Large surface proteins of hepatitis B virus containing the pre-s sequence.

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JOURNALOFVIROLOGY, Nov. 1984,p. 396-402 0022-538X/84/110396-07$02.00/0

CopyrightC 1984, American Society for Microbiology

Vol.52,No. 2

Large

Surface Proteins of Hepatitis

B

Virus Containing

the Pre-s

Sequence

KLAUS H. HEERMANN,' UDO GOLDMANN,1 WOLFGANG

SCHWARTZ,'

TORSTEN

SEYFFARTH,l

HORST

BAUMGARTEN,2 ANDWOLFRAM H. GERLICHl*

Departmentsof Medical

Microbiologyl

andImmunology,2 University ofGottingen, Gottingen, Federal Republicof

Germany

Received 6 June1984/Accepted 25 July 1984

Thesequenceof hepatitis B virus DNA containsan openreadingframe whichcodesforanot-yet-identified

protein ofatleast 389amino acids. Only the products starting atthe third(GP33/GP36)orthe fourth (P24/

GP27) initiation signal have been characterizedascomponentsofthe viral surface antigen. We foundalarger

protein, P39, and itsglycosylatedform,GP42, in hepatitis B virus particles and viral surface antigenfilaments. Immunological cross-reactions showed thatP39/GP42 is partially homologoustoP24/GP27and GP33/GP36.

The uniqueportion of itssequencebound monoclonalantibodies which had been induced by immunization with hepatitisB virusparticles. Proteolytic cleavage patternsand subtype-specific size differences suggested thatthe sequenceof P39startswiththefirstinitiation signal of theopenreading frame. Its amino-terminalpart(pre-s

coded)isexposed attheviral surfaceand, probably, is highly immunogenic. A model is presented of how the openreadingframe for the viral envelope leadstodefinedamountsof three different proteins.

Atypical consequence ofacute orchronic infection with

hepatitis B virus (HBV) is secretion of the viral surface

antigen (HBsAg) intotheblood of infected persons. Even in the presenceof efficientviral

replication, only

averyminor part ofthetotal HBsAg formsthe viral

envelope.

A larger

amountis foundonfilamentsof20-nmdiameter and variable

length. By far themostHBsAgis presenton

small,

noninfec-tious, 20-nm particles (20). These

particles

are used in

purified formas a vaccine

against

HBV.

There are two types of chronic carriers of

HBsAg.

A smallerfraction ofthecarriershas

high

titers of

HBV,

ahigh concentration of excess HBsAg, and soluble viral core

protein (e-antigen)in the blood. Most adult

HBsAg

carriers have, however, very littleor noinfectiousHBVorfilaments

intheblood andonly moderate concentrationsof

HBsAg

20-nm

particles

and antibodies

against

e

antigen.

The

mecha-nism for the

suppression

of viremia in these carriers is

unknown. Immune reactions

against

unidentified proteins

maybe involved (1, 5).

HBsAg from both types of carriers consists ofa

major

protein, P24, and its

glycosylated

form,

GP27

(19).

Purified

HBsAg20-nmparticles from viremic carriers

have,

in

addi-tion,afurther

glycoprotein, GP33,

andits

twofold-glycosyl-ated form, GP36 (24,

25).

The amino-terminal

protein

se-quence ofP24 has been

analyzed (19)

and

aligned

with the sequence of cloned HBV-DNA

(6a,

17, 29).

A

coding

sequence of 226

triplets

ending

with a stop codon was

identified forP24 (gene

s).

Gene s

begins only

at the fourth

possiblestartcodonofa

larger

open

reading

frame

(ORF),

so

it ispreceded in phase

by

163or174

codons, depending

on

the viral subtype (pre-s

region 28).

Recently,

itwas shown that GP33 consists of the P24 sequence and an

amino-terminalpartofca. 55 aminoacids

(13a, 24).

The

finding

of

anamino-terminal methioninein GP33

(14)

and the

transcrip-tion data(2, 22a)suggest thatthe sequenceof GP33 startsat

the third initiation signal of the

ORF,

which is 55 codons upstream of the fourth

signal (2).

