The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation.

The

Duck Hepatitis

B

Virus Pre-C Region

Encodes

a

Signal

Sequence Which

Is

Essential for Synthesis

and

Secretion

of

Processed Core Proteins but

Not

for Virus Formation

HANSJ. SCHLICHT,* JOCHEN SALFELD,t AND HEINZ SCHALLER

ZentrumfurMolekulare Biologie, University of Heidelberg, D-6900 Heidelberg, Federal Republic ofGermany Received 16 June 1987/Accepted 19 August 1987

Analysis of the serum of duck hepatitis B virus (DHBV)-infected ducks has revealed the presence of

C-terminally truncated viralcoreproteins(eantigens). These proteinsareglycosylated andthereforewerenot released from infectedcells by lysis but rather by active secretion, indicatingthat the DHBVcoreproteincan

besynthesized alternativelyas a cytoplasmicor asecretory protein. Transient expression of cloned wild-type DHBVDNA and ofaspecifically designed viralmutantinahuman hepatoma cell line (Hep-G2)showed that

theDHBVcore genepromoteris active in differentiated human liver cells and that synthesisand secretionof theprocessedcoreproteinsaredependent onthe expressionof the pre-C region,asmallopenreading frame

which precedes the core gene. In addition, these experiments showed that the mechanism ofcore protein

processingandsecretion isconserved between DHBV and the human hepatitis B virus and therefore might be important forthehepatitis B viruslife cyclein general. In spite of this, intrahepatic injection of the pre-C

mutantintouninfectedducks resulted in viremia without concomitant e-antigen synthesis, indicating that virus formation isindependent of pre-C expression.

Thehepatitis B virusesareenvelopedDNAviruses which

predominantly infect the liver (reviewed in reference 37). We have chosen duckhepatitis B virus (DHBV), which infects Pekinducks,to studythestrategies of hepatitisB virusgene

expression. Compared with the human hepatitis B virus (HBV), which only infects humans and chimpanzees, this animal model virus offers the advantage that systematic in vivo studiesonviralmutantscanbe performed. Inaddition,

since DHBV is the member of the hepatitis B virus family

mostdistantly relatedto HBV, acomparative study of both

viruses should give an insight into the principles ofgene

expression of all hepatitis B viruses.

Sequence analysis (19, 33) showedthat the DHBVgenome comprises three open readingframes (Fig. 1). The pre-S/S

open reading frame encodes the viral surface proteins (27, 31). It completely overlapswith the P open reading frame, which is believedtoencode the viral polymerase (38). The C

open readingframe encodes the viralcore protein(33). The

strategy by which the core gene is expressed is ofspecial interest,since thisgeneappearstoencodemultipleproteins,

as is suggested by the fact that more than one core gene

product can be detected in the liver and serum of HBV-infectedpersons.

One of these proteins, HBcAg, has a strong tendencyto

aggregate to viral core particles (8, 22, 34)and is

predomi-nantly present in the liver. The other, HBeAg, has been characterized as aC-terminallytruncated core protein (36). Itdoesnotformaggregatesand is encounteredespeciallyin the serum. Recently, it was suggestedfrom in vitro studies thatsynthesisandsecretion of theHBeAg mightbe linkedto

theexpressionof thepre-C region (21, 24, 28, 29, 40),asmall openreadingframeprecedingthecoregenein allhepatitisB

viruses sequencedtodate. Inaddition, itwasspeculatedthat thepre-C sequence mightbenecessarynot onlyforHBeAg

*Correspondingauthor.

tPresent address: Dana-Farber CancerInstitute,Harvard

Medi-calSchool, Boston, MA 02115.

secretion but also for virus formation by targeting nascent cores to the membrane of the endoplasmic reticulum (24, 40),wherebuddingofnewly synthesizedvirusesis believed

tooccur (15).

Here we show that C-terminally truncated core proteins

similartothe HBeAgcanbe detected in theseraof

DHBV-infected ducks. Since these proteins areglycosylated, they were not released from lysed cells but ratherwere actively

secreted. We also present evidence that the biosynthetic pathway which leads to the synthesis of these proteins is conserved between HBV and DHBV, suggesting that the productionofprocessedcore proteins confersanadvantage to the hepatitis B viruses. In addition, we show that the

formation and the secretion of the truncated core gene

products are dependent on a functional pre-C region,

whereasvirusformation is not.

MATERIALSANDMETHODS

Animals. Pekin ducks (1-day-old ducklings) were

pur-chased from commercial suppliers and tested for DHBV

infection by dot-blot analysis as described previously (32).

Animalsweresacrificedattheageof 3weeks, and liverand serum sampleswere storedat -70°C.

Immunoprecipitationand Western blotanalysisofcoregene

products. For Western blot (immunoblot) analysis, 150 to

500 ,ul ofserum, upto 10 ml of tissue culture supernatant, 700 ,ul of cell lysate (see below), or 100 mg of liver tissue

homogenized in 1 ml of TNE (150 mM NaCl, 20 mM Tris

hydrochloride, 10 mM EDTA, pH 8.0) were subjected to

immunoprecipitation with 10 ,ul of anti-core immune serum

preadsorbed to 25 ,lI of protein A-Sepharose (Pharmacia Fine Chemicals) preswollen in phosphate-buffered saline

(PBS; 140 mM NaCl, 10 mM phosphate, pH 7.4). The sampleswereincubatedat 4°C with slight agitation for4 to 16 h. Unbound material wasremovedby three washeswith TNE, and the specificallybound proteinswere dissolved in 50 pul of sample buffer (3% sodium dodecyl sulfate, 2% 2-mercaptoethanol, 10%sucrose,0.1%bromophenolblue,5

3701

Copyright C) 1987,American Society for Microbiology

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3702 SCHLICHT ET AL.

