Characterization and classification of virus particles associated with hepatitis A. III. Structural proteins.

0022-538X/81/040151-06$02.00/0 1

Characterization and

Classification

of Virus

Particles

Associated with

Hepatitis

A

III.

Structural

Proteins

JONDURI TRATSCHIN,' GUNTERSIEGL,'* GERTG. FROSNER,2AND FRIEDRICH DEINHARDT2

Institute of Hygiene and Medical Microbiology, University ofBern, CH-3010 Bern, Switzerland,1and Max vonPettenkofer-Institute of Hygiene andMedical Microbiology, D-8000Munich2,FederalRepublic of

Germany

Received 16 September 1980/Accepted22December1980

Hepatitis A viruswaspurified fromfecal samplescollectedatvarioustimes in

the incubationperiod of patients withnaturallyacquired hepatitisA. The proteins

ofparticles bandingataround 1.34 g/ml inCsClandsedimentingatabout 160S

wereradioiodinatedin vitro and separated by electrophoresisonpolyacrylamide

gels in thepresence of0.1%sodium dodecyl sulfate and8Murea. Under these

conditions, the capsid proteins resolved into four polypeptides with molecular weights ofapproximately 31,000, 24,500, 21,000, and9,000, respectively. A fifth protein of about 40,000 daltons in size and assumed to be equivalent to the

precursor polypeptide VPO of the picornaviruses was presentin particles

sedi-menting at only 150 to 155S and banding at around 1.33 g/ml in CsCl. The physicochemical characteristics ofthese particlesareconsistent with those of the provirion structures of picornaviruses. In several of the fecal samples, these particles representedaconsiderable fractionof all particlespresent. The signifi-cance of this finding with respect to the antigenicity of hepatitis A antigen extracted from stool specimens isdiscussed.

HepatitisAvirus (HAV) hasbeen identified repeatedly in samples of feces, bile, and liver collectedfrompatientsinanepidemic of hepa-titisorfrom

volunteers,

chimpanzees,

and

mar-mosets in controlled experimentalstudies. The virus particles measured 27 nm in

diameter,

bandedataround 1.34

g/ml

in

CsCl,

and sedi-mentedatabout 160S

(3,7,

16-18).

Furthernore,

thereis evidencetosuggest thatthegenome of HAV consists ofsingle-strandedRNA (8, 13, 17, 19). These physicochemical characteristics of thepurifiedparticlesstronglysupport the clas-sificationofHAVwith thepicornaviruses.

Intactpicornavirus

particles

containfour well-defined

proteins.

Theavailable dataonthe

poly-peptide spectrum of

HAV,

however,

are

only

partially in agreement with this

requirement.

Three

polypeptides

in the size range of

picor-navirus structural

proteins

VP1 to VP3 were

regularly detected (5, 8), but only very recent experimentsrevealedtraceamountsof afourth, minor

polypeptide

migrating

in theappropriate position ofVP4 (6). Onthe other hand, Fein-stone et al. (8)

reported

the presence of an

additional virus-associatedproteinwitha molec-ularweightof59,000.The size of thisproteinis muchlargerthan thatof theprecursor

polypep-tide VP0 (38 to 44 kilodaltons

[kD])

which is

present inimmaturepicornavirus capsids. Here weprovide evidencethat HAVparticles collectedatvarious times inthe incubation pe-riodofpatientswithnaturallyacquiredhepatitis Ashowaproteinspectrumcompatiblewith that of knownpicornaviruses.Our results alsosuggest that variations in the protein pattern can be relatedtothe relative concentrations of imma-ture, provirion-like structures andmature viri-onsintheHAV

samples

under

investigation.

MATERIALS AND METHODS

Origin and purification of viruses. The fecal specimens used as a source of HAV were collected fromanindividual involved inanepidemicofhepatitis

AinGermany (sampleno. 1) (9) and frompatientno.

2, whocontracted the infectionduringholidaysinthe Mediterraneanarea.Acompleteseries offecalsamples

from patient no. 2 was available from days -9 to 4

(day0wastheonset ofdisease) (8a).Samplesno.2 11/5,no. 2 11/6,andno. 211/9, collected 5, 4,and 1

daybefore theonsetofdisease,respectively,wereused asasource of HAV particles. Stool ofpatientno. 1 was obtained on the 6th day before the onset of jaundice. Theserological and physicochemical char-acteristicsofparticlesfromthe lattermaterial,aswell as the methodused for purification, have been de-scribed indetailpreviously (18, 19).