The translation

products

beginning

atthefirstorsecondstartcodon havenotyetbeen identified. The conservation of the pre-s

region

during

evolution(15)and the invitro

transcription

data

(13)

make it

*Correspondingauthor.

verylikely that at least one larger surface protein exists. In the search for this hypothetical protein, we analyzed

purified HBV particles and filaments in addition to 20-nm

particles. Immunization of mice with HBV particles led to

monoclonalantibodies,which selectively detectedtwolarge

viral surface proteins. Cross-reactions with P24 and GP33,

partial proteolysispatterns,glycosylation data, andsubtype

heterogeneities suggeststrongly that these twolarge surface

proteins are products of the total ORF and that the monoclo-nalantibodiesaredirected againstepitopes codedby the

pre-s region. We present models of how the six envelope proteins ofHBVarederivedfromonecontinuousORF and of how theirexpression may be regulated. Being

immuno-genic components of the viral envelope, the large surface

proteinsmay be ofgreatmedical importance.

MATERIALS AND METHODS

Purification of HBV and HBsAg particles. Plasma units (250 ml) from one HBV carrier were passed through a

column (10 by 120 cm) of Bio-Gel A5M (Bio-Rad

Labora-tories)with TNE (0.13 M NaCl, 0.01 M Tris-hydrochloride (pH 7.4), 0.001 M disodium EDTA). Samplesofthefractions close to thevoid volume were mixed with 0.3%

,B-mercap-toethanoland 0.5% Nonidet

P-40,

and the HBVcore antigen was determined by an enzyme immune assay (7). Positive

fractionswere combined and centrifuged in anangle rotor (6

by90 ml) through a layer of 20% sucrose-TNE for 20 h at 25,000 rpm and 10°C. The pellets were suspended in 0.5 ml of TNE and layered on an S-shaped sucrose gradient with best resolution between 35 and 45% (wt/wt) sucrose. After 20 h at

34,000 rpm in an SW42 rotor, fractions were assayed for

HBsAgand viral core antigen. In electron microscopy, peak

fractions of viral core antigen consisted of HBV particles with less than 10% filaments. Peak fractions of HBsAg

consisted of>95% long filaments. HBsAg 20-nm particles with much GP33/GP36 were purified from Bio-Gel ASM

fractions containing the highest HBsAg activity by banding

in CsCl (9). These preparations were free of HBV or long

filaments.

Antisera. For raising anti-P24, purified HBsAg without

minor proteins (27) was treated for 30 min with 1% dithio-threitol (DTT) and 1% sodium dodecyl sulfate (SDS) and

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thendialyzed for2hagainst0.01% DTT-0.01% SDS in 0.13 NaCl. Foranti-GP33, purified HBsAgwith much GP33 but with undetectable GP42 was used without denaturation. Portions (100 pg) of HBsAg subtype ad were mixed with Freund complete adjuvant andinjected intramuscularlyinto guinea pigs. The injections were repeated with incomplete

adjuvant after 4 weeks. Serawere taken 10days later. Monoclonal antibody. Ten micrograms of purified HBV was mixed with complete Freund adjuvant and injected intraperitoneally into BALB/c mice. After 4 weeks the

injections were repeated with incomplete adjuvant, and 10

days later spleens were obtained for fusion with myeloma cell line P3-X63-Ag8.653 as described previously (10). Su-pernatants of

immunoglobulin-producing

hybridomas were

placedon microtiterplateswhichwerecoatedwith 40 ng of HBV andfilamentsperwell.Bindingofmouse immunoglob-ulinwasdetected bytheaddition ofperoxidase-labeled anti-mouseimmunoglobulin(Dako, P161). Clone A18/7

(IgG,K)

was recloned and injected intoBALB/c mice forgrowth as

ascites tumors. Immunoglobulin G from ascites liquid was

purifiedby precipitation with 18%(wt/vol) sodium sulfate. Gel electrophoresis and immune staining (26). Purified