FIG. 1. Schematic representation of the DHBV genome. The

arrows representthe three openreading frames asdefinedby start

and stopcodons. The depicted RNA ofmore than genome length

encodes the viral core protein and also serves as a pregenome for

viral replication.

mM EDTA, 200 mM Tris hydrochloride, pH 8.8). When samples were assayed directly, 10,ul of serum or 10

RI

of

liver homogenate was mixed with 40

RI

of sample buffer.

After the samples were heated for 3 min in aboiling water

bath, they were applied to a 12.5% polyacrylamide gel.

Polyacrylamide gel electrophoresis and Western blotting

wereperformedessentially asdescribed previously (17, 39).

Aftertransfer, unoccupied binding sites were saturated by

incubatingthe filter foratleast 6 hinPBS-1% bovine serum

albumin (Miles Laboratories, Inc.). Core gene-encoded

pro-teins were detected by incubation of the filter with the

anti-core serum at a serum dilution of 1:2,000 in PBS-1%

bovine serumalbumin-0.01%sodium azide for 16 h atroom temperature. Excess antibody wasremoved by washing the

filter with three changes of PBS-0.1% Nonidet P-40 (total washing time, 30 min). Bound antibody was detected by

incubatingthe filter with '25I-labeled protein A (0.05

,uCi/ml;

Amersham Corp.) in PBS-1% bovine serumalbumin for3 h

at room temperature. Unbound protein A was removed by

washing with PBS-0.1% Nonidet P-40 as described above.

The air dried filter was exposed to X-ray film (XAR-5; Eastman Kodak Co.) at -70°C for 3 to 16 h with an

intensifying screen.

Endo F digestion. The serum-specific core proteins were

immunoprecipitated from duck serum(150 pdofserum, 350

[lI

ofPBS)asdescribedabove, washed, and solubilized in 50

[lI

ofKNP buffer, pH 7.0 (5.2 ml of 0.5 M KH2PO4, 16.2 ml

of 0.25 M Na2HPO4, 78.6 ml of H20) with 2% sodium

dodecyl sulfate and 2%2-mercaptoethanol by heating for 3

min in a boiling water bath. Then 700

RI

of KNP with 1% Triton X-100, 1% 2-mercaptoethanol, and 20 mM EDTA were added, the Sepharose was pelleted, and the

superna-tant was transferred toa fresh tube. Fordigestion 0.5 U of

endo-p-N-acetylglucosaminidase

F (endo F; Boehringer, Mannheim GmbH, Federal Republic of Germany) was

added, and the samples were incubated for 24 h at

37°C.

After digestion, sodium dodecylsulfate wasaddedtoafinal

concentration of 2% (75 of20% sodium dodecyl sulfate), andtheproteins wereprecipitated with trichloroacetic acid

(final concentration, 10%; 30 min on ice). The protein

precipitatewas pelletedand, aftercareful removal of

resid-ual acid,was dissolvedin 50

[lI

ofsample buffer.

Generation of theC terminus-specific peptide antiserum. A peptidecomprisingthe last 13aminoacidsofthe DHBVcore

gene (sequence, S-S-S-S-H-H-R-S-P-S-P-R-K) was

synthe-sized by using an automated peptide synthesizer. For cou-pling, 7 mg of tetanus toxoid and 2 mg of

peptide

were

dissolved in 1 ml of phosphate buffer (0.1 M, pH 6.8); with stirring, 0.5 ml of 21 mM

glutaraldehyde

wasaddeddropwise

over a period of 1 h at room temperature. After storage overnight at

4°C,

the mixture wasdialyzed againstPBS. For generation of an antiserum, twoNew Zealand Whiterabbits

were immunized with the coupled peptide. The first dose contained 300 pLg of coupled peptide in 2 ml of Freund

complete adjuvant. Booster injections (200

,ug

of coupled

peptide in 2 mlof Freund incompleteadjuvant)weregiven 3,

5, and 6 weeksafter thefirstinjection. After thethird booster

injection, blood was withdrawn and tested for an antibody titer against the peptide by enzyme-linked immunosorbent

assay asdescribedpreviously (25). The titerofbothsera was about 1:8,000. ForWesternblotting, the sera were usedat a dilution of 1:500.