All stepsin the purification andconcentration of

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Radioiodination of viralproteins. Forlabeling

of virionproteins with theluI-Bolton-Hunterreagent (1), virusparticlessuspended in0.1Msodiumborate (pH8.4)-imMEDTAweredisruptedin the presence of 1% sodiumdodecylsulfate (SDS) by boilingfor1 min. Samplesof50 to 100

Id

ofthe SDS-complexed

polypeptides were then iodinated with100 to 200,uCi

of the "nI-labeledBolton-Hunter reagent ina silicon-ized glass tube at 00C for 90 min under standard conditions(1). Labeledproteinswereseparated from low-molecular-weight componentsby gelfiltrationon a Sephadex G-10 column equilibrated with elution buffer(50 mMTris-hydrochloride [pH 6.8],0.2%

gel-atin) and prerun with 0.5 ml of a 10% solution of bovineserumalbumin.

For iodination of proteins by the chloramine-T method, virusparticles were dialyzed against 50 mM sodiumphosphate (pH 7.5) beforedisruption by boil-ing. Iodination under the conditions given by Carthew and Martin (4) was allowed to take place at room temperature for 1 min, and labeled proteins were separated from unreacted iodide by gel filtration as described above.

The"25I-labeledBolton-Hunter reagent (specific ac-tivity, 2,000 Ci/mmol) and ['251]iodide (carrier-free) wereobtained from the RadiochemicalCentre,

Amer-sham, England.

Gelelectrophoresis.Samplesoflabeled HAV pro-teins werepreparedin theappropriatesamplebuffers ofthe gel systems described below and, in general, wereheated to 90°C for 15 min beforeloading onto thegels. Three types of gel and buffer system were used forelectrophoresis:(i)SDS-polyacrylamidegels inthe discontinuousbuffer systemdescribed by La-emmli(12); (ii)SDS-polyacrylamide gels in the same discontinuous buffer systemsupplemented with either 1or 4M ureaby the method of Bowen et al. (2); and (iii)amodification of the continuous phosphate buffer systemcontaining8Murea asdescribed by Summers etal. (21). In the latter system, both the gel and the

runningbuffer contained 0.1 Msodium phosphate (pH

8.0)-0.1% SDS-8 M urea. Protein samples were pre-paredinthesame bufferplus 1%

,f-mercaptoethanol,

2%SDS, 8 M urea, 20% sucrose, and 0.002% bromo-phenol blue. Electrophoresis in discontinuous gels was carriedoutinslab gels (120 by 130 by 1.5 mm) at 80 V pergel, whereas electrophoresis in continuous phos-phate-urea gels was performed in glass tubes (5 by 130 mm)at aconstantcurrent of7 mA pertube. Proteins were fixed and stained in 1.5% Coomassie blue-1% amidoblack-45% ethanol-9% acetic acid and destained in 15% ethanol-7.5% acetic acid. Gels were finally

sliced into 1-mmsections, and the radioactivity was determined withoutfurther treatment of the slices in aPackard PRIASAuto-Gamma counter.

tions, only particles purified from stool no. 1 gave rise to a polypeptide spectrum that was more orless consistentwiththatof the marker

picornavirus (Fig. 1). It consisted of four poly-peptides tentatively labeled VP1 to VP4 and about35, 31, 26, and 7 kD in size. Moreover, two additionalpolypeptides with apparent molecular weights of 45,000 and 48,000 could be distin-guished. The latter polypeptides were also pres-ent inall HAVpreparations purified from fecal specimens of patient no. 2. In general, they con-stituted themajority of labeled proteins in these samples, whereasthe polypeptides migrating in thepositions of polioviruspolypeptides VP1 and VP4could barely be detected or were not

pres-ent atall.

A variation in several experimental param-eters (e.g., the technique usedfor

radioiodina-tion, treatmentofsamplesbefore electrophore-sis, origin and concentration ofacrylamide,and

addition of 1 and4 M urea to the discontinuous

gel system) hadnosignificanteffect onthe qual-itative andquantitative aspectsofthe polypep-tide pattem of individual samples. However, electrophoresisin acontinuousphosphatebuffer system containing 0.1% SDS and 8M urea

re-sulted in thefollowingdrasticchanges (Fig. 2). Besides ahigh-molecular-weight peak,three ma-jor proteinsand afourth minorproteincould be detected regularly in all HAV samples. All of thesepolypeptides appearedtobesmaller than the respective structural proteins ofpoliovirus (Table 1). Theonlyhigh-molecular-weight poly-peptideresolved in thepresence of 8 M ureahad an apparent molecularweight ofabout 40,000. This is clearlywithin thesize rangeof the pre-cursorpolypeptideVPO ofwell-characterized pi-comaviruses.