HBV orHBsAg was denatured by 2.5% SDS and 5% DTT

for 5 min at 100°C. Proteins were separated in 12 or 15%

polyacrylamide-N,N'-methylenebisacrylamide gelswith the

Laemmli buffersystem and 2 to 5 V/cm (23). After

electro-phoresis,theseparated proteinsweretransferredtoaporous

membrane (GVHP; Millipore Corp.) bytransversal

electro-phoresisat40 V in 0.025 M Tris(pH 8.3), 0.192Mglycine, and 15% (vol/vol) methanol. Nonspecific protein binding

was saturated with 20% fetal calf serum-TNE for 1 h.

Thereafter, asuitableamountofantibodywas addedfor1 h underagitation. After a thorough washing, 1251_ or

peroxi-dase-labeled second antibody against the first antibody in

20% fetal calf serum was added for 1 h. The presence of

antibody was visualized either by autoradiography or by

enzymatic staining with 0.01% diamino benzidine-0.06% H202-0.05 MTris-hydrochloride (pH 7.4).

RESULTS

Proteincomposition of HBV particles, filaments, and 20-nm particles. All three morphological forms ofHBsAg consis-tently containedsixprotein bands: P24,GP27, GP33, GP36,

a39-kilodalton (kDa) protein, and a42-kDaprotein.

Experi-mentstobe described later showed that the 42-kDaprotein

was glycosylated, but the 39-kDa protein was not. Thus,

theyarereferredtoasP39 and GP42(Fig. 1). HBV particles

contained,inaddition, the P22 bandof the viral core protein

(7) and several weaker protein bands of>45 kDa (Fig. 1, lane 1) Mock preparations of HBV particles from negative human plasma also showed several high-molecular-weight

bands, but there were no bands atthe position of the viral

proteins.

The relative staining intensities of P24,GP27, GP33, and

GP36 were very similar in the three morphological forms.

Theintensities ofP39 andGP42 differedsignificantly, how-ever, among the three forms. In HBV particles and fila-ments,theGP42 band was more intense than the GP33 band; in 20-nmparticles, the GP42 band was weaker than the GP33 band. P39 and GP42 occurred in all isolates as pairs; GP42

was always more intense than P39. This typical staining

behavior of the three morphological forms was confirmed with samples from six further HBV carriers. The intensity of

the protein bands after silver staining suggests that HBV

particlesmaycontain up to 20 times more P39 and GP42 than do 20-nmparticles fromthe same plasma source. According

to the particle mass of 3 x

106

daltons, 20-nm particles

1

GP

42-P

39-GP

36-GP

33-2

:

a.

_Em

a.:9

GP

27-P

24-P

22-FIG. 1. Protein compositionof HBV particles (lane 1), HBsAg

filaments (lane 2), and20-nmparticles(lane 3). Theparticles were purified from the plasma ofa chronic HBV carrier (subtype ay). Sampleswith similarproteincontentswereelectrophoresed through 12%polyacrylamide,andtheproteinswerestained with silver(16). Size markerswerelysozyme(14.3kDa),trypsininhibitor(20kDa),

carbonic anhydrase (30kDa), ovalbumin(45 kDa),serumalbumin

(68kDa), and phosphorylase B (94kDa).

consist of ca. 100 protein subunits. The weak staining

suggeststhat only one oroccasionally twoGP42/P39 mole-cules may be present in 20-nmparticles. In contrast, HBV,

with itsfour-times-larger surface, may contain 40to 80 P39

orGP42 molecules per virion.

Monoclonalantibody againstHBV. Five micewere

immu-nized with purified HBV particles, and all developed high

serum

antibody

titers against HBsAg. Several thousand

hybridoma cloneswere derived fromthe spleencellsofthe

mice,butonlytwoofthe clonesproducedantibodiesagainst

HBV. Neither antibody bound to mock preparations of

HBV. The antibodies were apparently directed

against

the

same or very

closely

since mixtures of the

antibodies did not

produce

stronger

binding

than

single

antibodies. Oneofthemonoclonal antibodies

(MA18/7)

was

usedfor further

experiments.