Site-directed mutagenesis. The pre-C stop mutant was generated by site-directed mutagenesis with a DHBV 16 full-length genome cloned into the BamHI site of the vector

M13mp8 (3). For site-directed mutagenesis, a single

mis-match 18-mer oligonucleotide comprising nucleotides 2523 through 2540 of the DHBV minus strand (sequence, G-C-A-A-G-A-A-T-C-T-T-A-A-A-C-A-A-T) was synthesized by us-ing an automated oligonucleotide synthesizer. The melting temperature of this oligonucleotide was determined to be

58°C

for the wild type and

63°C

for the mutant in 3 M tetramethylammonium hydroxide. Mutagenesis was carried out by a standard protocol (43). A phage clone, which strongly hybridized to this oligonucleotide after stringent washing

(61°C,

3 M tetramethylammonium hydroxide), was selected by screening of the recombinantphage plaqueswith

the 5'-labeled mutagenizing oligonucleotide. This phage clone was plaque purified, and the T-to-A exchange

(nucle-otide 2531) was confirmed by sequence analysis.

Preparation of DHBV circles for in vivo and in vitro transfection. A DHBV 16 full-length genome excised from plasmid 16t27 (32) was cloned into the

EcoRI

site of the vector pUC13. CsCl gradient-purified plasmid DNA (DHBV 16, wild type) or M13 replicative form (DHBV 16 pre-C mutant) were prepared by standard methods (see reference 43 for preparation of M13 replicative form). The DHBV genome was excised by cutting with the respective restric-tion enzyme and circularized by ligarestric-tion at a DNA concen-tration of 3 pmol/ml (4 U of T4 DNA ligase per ml, 16 h at

15°C).

After ligation, proteins were removed by phenol extraction. The DNA was ethanol precipitated, dissolved in water, and dialyzed against 10 mM Tris hydrochloride-0.1 mM EDTA (pH 7.0).

Transient expression of DHBV DNA in tissue culture. The human hepatoma cell line employed (Hep-G2) (1) was culti-vated in Dulbecco minimal essential medium (GIBCO Lab-oratories) containing 10% fetal calf serum (10 ml of medium per

25-cm2

flask). Cells were grown to 30% confluency in

25-cm2

tissue culture flasks and transfected with 5 to 10

,ug

of circularized DHBV DNA by using the

Ca3(P04)2

precipitate technique (13). After 4 to 6 h, a dimethyl sulfoxide shock was given (10% dimethyl sulfoxide in Dulbecco minimal essential medium-10% fetal calf serum, 20

min

at

37°C).

The dimethyl sulfoxide medium then was discarded, and 10 ml of fresh medium was added. After 48 h, the medium was removed, and the cells were lysed in 700

,ul

of TNE-1% Nonidet P-40-0.5 mM phenylmethylsulfonyl fluoride. Insoluble

mate-J. VIROL.

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[image:2.612.107.265.76.240.2]

rial was pelleted and discarded, and the clear lysate was

subjectedtoimmunoprecipitationand Western blotanalysis

as described above. Usually half of the DHBV proteins obtained from the cells or the medium of a 25-cm2 tissue culture flask was applied to a slot for gel electrophoresis and

subsequent Westernblotting.

Transfection ofducks with cloned DHBV DNA.

Four-day-oldDHBV-negativeducklings were transfected with 10,ug of

circularized DHBV DNA dissolved in 0.3 ml of PBS by

injecting the DNA solution into multiple sites of the liver.

After 3 weeks the animals were sacrificed, and liver and

serumsampleswere examined for DHBV DNA by dot-blot

analysiswith32P-labeled,cloned DHBV DNA. Usually50 to

80% oftheducks became DHBV positive after transfection.

Purification of virus by CsCl equilibrium centrifugation.

CsCl(971mg)wasdissolved in a mixture of 0.4 ml of serum and 3.2 ml of water. This solution was centrifuged in an

SW60rotor(Beckman Instruments, Inc.) for 19 h at 60,000 rpm at 20°C. Fractions of 200 ,ul were collected from the

bottom andassayed for DHBV DNA by dot-blot analysis.

ADH

BV-3

E111411..

....I

SSO,

eo

zc,6

ja

I

W 69k

_. 46

BDHBV-16

ll.~~~~~~~~~~~

l l l<

cD

,~'e~lOt

_DI

14

RESULTS

Western blot analysis of core gene products in serum and liver. To test whether proteins similar to the HBeAg are produced during DHBV infection, serum samples ofducks

endogenouslyinfected withDHBV were subjected to

immu-noprecipitation and Western blot analysis by using a rabbit

antiserum raisedagainst bacterially synthesized DHBVcore

antigen (DHBcAg; J. Salfeld, Ph.D. thesis, University of

Heidelberg, 1985). Two proteins with

molecuiar

sizes of

about30 and 33kilodaltons(kDa) could be detected with this

antiserum only in DHBV-positive serum samples (Fig. 2,

lane 1). In somesera, two additional proteins with molecular

sizes of about 27 and 27.5 kDa were also present in small

quantities. Asis the case with HBV, theseproteins existin

a nonparticulate form, since the protein pattern remained

unchanged after preclearing ofthe serumby

ultracentrifuga-tion (data not shown). The soluble core proteins could be

easily detected in all serum samples from ducks infected

with wild-type DHBV (27 samples tested). In the liver,

multiplecoreproteins with molecular sizes ranging from32

to 46kDaweredetected(Fig.2,lane2).These proteinswere

located in particles, as could be shown by sedimentation

analysis (datanot shown). Adetailed analysis ofthe

struc-ture oftheliver-specificcore proteins will bethe subject of

aseparate report.

