not

nec-essarily correspond to a single tryptic peptide.

Dependinguponthe number oflysine residues

withfreeepsilonaminogroups in a givenpeptide

andtheextent to which these groups are

acyl-atedbytheBolton-Hunterreagent, thisreaction

introduces somechemicalheterogeneity into a

populationofotherwise identicalprotein

mole-cules. The relatively large number of spots,

therefore, isareflection ofthischemical

heter-ogeneity introduced by the acylationprocedure.

This is in contrast to the relatively few spots generated from direct iodination with chlora-mine-T oriodogenwhere thischemical

hetero-geneity isnotpresent. This isespecially

impor-tant when the differences involve spots which areminorbyautoradiography. Underidentical

reaction conditions the heterogeneity intro-ducedby this method isquitereproduciblesince

different radiolabeledpreparationsof aprotein

giveidenticalpeptide maps.

Thecomposite peptide mapsinFig. 7band d

define therelationship between the major struc-tural polypeptides of GSHsAg (Fig. 7a) and

HBsAg (Fig. 7c). InFig. 7b, about one-third of

thespotsinthemapof theGSHsAgpolypeptide

(P-23) werealso in thepeptide mapof the

ap-propriate HBsAg polypeptide (P-25). Nearly

half of the spotsin themapofHBsAg P-25were

also in themapofGSHsAg P-23 (Fig. 7d). That

common tryptic peptides signify considerable

sequence homology has recently been

demon-strated inthecomparison of simian virus40and BK virus T antigens (30). These different

pa-povavirus antigens share 7 of 20 or 21 tryptic

peptides, andyetby DNA basesequence

anal-ysis, they share 70% homology. This suggests

that the percent homology derived from the

peptide maps described here probably

repre-sents aminimumestimate. These resultsarein apparent contrast to the minimal serological

cross-reactivity of HBsAg andGSHsAg, which

was undetectable or less than 10-4 with the

antisera used here. Itappearspossible that the

selectivepressureof the hostimmune response

results in greater variability in the partof the

proteinmanifestingsurfaceantigenic reactivity

than in the overallsequence.Thecoreantigens

from ground squirrel hepatitis virus and HBV

havemore serological cross-reactivitythan the

surfaceantigens (W. Gerlichetal., unpublished

data). This observation confirms for thisgroup

ofviruses thegreatervariabilityof virionsurface

than internalantigens found formanyenveloped

viruses. Asthe molecularbiologyof theground

squirrel virus is further elucidated and compared

with that ofHBV, theextent towhich this

virus-host system can be used as a model for HBV

infection in humans will becomemoreapparent.

ACKNOWLEDGMENTS

This studywas supported by Public Health Service

re-search grant AI13526from theNational Institutes of Health. M.A.F. was supported by postdoctoral fellowship PF-1792

from theAmerican CancerSociety.W.H.G. received grant Ge 345/5from theDeutscheForschungsgemeinschaft.

LITERATURE CITED

1. Bancroft, W. H., F. K. Mundo, and P. K.Russel.1977.

Detection ofadditional determinants ofhepatitis B antigen.J.Immunol.109:842-848.

2. Bayer, M. E.,B.S.Blumberg, and B. Werner. 1968. Particles associatedwithAustralia antigen in the sera

ofpatients with leukemia, Down's syndrome and hep-atitis.Nature(London)218:1057-1059.

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3. Bolton, A. E., and W. M. Hunter. 1973. The labelling of proteinsto high specificradioactivities by conjugation to a l25I-containingacylating agent. Biochem. J. 133: 529-539.

4. Courouce, A. M., P. V.Holiand,J.Y. Muller, and J. P.Soulier. 1976. HB. antigensubtypes.Proceeding of theinternationalworkshop on HB. antigensubtypes. Bibl. Haematol. (Basel) 42:1-158.

5. Courouce-Pauty,A. M., and J. P.Soulier.1974. Fur-therdata onHB.antigen subtypes-geographical dis-tribution. Vox Sang. 27:533-549.

6. Dane, D. S., C. H. Cameron, and M. Briggs. 1970. Virus-likeparticles in serum of patients with Australia antigenassociatedhepatitis. Lancetii:695-698. 7. Dressman, G. R., R.Chairez,ILSuarez, F. B.

Hollin-ger, R. J. Courtney,and J. LMelnick. 1975. Pro-duction of antibody to individual polypeptidesderived frompurffiedhepatitisBantigen. J. Virol. 16:508-515. 8. Fraker,P.J., and J. C.Speck. 1978.Protein andcell membrane iodinations with asparinglysoluble chlor-amide, 1,3,4,6-tetrachloro-3a,6a-diphenyl-glycoluril. Biochem.Biophys.Res.Commun. 80:849-857. 9. Galibert,F., E.Mardart,F.Fitoussi,P.Tiollais,and

P.Charnay. 1979. Nucleotide sequence of hepatitis B virusgenome (subtype ayw) cloned in E. coli. Nature (London)281:646-650.