Inatwo-site enzymeimmune assaywith MA18/7at thesolid

phase

and

peroxidase

conju-gated

aslabeled

antibody,

HBV

particles

gavethe strongest

signal

of the three

morphological

forms. A

2-fold-higher

protein concentration of

purified

filamentsora

20-fold-larger

amount of 20-nm

particles

was necessary to bind the same amount oflabeled

antibody

to the solid phase

(Fig.

2). The HBV particles used for immunization had been ofHBsAg subtype ayw. MA18/7 reacted, however, with

subtype

adw

as well.

The part of the viral envelope which bound MA18/7 was

sensitive to proteolysis by trypsin. A limited digestion of

HBsAg 20-nm particlescompletely abolished the bindingof MA18/7. In contrast, the binding ofaconventional anti-P24

antibody

increased slightly after the digestion (Fig. 3A).

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398 HEERMANN ET AL.

1-l1 ng/ml

160

idoo

FIG. 2. Binding of labeled monoclonal antibody MA18/7 to in-creasing amounts of HBV (U), HBsAg filaments (0), or 20-nm particles(A).Wells of microtiterplates were firstcoatedwith 0.5 ng of MA18/7 each and thenincubated with the indicated dilutionsof purified particles for1 h at 37°C. Afterbeing washed, peroxidase-labeled(8)MA18/7 in1% bovineserumalbumin-phosphate-buffered saline was added for 1 h, and after further washings, ortho-phenylenediamine-H202assubstrate wasadded. Thecolored reac-tionproduct wasassayed at493 nm(E493).

Reduction oftheHBsAgparticles didnotchangethebinding of MA18/7, but most of the conventional anti-P24 did not

bind anylongerto theparticles (Fig. 3B).

Immune reactivity of isolated HBsAg proteins. When the

immune blot technique was used after gel electrophoresis, MA18/7bound only to P39 and GP42

(Fig.

4, lane

C).

This

finding showed that P39 and GP42 sharedan

epitope

which was not present onthesmallerHBsAgproteins.The

finding

also excluded the possibility that the P39 and GP42 bands were mere aggregation artifacts consisting ofP24 orGP27. The epitope of MA18/7 was completely resistant to the

combined effects ofreduction,

detergent,

and

heat,

which were employed beforeelectrophoresis.

The sizes of P39 and GP42 were consistent with the

assumption that they might be

products

of the whole ORF

for HBsAg.

According

to that

hypothesis,

P39 and GP42 would have asequence uniquetothem of108or 119amino acids, and MA18/7 would bind to this

region.

In addition, theywould have all sequences and the denaturation-resistant

epitopes of P24 or GP33. Two conventional

guinea

pig

antiserawereusedto testthis

hypothesis,

because monoclo-nal antibodies against denatured P24 or the pre-s part of

GP33 were not available.

An antiserum against P24 and GP27 was raised

by

the

injection ofreduced and denatured

particles

which did not

have detectable

GP33, GP36,

P39,

or GP42. This anti-P24

antibody

bound

equally

well to all six

HBsAg

proteins,

includingP39 andGP42

(Fig.

4,lane

A).

Asecond antiserum

was produced by the

injection

of 20-nm

particles

which

contained much GP33 and GP36 but no detectable P39 or

GP42 (23). The injected animals

produced

more

antibody

against the pre-s part ofGP33 than

against

denatured P24,

although the inoculum contained more P24 than GP33. At

the dilution used for the

experiment

shown in

Fig.

4,lane B,

immunoglobulinG fromthis antiserum bound wellto GP33

and GP36 butweaklytoP24andGP27. P39and GP42were also well immune stained

by

thisantiserum.

Insummary, GP42 andP39are

indistinguishable by

immu-nological methods. The two

proteins

have three

antigenic

regions: oneisdefinedbythe

HBV-specific antibody

MA18/

7, the second is coded

by

the pre-s part of

GP33,

and the

third iscoded bygenes.