Serum Liver

6 kD

Amme~~~~~A

3i_

30

FIG. 2. Western blotanalysis ofcoregene-encoded proteins in

serumand liver.Samplesof 250,ulofserum(lane 1)and 1 ml of liver

homogenate (lane2)weresubjectedtoimmunoprecipitationwith the

anti-core serum as described in Materials and Methods, and the immunoprecipitates wereexamined forcoregene-encoded proteins

byWestern blotanalysis.

FIG. 3. Endo F digestion of the serum-specific core proteins. Serum samples from ducks (250 Ill) infected with either DHBV 3 or

DHBV 16 weresubjected toimmunoprecipitation with the anti-core

serum. The precipitated proteins were then analyzed by Western blotting either directly, after mock digestion, or after digestion with endoF. Inthe upper partthepotentialglycosylation sites within the

coregenes of therespective virus strains (amino acid positions4and 160 relative to the methionine encoded by the ATG at nucleotide 2647)areshown. MWM,Molecularweight marker.

The major core proteins in the serum are glycosylated.

Since the core

protein

ofDHBV 3, avirus strain which is

endemic in Germany(33), contains two potential glycosyl-ation sites (Fig. 3A), we tested whether the secretedcore geneproducts detected in serum were glycosylated, asisthe casewith many secretoryproteins.Forthis purpose,

immu-noprecipitates obtained with the anti-core serum from

DHBV-positive sera weresubjected to digestionwith endo

F, an enzyme which cleaves both high-mannose and

com-plex-type, N-linked carbohydrate side chains (9). Afterendo

F treatment, twoproteins withmolecular sizes of about27

and 27.5 kDa could be detected (Fig. 3A, lane 3). Mock

digestion had no influence on the protein pattern (lane 1

versus lane 2). Thus, these proteins, which were present

onlyin smallamountsinuntreatedsera, appeartorepresent

theprimary(i.e., unglycosylated) secretory core gene

prod-ucts which become modified during

biosynthesis by

the

attachment ofone or twocarbohydrate side chains.

To support this interpretation, we transfected

DHBV-negative ducklings with cloned DNA from DHBV 16, an

Americanisolate(19, 20)in

which,

dueto apoint mutation,

the internal glycosylation site is

missing (Fig. 3B),

and

thereforethe virusshouldproduce onlyamonoglycosylated

coregeneproduct.The results obtained withserafrom these

animalswith respecttothe secretedcoreproteinsareshown

in

Fig.

3B. As

expected,

the 33-kDa

protein

was

missing.

The coregeneproductswith molecular sizes of about 30 and 27 kDa werefound in about

equal

amounts,

indicating

that

theN-terminal

glycosylation

site isnotusedas

efficiently

as

theinternalsite. After endo Ftreatment,

only

protein

bands atabout 27 kDa could beobserved

(lane 3),

ashad been the case with the serum

samples

obtained from DHBV 3-infected ducks.

Takentogether,these resultsdemonstrate that the 33- and 30-kDacore

proteins

represent

doubly

and

singly

on November 10, 2019 by guest

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3704 SCHLICHT ET AL.

~:1

2

3

T

4

5

6

I'

F- C ,uG t'T1 (o

fV~c-r

'-4.

__~~~~e

FIG. 4. The secreted core proteins are C-terminally truncated. Samples of1mlof liverhomogenateor250,ul ofserumofaDHBV-3 infected duck were subjected to immunoprecipitation (IP) with antisera raised against either the whole core protein (aC) or a C-terminalpeptide (aCT). Theimmunoprecipitates werethen ana-lyzedbyWesternblotting (WB) with eithertheanti-coreserumor theanti-C terminusserum.

atedforms ofa secreted 27-kDacore

protein,

respectively.

In this paper the two glycosylated core gene products are termed gp33c and gp3Oc, whereas the unglycosylated, se-cretedforms arereferred to asp27c. By

analogy,

themajor

nonsecreted core proteins detected in aparticulate formin the liveraredesignated asp30c.

The major core gene products in serum are C-terminally

truncated. Since it has been shown for HBV that the secreted core gene product, the HBeAg, represents a

C-terminally truncated core protein, we raised an antiserum

againstachemically synthesized

peptide

comprising

thelast

13 C-terminal amino acids of the DHBV core

protein

and

examined whether the same was true for the respective

DHBVproteins. This C

terminus-specific

antiserum

precip-itatedthemajorcoreproteinspresentin liver(Fig. 4,lane5)

and also recognized these proteins after Western blotting

(lanes 5 and 6). However, no reaction with the

serum-specificcore proteinscould beobserved(lanes 1, 3,and 4).

These dataindicate thatgp33c,gp3Oc,andp27c all represent

C-terminally

truncated core proteins. Thus, in analogy to

HBV, these proteins will be referred to as DHBeAg. In

addition, thefact that undissociated cores could be

immu-noprecipitated fromliverhomogenatewith the anti-C

termi-nus serum shows that the C terminus ofthecapsid protein

must belocated at theoutside ofthecoreparticle.

Afunctional pre-C region is essential for thesynthesisand secretion of the processed core proteins. Since gp33c and

gp3Oc were sensitive to digestion with endo F, these two

proteins

have

been glycosylated during biosynthesis. It is

wellestablishedthat allproteinswhich becomeglycosylated

mustbe translocatedthrough themembraneof the

endoplas-mic reticulum, a process mediated by a signal sequence

which,in most cases,is located at the very N terminus of an

exportedprotein (30).