PolypeptideVPOofpicomavirusesis present insignificant quantities onlyin"empty" capsids or in immature virusparticles. Thesetypes of particle differ from the mature virion in their buoyancy andsedimentation behavior (14). As buoyantdensity centrifugationofaliquots ofthe HAV samples in shallow CsCl gradients indi-cated (Fig. 3), only particles purifiedfromstool no. 1and knowntobealmost devoid ofthe VPO-likepolypeptidehad abuoyancy behavior com-parableto that ofpoliovirus; yetpeak

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STRUCTURAL PROTEINS OF HAV

m₀ x

E Ur

In

SUCE NUMBER

FIG. 1. SDS-polyacrylamidegel electrophoresis of the capsid proteins ofpoliovirustype2(A) and of HA V particles extractedfromstoolsamplesno.1(B),no.211/5 (C), and no. 211/6 (D). The purified viruses were disrupted in the presenceof1%SDSby heatingto100°C for 1 min before radioiodination with the Bolton-Hunter reagent.Labeledproteinswereagain heatedto100°Cfor2min(1%SDS,2.5%

,8-mercaptoethanol),

and

polypeptides

wereseparated byelectrophoresisinadiscontinuous 15%SDS-polyacrylamide slab gel by the methodofLaemmli(12).Arrows indicate thepositions ofpolypeptidesVPOtoVP4ofpoliovirus.

trations ofhepatitis A antigen accumulatedata

slightly lower density (1.336 g/ml) than did the marker picornavirus (1.34 g/ml). In contrast,

virussamples extractedfrom the fecal specimens of patient no. 2 and containing the VPO-like protein in considerable quantity regularly bandedatdensities of below 1.336 g/ml. Insome of these samples, there wasalso evidence that suggested thepresenceofmorethanoneparticle

species (e.g.,no.211/6inFig. 3). Sedimentation studies yielded consistent results (data not

shown indetail).Particles extracted from sample no. 1sedimented at 157S,whereas 150to 154S wasrecorded for HAVcontained in the stools of patientno.2.

DISCUSSION

The resultsreported in this paper arein

ac-cordancewith the observations of Coulepisetal.

(5) and Feinstone et al. (8) in as far as they

confirm the existence of three major protein

components in HAV capsids with molecular weights similarto those of the structural

poly-peptides

VP1 to VP3 of

picomaviruses.

More-over,there isevidencetosuggestthat theHAV particles also containaminorpolypeptide com-ponent

migrating

inthe

position

of

picomavirus

polypeptide

VP4, as well as a

high-molecular-weight

protein

comparable

insizetothe precur-sor

polypeptide

VPO. Theconcentration ofthe latter

proteins,

however, varied withthe

origin

of thevirus

particles.

Rigorous

experimental

conditions

proved

to

benecessaryfor

resolving

the

polypeptide

spec-trum of HAV in a reliable and

reproducible

manner. At least one of the

high-molecular-weight

peaks

of the spectrum

displayed

in dis-continuous

gel

systems

disappeared

upon elec-trophoresis in the presence of 0.1% SDS-8 M urea, and radiolabeled material lost from that region

apparently

addedtotheconcentrationof

polypeptides

VP1toVP4

(e.g.,

no.2

11/6

in

Fig.

1 and 2).

Therefore,

it may be concludedthat the

formation

ofaggregatesofone or more

struc-tural

polypeptides

ratherthancontaminationof virus

samples

with unrelated or

virus-specific

VOL.

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w-E

a.