Proteolytic cleavage between the pre-s and gene s coded

sequences. Evidence on the

primary

structure of P39 and

GP42 was obtained by digestion with the glutamic acid-specific protease from Staphylococcus aureus V8. Gene s has only two glutamic acids at positions 2 and 164; the pre-s sequence does not have any glutamic acid. Previously, we have shown that GP33 or GP36 is slowly cleaved by V8 protease into P24 or GP27 and an 11-kDa fragment which consistsof the 55 pre-s-derivedamino acids and of N-linked

glycan (24). If P39 and GP42 contain the whole pre-s sequencelinkedtothegene s sequence, V8 protease should generate a fragment of164 or 175 amino acids which binds

MA18/7. Figure 5, lanes B, shows that such a fragment of 18 kDa was generated. Since both GP42 and P39 were cleaved by the protease, the production of only one fragment react-ing with MA18/7 showed that P39 and GP42 were completely identicalin their pre-s part.

Subtype heterogeneity of the pre-s sequence. All known

DNA sequences of different HBsAg subtypes specify a constant size of gene s. The pre-s sequences of the two

sequencedaywsubtypeshave 163codons(6a, 17). The adw2

subtype is largelya homologof subtype ayw, but it has an

additional 11 codons at the 5' end of the ORF (29). In

agreementwith the DNA sequence data, P39 and GP42 from four adwsubtypeisolates were consistently larger than those from two ayw subtypes (Fig. 6, lanes B). The difference was ca. 1.0 to 1.5 kDa.

Asubtype-independent microheterogeneity of the HBsAg proteins has been described previously. This heterogeneity resides inthe gene s part (23) and is also visible in P24/GP27 ofthe isolates shown here. A smaller GP33 or GP36 from ayw subtypes was not noted (Fig. 6, panel A), and so the observed subtype-specific size heterogeneity is not due to the sequences present in the gene s part. The subtype differences between adw and ayw suggest that the sequence of P39 andGP42 starts with the very first codons of the ORF for HBsAg.

Glycosylation of the HBsAg proteins.Asensitive,

glycopro-1'

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6o

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1i0

- +

FIG. 3. Effects of trypsin (A) and DTT (B) on the bindingof MA18/7(0) and anti-P24(0). PurifiedHBsAg20-nmparticles (0.8 ,ugin100,ul) with relatively much GP42wereadsorbedtomicrotiter

wells(NuncII) for 4 hat20°C. The wellswerewashed,and 100p.lof

differenttrypsin dilutionsorof0.1 M DTTwasadded for 30minat

37°C.ReducedSHgroups wereblocked with 0.1 M iodacetamide for 16 h at4°C. Wellswere washed, anda 1:8 dilution ofhybridoma

supernatant MA18/7 or a 1:8,000 dilution of anti-P24 guinea pig

serumin1% bovineserumalbumin-TNEwasadded for 1 hat37°C.

Afterathorough washing, bindingof the antibodieswasquantitated by the addition of peroxidase-labeled second antibody (1:1,000 Dako) and by the measurement of bound peroxidase at 493 nm

(E493)-E493 7

/-,/

A A B

jig/ml

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PRE-s PROTEINS OF HEPATITIS B VIRUS 399

-A

B

C

GP42-

P39-

GP36-GP33- A

P

GP 27-

A

P24-

A

FIG. 4. Immune staining ofHBVsurface proteinswithanti-P24 (laneA),anti-GP33 (lane B), and MA18/7(laneC). PurifiedHBsAg

filaments(ayw) weredenatured,and theproteinswereseparated by

electrophoresisandtransferredtoporousmembranes. LaneAwas stained with a 1:50 dilution of anti-P24 serum, lane B wasstained with a 1:400dilution of anti-GP33 serum, andlane Cwas stained with a 1:10 dilutionofhybridomasupernatant.

tein-specific stain (3) showed that GP42 was glycosylated,

but P39 was completely glycoside free (Fig. 5, lane C). In

agreement with the conclusion drawn before, the 18-kDa

fragment of the glu-specific cleavage also did not contain glycan. Thus,theglycanof GP42 is boundtothegenespart, mostprobablyatthesamesiteasin GP27 (18)orGP36(24).