In the case of all hepatitis B virus core proteins, a

candidate for such a signal sequence is the hydrophobic

peptide which is encoded by the pre-C region. To test

whether expression of the pre-C region was essential for

DHBeAg secretion, we introduced a stop codon into this

region by site-directed mutagenesis with a cloned DHBV 16

genome (Fig. 5A). Since sequence elements important for viralreplicationarelocated in the pre-C region, even a single

base change within this region might interfere with the

B..

Celis

i

1 2 3

d4 A

.,

e.

1.

AIed

rum

4 5

k 1- N.I

+. Er, of") F

FIG. 5. Transient expression of the DHBV 16 wildtype and a

DHBV 16 pre-C mutant in a human cell line. (A) Schematic representation of the core gene 5' end. The eight pre-C codons preceding the core-ATG aregiven. The indicated pointmutation, whichresulted inan in-frame stopcodon within the pre-Cregion,

wasintroduced by site-directedmutagenesisofaDHBV16genome

cloned into M13mp8. (B) Comparison of the core gene products obtained after transient expression of the DHBV 16 wildtypeand the pre-C mutant in Hep-G2 cells. Cell lysates and tissue culture

supernatantswere subjectedtoimmunoprecipitation with the

anti-core serum asdescribed inMaterialsandMethods,andthe

respec-tive immunoprecipitates were analyzed by Western blotting. (C)

Western blotanalysisof the secreted coreproteinspresent in DHBV 16-positive duck serum(S), or in tissue culture medium(M) after transientexpression of DHBV 16wild-typeDNAinHep-G2cells. The samples weresubjected toimmunoprecipitationwith the anti-core serum, and the respective immunoprecipitateswereanalyzed by Westernblotting. Endo Fdigestion wasperformed where indi-cated. Lane 5 is a shorterexposure of lane 4.

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DHBV PRE-C FUNCTION 3705

infectivity oftheclonedDNA. Therefore,wefirsttested the

influence ofthepre-C stop mutationon DHBeAg secretion

in tissue culture. For these experiments the human

hepa-toma cell line Hep-G2 was employed, a cell line which has

been shown recently tobe competent for HBV production

and HBeAg secretion (6, 35).

Theresult of thisexperiment is showninFig. SB. Whereas

the intracellular core proteins were qualitatively and

quan-titatively unchanged after transient expression ofthe

wild-typeand thepre-C mutant(lanes 1 and 2), secretionproved

to be completely dependent on a functional pre-C region,

since after transient expression of the pre-C mutant no

DHBeAg could be detected in the medium (lane 4 versus

lane 3). In neither case could the DHBeAg be detected

intracellularly. Thus, the pre-C region appears to encode a

signal peptide which is essential for the formation and

secretion of theprocessedcoreproteinsbut has noinfluence

on the synthesis oftheintracellular forms.

The high

efficiency

by which the DHBV C gene was

expressed in the

Hep-G2

cells also enabled us to test

whether theprocessingstepswhich leadtothe synthesisand

secretion of modified core gene products are conserved

between human and duck cells. With the

exception

of a

slightly

different

glycosylation,

the

processed

core gene

products which were secreted by this cell line

during

tran-sient

expression

of DHBV DNA

(Fig.

5C, lane

2)

are

identical with those which were detected in the serum of

infected ducks (lane 1). In

particular,

the C-terminal

cleav-age appears to take place at the same sites within the core

protein, since after endo F

digestion

no difference between

thep27c derived from duckserum

(lane 3)

and the

p27c

from

the tissue culture supernatant

(lanes

4 and

5)

could be

observed.

A functional pre-C region is dispensable for virus forma-tion. To test the influence of the

pre-C

mutation on the

infectivity of the viral DNA, three

DHBV-negative

duck-lings were transfected with the mutated DHBV genome

by

intrahepatic

injection

of cloned DNA. After 3 weeks the

animals were sacrificed and tested for DHBV infection. As

shown

by

dot-blot

analysis

of liver

homogenate

and serum, twoofthe threeanimals had becomeDHBV

positive

atthis

time (data not

shown).

Toprove that the DHBV DNAdetectedin the serumwas

located in viral

particles,

serum

samples

were

subjected

to

CsCl

equilibrium

centrifugation,

and the

gradient

fractions

were examined for DHBV DNA

by

dot-blot

analysis.

The

virus-DNA

peak

wasdetected in thefractions

corresponding

to a

density

of 1.19 to 1.20

g/cm3,

the

density

of DHBV

particles (31;

Fig.

6A).

Wethen examined

whether,

aftertransfection of the

pre-C

mutant, viremia had

developed

in the absence of

DHBeAg

synthesis,

as should be

expected

from the tissue culture

data. Forthis purpose, serum and liver

samples

ofthe two

ducks which became DHBV

positive

aftertransfection with

the

pre-C

mutant, as well ascontrol

samples

takenfroman

animal transfected with

wild-type DNA,

were examined for

core gene

products

by

Western

blotting

(Fig. 6B).