SUCE NUMBER

FIG. 2. Polypeptides ofHAVparticles extracted fromstoolsamplesno.211/6(A) andno.211/9(B)

as revealed by electrophoresis incontinuous 12.5%

polyacrylamidetubegels containing8Murea.HAV

particlesweredisruptedasdescribedinthelegendto

Fig. 1, radioiodinated by the chloramine-T

proce-dure,andheatedto90'Cin thepresenceof2% SDS-1%f3-mercaptoethanol-8Mureafor15min.Arrows indicatethepositions ofunlabeledpoliovirus poly-peptides coelectrophoresed in the same tubes and

locatedbyproteinstaining.

butnonstructuralproteins (e.g., the equivalents of the 52- and 57-kDproteins detected in foot-and-mouth disease [15] or Mengo virus [22] particles, respectively) might be responsible for the appearance ofhigh-molecular-weight com-ponentsintheprotein pattem of HAVas occa-sionally revealed in Laemmli gels (8).

Ifwe assume thatHAV is atypical picorna-virus, it is obvioustorelate the 40-kD polypep-tideresolved in SDS-ureagelstotheprecursor polypeptide VPO. This protein is known to be

present inprocapsids (empty capsids) of picor-naviruses and, in the process of maturation of thevirus, is cleaved intoVP2 and VP4 (14). The onlytype of particle in which VPO, VP2, and VP4 have been found simultaneously and in

various concentrations isanimmediate precur-sor, the provirion, ofmature enterovirus parti-cles (10, 11, 20). Provirions contain a full com-plement of viral RNA, sedimentat130to150S,

VP4 9,000±2,000

aTentative classification.

b Polioviruspolypeptides

(VP0,

41kD;VP1,32kD;

VP2, 28 kD; VP3, 25 kD; VP4, 8kD) were used as

internalmarkers and detectedby stainingwith Coo-massie blue. Additional molecular weight markers werebovineserumalbumin(68kD),gammaglobulin,

H-chain(50kD), ovalbumin(43 kD),pepsin (35kD), chymotrypsinogen (25.7 kD), papain (23 kD), cyto-chrome c (11.7 kD), and insulin B chain (3.4 kD). Numbers representmeanvalues ofseven determina-tions.

and band atlower densities inCsCl than does the mature virion. The analogies between the proteinpattern and the buoyancy and sedimen-tationcharacteristics ofthese structures and the HAVparticlesunderinvestigationarestriking. Therefore,it isconceivable that the 40-kD pro-tein indeedrepresentstheequivalent of the pre-cursor

polypeptide

VPO. At the same time it must be concluded that the

majority

of HAV particlespresent in the fecal

samples

collected frompatientno. 2 areprovirionstructures.Since provirions of picornaviruses probably are not infectious (11), the concentration of physical particles allows no reliable conclusion to be drawn about the infectivity of a certain stool sample. This pointmustbeconsidered when the many unsuccessful attempts to infect cell cul-tures with HAV-containing fecal extracts are evaluated.

Finally, itiswellknown thatmature

picorna-viruscapsidscarry acharacteristic immunolog-icalreactivity definedasD (orN) antigen. The absence or loss ofVP4 from these particles is

equivalent

totheappearanceofanentirelynew setofsurfacedeterminants,theC(or H) antigen (14). Theantigenicityof provirionparticleshas not been determined. In light of our protein data, however,it istemptingtospeculateon the extenttowhichthe use of uncharacterized fecal extracts as asourceofhepatitisAantigen could affectthesignificance and reproducibility of the available serological tests for HAV anti-bodies.

ACKNOWLEDGMENTS

We thank Giovanna CattaneoandDanielWasem for ex-cellent technical assistance.

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OF HAV

0

x

a.

u I

5

0

5

4 X

K

3

YI

2

FRACTIONS

FIG. 3. Buoyantdensity centrifugation of mature poliovirions (A) and HAVparticles (0)purifiedfrom

stoolsamples no. 1(A), no.211/5 (B),and no. 211/6(C) inCsCL.[3H]uridine-labeled poliovirus(10

fi)

and unlabeled HAVparticles (50

id)

weremixed with 7.9mlof CsCI (p= 1.34g/ml;made up in 50 mM

Tris-hydrochloride[pH7.41-0.1% Sarkosyl NL97)andcentrifugedtoequilibrium in a Beckman 50 Ti fixed-angle rotor at40,000 rpm at 15°C for 60 h. A total of 63fractionswere collectedby bottom puncture of the tubes.

Samples of each fraction were assayed for3Hradioactivity by liquid scintillation counting and for the presence of hepatitis A antigen in theradioimmunoassay. Poliovirus was assumed to band exactly at a

density of1.34g/ml.

This studywas supportedby grant 3.945-0.78 from the Schweizer Nationalfonds and grant De 261/1 from the DeutscheForschungsgemeinschaft.

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