The amino-terminal (24) mannose-rich glycan (22, 23) of

GP33 or GP36 is apparently nonexistent in the products of

the whole ORF.

The nature of the glycan in GP42 was studied further by digestion with endoglycosidase F. This enzyme removes

asparagine-linked glycans from their protein part (4). The

electrophoretic mobility of

glycoproteins

is shifted

by

3 kDa for everyglycanremoved. Figure 5,lanesD,show that GP42

decreased after digestion and P39 increased. GP36

disap-pearedcompletely, GP33 decreasedstrongly,andanintense

P30 band appeared. This

finding

supports the conclusions thatGP42 contains oneasparagine-linked glycan group and that this group is the only difference from P39. The results shown in Fig. 5, lanes D, directly confirm our previous

report(24)thatGP33 hasoneglycangroupandGP36hastwo

glycans.

DISCUSSION

Theprotein composition of HBsAghasbeenthe

subject

of

numerous studies. Due to the variability of the HBsAg proteins (23), discrepant results onthe minor

proteins

were

reported. Theelectrophoretic components largerthan GP42 are now understood asdimers (11, 23), and inthis

study

no

suchproteinscould be

reliably

identified as

HBsAg

orHBV components. Feitelson et al. found the minor HBsAg

pro-teinsp43,p35,andp32,whichprobably correspondtoGP42, GP36, and GP33. These

proteins

shared many

tryptic

pep-tides with P24 or GP27, but they also had unique peptides

(6). Sanchezetal. demonstrated serological cross-reactions

between theminorproteins (p27, p31, p35,andp40)andthe

major proteins (p22 and p25). They suggested "repeating antigenic determinants" in all HBsAg proteins (21). These findings are consistent with our data. Stibbe and Gerlich (24) showed that limited proteolysis of GP33orGP36generated

the same fragments as proteolysis of P24 or GP27, but the

twolarger proteins hadanamino-terminal extension of 50 to 60aminoacids,with threeproteolytic cleavagesites predict-ed by the pre-s(2) sequence (see Fig. 7 for definition).

Machidaetal. (14) alsofoundtwominorglycoproteins, p31

and p35, to be coterminal with P24. They showedbyamino acid analysis that these proteins contained a cyanogen bromide fragment which was coded by thepre-s(2) region.

Thefragment carries the HBV-associated receptor for

cross-linked human albumin (13a). Neurath et al. synthesized a

peptide

containing the 26amino-terminal aminoacids of the pre-s(2) region. Antibodies against this peptide bound to GP33 and GP36 (16a).

The major mRNA of HBV in infected liver starts (2, 22a)

closelyupstreamof the thirdinitiation codon oftheORF(17, 29), and soit is very likelythatGP33 and GP36 also start at