Whereas the secretory core

proteins

could be

easily

detected in the serum of the control animal

(duck

C),

no

DHBeAg

was detectable in the sera of the two ducks which had been

transfected with the

pre-C

mutant

(ducks

Aand

B),

indicat-ing thatthe infection was due to the mutant. No

DHBeAg

was visible even after a

10-fold-longer

exposure

(data

not

shown).

No differences could be observed with

respect

to

the

liver-specific

core

proteins

aftertransfection of

wild-type

andmutant DHBV DNA.

AFraction

Density

Duck A

Duck B

-.9

8

1i

, t t t

? 3 1 :;:.

,

1 8

g/cmi93

* v

l*

BuSerum

B Duck A B C A vBeCC

-4t.ikD

mm-FIG. 6. (A) Serum

samples

oftwoducks which becameDHBV

positive

aftertransfection with the

pre-C

mutantwere

sUbjected

to

CsCl

equilibrium

centrifugation. Samples

of the

gradient

fractions then were tested for the presence of DHBV DNA

by

dot-blot

analysis

with

32P-labeled,

cloned DHBV DNA as a

probe.

The

densities of the

gradient

fractions which contained thevirusDNA

are

given.

(B) Examination of serumand liver

samples

ofducks,

eithertransfected with DHBV 16

wild-type

DNA

(duck

C),orwith aDHBV 16mutantwhich carriedanin-framestopcodon withinthe

pre-C region

(ducks Aand B), for DHBV core gene

products by

Westernblotting. Serum

samples

(150 ,ul)were

subjected

to

immu-noprecipitation

with the anti-coreserumbefore

analysis.

The liver

samples

(1mgof livertissue) were

assayed

directly.

To test whether the viral particles

produced

after

trans-fection withthe

pre-C

mutantwere

infectious,

weinoculated

three

DHBV-negative

ducklings

with serum obtainedfrom

the two ducks which became DHBV

positive

after

transfection withthe mutantDNA

(200

,ul

ofserum

injected

intrahepatically).

After3 weeks theanimals were

sacrificed

and tested for the presence of DHBV DNA in liver and

serum

by

dot-blot

analysis

as well as for the presence ofe

antigen

inthe serum

by

Western

blotting.

Aswasevidenced

by

the presenceofDHBV DNAinserumand

liver,

all three

animalshad becomeDHBV

positive

within

this

period,

but

again

no

DHBeAg

could be detected in the serum

(Fig.

7,

lanes

3, 4,

and

5).

The amount ofviral DNA in liver and serum was

compared

between

samples

taken from ducks

which became DHBV

positive

after transfection with the

pre-C

mutant DNA

(lanes

1 and

2),

after infection with

mutant virus

(lanes 3,

4,

and

5),

and aftertransfection with

wild-type

DNA

(lanes

6 and

7);

no

significant

differences

could be

observed,

irrespective

ofthe fact thattransfection

or infectionwith themutantledtoviremiain the absence of

any

e-antigen

synthesis.

In this context it should be

men-tioned that

endogenously

DHBV-infected ducks or ducks

which were

experimentally

infected withserum takenfrom

endogenously

infected animals showed serum titers which

were about 5to 10times

higher

thanthose shown in

Fig.

7,

whereas theamountsof viral DNA in the liverwere

compa-rable.

However,

since virus titers were

equally

low after

transfectionwith the mutantorthe wild

type,

thereappears

to be no correlation between

e-antigen synthesis

and virus

titer. That the virus titerwasalsolow afterinfectionwith the

VOL.61, 1987

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[image:5.612.324.545.71.284.2]

3706 SCHLICHT ET AL.

A

[,z1

fc!c:to T

ranrfst

M tjtinTFl Auitiu1t )Pv$

**,* * *

M

t

M

tci

t

_

C-JS.)HP1\/- DN A

if,H:-'

DN

DHV D tA;

r

n s rLJ m

DHF3BA. E S r

uru

rn

FIG. 7. Detectionof DHBV-DNA in liverhomogenate and

se-rumby dot-blotanalysis (A) and of DHBeAg inserumbyWestern

blotting (B). Liver tissue (100 mg)washomogenizedin1mlofTNE

buffer, and 1 ,ul ofhomogenate wasspotted onto anitrocellulose filter. Serum samples (1 ,ul) were spotted directly onto the filter. ViralDNA wasdetected by hybridization with32P-labeled, cloned DHBV DNA. For Western blot analysis, 250 ,ul of serum was

subjectedtoimmunoprecipitation with the anti-core serum, andthe

immunoprecipitates thus obtained were analyzed as described in Materials and Methods. Lanes: 1 and 2, samples from ducks transfected with thepre-CmutantDNA;3 to5, samples fromducks

infected withmutantvirusbyintrahepatic injection of 200 p.1serum

taken from the ducksAand B inFig. 6;6and 7,samples from ducks transfected with wild-type DNA.

During transient expression of DHBV DNA in the human

hepatomacell lineHep-G2,acell linewhich is competent for

HBV production, large amounts of DHBV core gene

prod-ucts(gp33C, gp3Oc, p27c, andp30c) were produced. Thus it

appears that the cellular factors which regulate C-gene

expression act on the DHBV genome as on the HBV genome and that the species specificity of the hepatitis B viruses might not be due tospecies-specific regulatory proteins. We are currently testing this possibility by examining whether infectious DHBV particles are producedby the Hep-G2 cells after transfection with DHBV DNA.