10.Gerin, J.L.1974.Structure ofhepatitis B antigen (HB Ag), p.215-224.In W. S. Robinson and C. F. Fox (ed.), Mechanisms of virusdisease. W. A. Benjamin, Menlo Park,Calif.

11.Gerlich,W., andR.Thomssen. 1975. Standardized de-tectionofhepatitisBsurfaceantigen: determination of its serum concentration inweight units per volume. Dev.Biol. Stand.30:78-86.

12.Gold,J. W.M.,J.W.Shih,R. H.PurcelLand J. L GeriD.1976.Characterizationof antibodiestothe

struc-turalpolypeptides ofHB.Agevidence for subtype-spe-cificdeterminants. J.Immunol. 117:1404-1406. 13.Greenwood,F.C.,W. M.Hunter,and J. S. Glover.

1963. Preparationof'3'I-labelledhuman growth hor-moneofhigh specificradioactivity.Biochem. J. 89:114-123.

14.Laemmli, U. K. 1970. Cleavageof structural proteins during theassemblyof the headofbacteriophageT4. Nature(London)227:680-685.

15.LeBouvier,G.L 1971. TheheterogeneityofAustralia antigen. J. Infect. Dis. 123:671-675.

16.Marion,P.L,LOshiro,D.C. Regnery,G. H. Scul-lard,and W.S. Robinson. 1980.Avirus inBeechey groundsquirrelswhich is relatedtohepatitisBvirus of man.Proc.Natl. Acad. Sci. U.S.A. 77:2941-2945.

17. Marion, P.L, F. H. Salazar, J. J. Alexander,andW. S. Robinson. 1979. Polypeptides of hepatitis B surface antigenproduced by a hepatoma cell line. J. Virol. 32: 796-802.

18. Means, G. E*, and R. E. Freeney. 1968. Reductive alkylation of amino groups in proteins.Biochemistry 7: 2192-2201.

19. Peterson, D. L, L. M.Roberts,and G. N.Vyas. 1977. Partial amino acid sequence of two major component polypeptides ofhepatitis B surface antigen. Proc. Natl. Acad. Sci. U.S.A. 74:1530-1534.

20. Robinson,W. S. 1977. The genome ofhepatitis B virus. Annu.Rev. Microbiol. 31:357-377.

21. Schober, A., R. M. Bigawar,and R.ThomRsen. 1975. Direct solid phase radioixmunoassay (dsp-RIA) for detection ofantibody to hepatitis B surface(HB,) an-tigen. Dev. Biol.Stand. 30:93-102.

22. Shih,J.W.,and J. LGerim. 1975.Immunochemistryof hepatitisBsurfaceantigen(HB.Ag):preparation and characterization of antibodies to theconstituent poly-peptides. J. Immunol. 115:634-639.

23. Shih,J.W., and J. L Gern. 1976. Proteins of hepatitis B surfaceantigen. J. Virol. 21:347-357.

24. Shih, J.W.,andJ. LGern. 1977. Proteins ofhepatitis B surfaceantigen: amino acid composition of themajor polypeptides.J.Virol. 21:1219-1222.

25. Summers, J.,J. M.Smolec, and R.Snyder.1978. A

virussimilar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc. Natl. Acad. Sci. U.S.A.75:4533-4537.

26. Swanstrom, R., andP.R.Shank.1978.X-ray intensi-fyingscreensgreatlyenhance thedetectionby

autora-diographyof theradioactiveisotopes'Pand "1I.Anal. Biochem.86:184-192.

27. Vyas, G. N., K. R. Rao, and A. B. Ibrahim. 1972.

Australia antigen (hepatitis B antigen): a conforma-tional antigendependentondisulfidebonds. Science 178:1300-1301.

28.Vyas,G.N.,E. W.Williams,G. G. B.Klaus,and H. E. Bond. 1972. Hepatitis-associated Australia antigen: protein,peptidesand aminoacidcompositionofpurified antigenwith tissue in determiningsensitivity ofthe hemagglutinationtest.J.Immunol. 108:1114-1118. 29. Werner,B.G.,J.M.Smolec, RI Snyder,and J.

Sum-mers.1979.Serologicrelationahipofwoodchuck hepa-titis virus and humanhepatitisBvirus. J.Virol.32: 314-322.

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Figure

FIG.1.HBsAg-containingrespectively,mAequilibriumserum;andacetic Electroimmunodiffusion of GSHsAg and HBsAg with sheep anti-HBs
FIG. 3.HBsAgHBsAgmlantigen-containingSW40tratedandscribedminedofsioncentrifugationwereandance the Isopycnic centrifugation ofGSHsAg (a) or (b) in CsCl
FIG. 4.GSHsAg. UV Fraction
FIG. Polypeptides4ticle 6. of GSHsAg an preparations. Purifiedpreparatic)ns
+2

References

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