A

B

1 2

*iv-i

42-_

39--

M

--

___

~~~-

- 42 -

___--39

10

36- AN

-33_-__w

-42 -39 -36 -33 -30 -27 -24 27

24

- 18 >

FIG. 5. Generation ofan 18-kDa pre-s-coded protein fragment by V8 protease(lanesB), concanavalin A binding of HBV surface glycoproteins(lane C), and removal of glycan by endoglycosidase F (lanes D). (A) Silver stain. (B) HBsAg filaments (adw2) were digested with6 ,ugof V8 protease (Bio-Rad) in 25,ulof0.1% SDS-1% DTT-0.1 MTris-hydrochloride (pH 7.4) for 72 h at 37°C. Each 24h,newprotease was added. The digested proteins were separated by SDS-gel electrophoresis, transferred to a membrane, reacted with MA18/7 and 125I-labeled anti-mouse immunoglobulin G, and autoradiographed. Lane

Bi,

Control incubation without protease; lane B2, protease digest. (C) Before treatment with MA18/7, the membrane withthe separated proteins was incubated with 100u.gof concanavalinAperml and10 mgofbovine serum albumin per ml in 0.13 M NaCI. After 30 min at 37°C, the washed membrane was agitated with 30 ,ug of horseradish peroxidase (type VI; Sigma Chemical Co.)perml in 0.13 M NaCl. After a further 30min and subsequent washings, the bound peroxidase was detected as de-scribed in thetext. LaneCshows the staining of lane B2. Note the absence of stainingin P39 and the 18-kDa fragment (-). (D) In a parallel experiment, filamentsweredigestedwith5

,u1

of endoglyco-sidaseF(Bio-Rad) in35 ,ul of 0.1 M sodium phosphate (pH6.1)-S50

mM disodium EDTA-0.1% SDS-1% Nonidet P-40-1% mercap-toethanol for16h at 37°C. The proteins were separated, transferred

to amembrane, and immune stainedwith anti-P24 serum. LaneDl, Control incubation; lane D2, endoglycosidase F digest. Note the increase ofP39and theformation ofP30 in the digest.

VOL. 52,1984

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400 HEERMANN ET AL.

l 2 3 4 5 6

(- fv&4* R;--42

*ift'" Oa0080 A -39

27_-inI

P

24-A

B'.

FIG. 6. Different sizes of GP42 and P39 in HBsAg samples of different subtypes. Proteins ofpurified20-nmparticleswere separat-ed induplicate bySDS-gelelectrophoresis. Panel A of thegelwas stained with silver. Panel B was transferred to a membrane and immune stained with MA18/7.Lanes1 and3,Subtypeay;lanes2,4, 5, and 6, subtype adw. Note the smallerdistance between GP33/ GP36 and P39/GP42 inpanelAand the fastermigrationof P39/GP42 inpanelB withtheaysubtypes.

this translation signal (Fig. 7). The first AUG codon of this mRNA,however,lacks thetypicalflankingbases ofastrong

initiation codon (12),soproteinsynthesiswillprobablymore

often begin at the start of P24 or GP27 with its typical

initiation codon. By such a mechanism the two coterminal

proteins with different amino termini may be translated in

defined proportions from one mRNA. All results of this

study confirm the hypothesis that P39 and GP42 are the

translation products of the whole ORF. Thus, atleastsmall amounts of a mRNAstartingupstream of the first initiation codon of theORFmustbepresentinHBV-producingcells,

although it has not yet been found in

HBsAg-positive

liver

samples (2). Such a mRNA was, however, presentin COS

cellstransformed withtheappropriate DNAfragments(13). Sincethefirst initiation signal isstrong, thesmaller HBsAg

proteins

are probably not derived from this larger mRNA. The second AUG in the ORF ofsome HBV isolates is not

conserved andisprobablynotaninitiationsignal.Thelarge

mRNA has a promoter with a TATA box (13), but the

promoter of the small mRNA is more like the late simian

virus40promoter(2). Thus, expressionofP39/GP42maybe

completely independent

ofexpression ofthesmallerHBsAg

proteins.

Since designations based on electrophoretic sizes are

ambiguous

formany reasons, itmaybe preferableto

distin-guishthe threetranslation products ofthe ORF forHBsAg

as

large, middle,

and small(ormajor) surface proteins. The

sequenceoftheORFmaybedivided into three

independent-lyexpressed parts:pre-s(1),presentonlyin the largesurface

protein;

pre-s(2),presentalsoin themiddle protein;andgene s, as suggested previously (28). The expression of three

different envelope proteins by the variableuse ofinitiation

codons inoneORFcertainlysavesmanyhundreds of

coding

capacity triplets

and regulating signals. The genetic