The application of this system to the analysis of the expression of the DHBV core gene showed a variety of

interesting results.

(i) The DHBV core gene products can pass through two differentbiosynthetic pathways. In addition tothe structural coreproteins, p3oC, whichweredetected inlarge quantities in theliver, considerableamountsofC-terminally truncated coreproteins weredetected in the serumofDHBV-infected ducksaswellasin the medium of cells transfected withviral DNA. Inanalogy to the HBeAg, a similar protein known to be present in the serum of HBV-infectedindividuals, these proteinswere termedDHBeAg.

We have shown here that the bulk of the DHBVeantigens are glycosylated (the HBV eantigen contains no glycosyl-ation site). This is animportant finding, since it proves that thepresence of theseproteinsin the serum is not due to cell lysis but ratherto active secretion. Thus, as demonstrated here for the duckvirus, hepatitis B viruscoregeneproducts can enter twodistinctly different biosynthetic pathways (Fig. 8). One product, p30C, is synthesized in the cytoplasm and aggregates to form coreparticles, whicharethenexportedas acomponent of the virusparticles. In fact this protein might also be modified, asis indicatedby the pronounced hetero-geneity of the different core gene products which were detected in the infected liver. Anotherproduct,thepre-C/C

mutantvirusmaybedue to the low virus titeroftheserum

that was used for the infection. The question of whether

thereare anydifferences between the mutant andthe

wild-type virus with respect to pathogenicity is currently the

subject ofa systematic study.

Insummary, the data presented above prove that

expres-sion ofthepre-Cregionis notnecessaryforthegeneration of

infectious virus.Furthermore, the data show that the

inabil-ity to synthesize e antigens can be transmitted as a stable

trait which ischaracteristic for the generated DHBV mutant

andthat thereisnostrongselectivepressure withrespect to areversion to the wild type.

DISCUSSION

Inthework described here we present a system which for

thefirsttime allowed thecombined analysis of a hepatitis B

virusmutant in tissue cultureand in vivo. This was possible

because of the unexpected finding that the DHBV C-gene promoter is activein thehuman hepatoma cell line Hep-G2. In several reports it has been described that the HBV core gene is only poorly expressed in most cell lines after

transfection with viral DNA, whereas the surface proteins

are usually expressed at a high level (2, 5, 7). Thus it has

been proposed that transcription of the C gene is strictly

regulated and that the respective regulatory elements may

be, atleast in part, responsible for the species specificity of

thehepatitis B viruses.

I

C-Gene

DGlycosylation

Core asse y 30C

Core assernbly

I

L ~ Ip27C Export

I :z:::::JIg9p30C

f ?

I I9~~P33c

Secretion

FIG. 8. Schematic representation of the two different biosyn-theticpathwayswhichare entered by thehepatitis B virus core gene productsas deducedfromthe experimental results presented in this report. The pre-C regionis shaded; the two different mRNAs are depicted as wavy lines with different thickness according to the different abundance of these mRNAs. Further details are given in

the text. ER, Endoplasmic reticulum.

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protein, is translocated into the lumen of the endoplasmic reticulum, modified by glycosylation, truncated by C-terminal and, most likely, N-C-terminal proteolytic processing, and eventually secreted.

(ii) The pre-C region encodes a signal sequence which is essential for the synthesis and secretion of theprocessed core gene products. As mentioned above, aprocessed core pro-tein, the HBeAg, can be detected in serum during HBV infection. The process by which this protein reaches the serum remained unknown for a long time. Very recently, evidence was obtained indicating that partial deletion of the pre-C region abolished HBeAg secretion from mammalian cells (21, 24, 28, 29). However, many aspects, such as whether expression of thepre-C region was also essential for the synthesis of a correctly processed HBeAg, remained a matter of dispute.

In this study, we have constructed a pre-C mutant by introducing a stop codon into this region by a single base exchange. The expression of this mutant in tissue culture and in vivo showed that noDHBeAg was produced, either in a secreted form or in an intracellular form, when the pre-C region was interrupted by a stop codon. Consequently it appears that afunctional pre-C region is essential not only forthe secretion but also for the synthesis of the correctly processed e antigens. Since the synthesis of thecytoplasmic core proteins in tissueculture and in vivo was not affected by the pre-C mutation, most likely two different proteins are synthesized, a pre-C/C protein which is processed and secreted and a C protein which remains primarily intracel-lular and isonlyexported as acomponent of a virus particle. Thus, the signal sequence which is encoded by the pre-C region is only used optionally.

To date, only a few proteins have been described which are alternatively synthesized with or without a signal se-quence.Among these are the gag protein of Moloney murine leukemia virus (26), theplasminogenactivator-inhibitor (12), and the SUC2 gene product of Saccharomyces cerevisiae (4). In the last case it has been shown that the secretory and cytoplasmic proteins are encoded by the same structural gene but originate from two mRNAs with different 5' ends controlled by two differentially regulated promoters. It seems very likely that,inthe caseof the hepatitis B viruses, a similarmechanism leads to thedifferential synthesis of the pre-C/C and C proteins (Fig. 8). If so, the regulatory mech-anism which enables the synthesis of two different mRNAs from the core gene is conserved between human and duck liver cells, since expression of the DHBV core gene in the human hepatoma cell line Hep-G2 leads to theproduction of the same proteins that are synthesized in the infected duck

liver.

Major RNA species with heterogenous 5' ends which could be translated eitherinto the C- or thepre-C/C protein have beendescribed for the woodchuck and ground squirrel hepatitis B viruses (10, 23). However, no pre-C mRNAs have yet been detected for DHBV and HBV in RNA preparations obtained frominfected livertissue (3, 42). This failure is most likely due to the fact that, for unknown reasons, theabundance of this mRNA species is much lower in HBV- and DHBV-infected tissue than in the case of woodchuck and ground squirrel hepatitis B viruses. How-ever, since the pre-C mRNA is not packagedinto viral cores, as occurs with the C mRNA (11), even a low amount of pre-C mRNA could direct the synthesis of large amounts of e antigens.

(iii) The process of e-antigen formation appears to be conserved between the human and theduckhepatitis Bvirus.

Sofar, theprocessofe-antigen formation has been studied

only for HBV. This report describes for the first time the

structureand the biosynthesisof the processed core proteins

ofoneoftheanimalhepatitisBviruses and thus allowsus to deduce principles which underly the process of core protein secretion.

Ithas been shownforHBeAg that 34 or 36amino acidsare cleaved from the C terminus of HBcAg during e-antigen

biosynthesis (36). Despite the fact that DHBcAg is much larger than HBcAg (30.3 versus 21 kDa), the C-terminal

portion of DHBcAg whichismissinginDHBeAg appearsto

be of the same size, since the molecular size difference between DHBcAg (30.3 kDa)and unglycosylated DHBeAg

(27kDa)isabout3 to 4kDa. This calculation is based on the

assumption that most of the pre-C sequence has been

removed during DHBeAg biosynthesis, as is the case with most signal sequences (30).

When the DHBcAg amino acid sequence in the vicinity of

the putative cleavage site is inspected, a sequence motif is

found which is highly homologous to the known HBeAg

cleavage site (HBV, T-T-V-V-R-R-R; DHBV,

T-T-V-V-Y-G-R-R-R). This homology is especially striking since the

overallhomology between HBV and DHBV in this region is very low, i.e., less than 20% (33). Since a similar sequence is also present within the same C-gene region in woodchuck and ground squirrel hepatitis B viruses (H-T-V-I-R-R-R), it

is likely that this sequence motif represents a processing signal which is recognized in liver cells in a

species-independentmanner.Together with a conserved mechanism

for the generation of the C and the pre-C/C protein, this would explain why the DHBV core proteins, which are synthesized by the Hep-G2 cells, are identical with those that can be detected in infected ducks. However, the possi-bility that the cleavage might not take place at a single site is suggestedbythe presence of two primary secreted core gene products with slightly different molecular sizes (27 and 27.5 kDa, respectively).

Taken together, all of the dataindicate that themechanism which leads to the generation and the secretion of the truncated core proteins is conserved between HBV and DHBV, the most distantly related members of the hepatitis B virus family. This suggests that e-antigen formation con-fers an advantage to these viruses and may have played a role in their natural history.

(iv) Virus productionisindependent ofpre-C expression. In previous reports suggesting that the pre-C region was essen-tial for e-antigen secretion, it had also been speculated that the expression of thepre-C/C protein might beimportantfor the budding process by targeting nascent core particles to the endoplasmic reticulum membrane (24, 40).

We havechallenged this view by expressing aviralmutant with a defective pre-C region in vivo. Transfection of ducks with this mutant resulted in viremia without concomitant synthesis of DHBeAg, asensitive marker for pre-C expres-sion. Although we cannotrigorously exclude thatrevertants tothewild type were produced at a very low

frequency,

the fact that comparable virus titers were obtained after transfection with the mutant orthe wild-type DNAstrongly argues against afunctional role of the pre-C/C proteinin the process of virus formation. This view is also strongly sup-ported by the fact that the inability to synthesize eantigens

is a stable trait, as could beshownby the transmission of the virus mutant.

The finding that production of infectious virus is not necessarily coupled with e-antigen synthesis is also of med-ical importance since, in HBV, the HBeAg is the most

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3708 SCHLICHT ET AL.

commonly used marker for determining the infectivity of

serumsamples (18, 41). InDHBV,outof27DHBV-positive

duck sera which were examined forDHBeAg by Western

blotting, none was DHBeAg negative. However, the fact

that this marker is not completely reliable has also been

suggestedforHBV,sincein afewcasesviral DNA hasbeen

detected in serawhich were negative forboth HBeAgand

anti-HBeAg (14, 16). It will be interestingto seewhetherat

leastsomeof thesecases aredue toaninfection withaHBV

pre-C mutant.

ACKNOWLEDGMENTS

We thankR.Pipkornforsynthesisof thepeptide,M.Nassaland

E. Lehmann for synthesis of the oligonucleotide, S. Selzer for providing Hep-G2 cells, P. Galle for help with the transfection experiments, C. Kuhn and E. Schlichtmann for artwork, and V. Boschforcritically readingthemanuscript.

This work was supported by the Deutsche Forschungsgemein-schaft (SFB 229), the Bundesministerium fur Forschung und

Technologie (BCT0381-5),and the Fond derChemischenIndustrie.

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