organi-zation of thisORFis another example-in additiontotheuse

ofoverlapping ORFs-thatthe HBVgenomeis

evolutionari-ly

selectedto minimum size.

The glycosylation patterns of the three surface proteins

show interesting differences. Approximately 40% of the smallproteinisglycosylatedatthe carboxy-terminal

binding

site for N-linked glycans. As suggested by the staining intensity oftheprotein bands,thesamesiteis used less often in the middle protein, but probably moreoften inthe large

protein. The single binding siteof the middle protein in the

pre-s(2)region is alwaysoccupied by amannose-richglycan,

andnoglycoside-free middle proteinP30isfound in serum.

The same site, however, is not used at all in the large

protein. This observation demonstratesthe stronginfluence of an amino-terminal sequence on the processing of the

following

sequences.

Ourfindings with sevendifferent isolates suggestthat the

large surfaceproteinsareessentialcomponents of

complete

-HBV-DNA

2104bases -I

226codons

strong initiation signal stop

I

....D...large

mRNA... F

TA TA-box

V8-Protease

P39

Gc 389 amino

acids

389

weak strong

.-t.... ...>...small mRNA(>1otimes

more)..-*+--late promoter,

04

9~~~~~~~c

281

GP36 c

GP33 281

GP27

P24

YC

226

226 l

FIG. 7. Regulated expression ofsurfaceproteinsfrom HBV DNA andstructureof theHBsAg proteinsinrelationtotheircodingORF.

TheDNAsequenceof the ORF (top line) is from Paseketal.(17). The positionof the four5'-proximalinitiation sites(strong [>]andweak

[>]) for translationand the numbering of codons in relationtogenesareshown in the second line. The secondsignalisnotconserved inother HBVDNAs(6a, 29).Mappingof thetwopostulatedmRNAsisexplainedin thetext.Thebindingsitesof N-linked mannose-richglycans (Gm)

andcomplex glycans(Gj)weremappedpreviously (24).Theexactterminalmappositionsof P39/GP42 in theHBsAg particlesorHBVarenot

yetknown, but theyareprobably identicaltoorclosetotheproposedsites.

1 2 3 4 5 6

GP42- _..

P

39-GP36- _

GP 33- -_ __

GP

947 1261 1426

I pre-s(1)

1--s(2)

ss-gene,

-163 (-90) -55

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HBV orof filaments, butnotofHBsAg20-nmparticles. The binding ofMA187 to native HBV or HBsAg in an enzyme immune assay shows that at least parts of the pre-s(1)

sequencearelocatedatthe surface. Therapidinactivationof

the MA18/7 epitope by trypsin is consistent with its exposed position, and it is also inagreement with the large numberof basic amino acids in the pre-s(1) region. Previously, it was shown that the pre-s(2) region ofGP33/GP36 is also at the

outer side of HBsAgparticles (24, 25), and the data on the albumin receptor coded by the pre-s(2) region confirm this

observation (13a).

As seen in the immune blots, both theepitope ofMA18/7 and at least one major epitope of pre-s(2) are resistant to

denaturation by heat, SDS, and DTT. These epitopes are apparently more immunogenic than are the epitopes of

denatured P24. It is likely that the denaturation-resistent epitopes are defined by the primary sequence, irrespective

of the protein conformation. This is insharp contrast tothe

antigenicityofP24, which is highly dependentonthe confor-mation specified by its synthesis in eucaryotic cells. In the

search for immunogenic and protective poly- or oligopep-tides as analternate vaccine against HBV, sequences of the pre-s region may be of interest. Recently, Neurath et al. (16a) showed that the 26 amino-terminal amino acids in the pre-s(2) region act as ahighly efficient immunogen. Testing

cellular(5) and humoral(1)immunity in hepatitisB

convales-cents, itwasnoted that HBV hadanantigenic component(s)

which was absent or rare in HBsAg 20-nm particles. Most

interesting, the immune reaction against the HBV-specific antigenwas loworabsent in viremic HBsAg carriers, butit

waspresentin nonviremic carriers. Accordingtoourresults,

the pre-s(1) part ofthe large surface protein could be that

antigenic component.

ACKNOWLEDGMENTS

We thank R. Thomssen and 0. Gotzeforgenerous support, K. Lechte for technical asistance, and R. Stute for supplying HBV-containing plasma.

Thisworkwaspartiallysupportedby theDeutsche Forschungsge-meinschaft.

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