The hepatitis B virus enhancer modulates transcription of the hepatitis B virus surface antigen gene from an internal location.

0022-538X/88/041437-05$02.00/0

Copyright

©

1988, AmericanSociety for Microbiology

The Hepatitis B Virus Enhancer

Modulates Transcription of

the

Hepatitis B Virus Surface Antigen Gene from

an

Internal Location

GARY A. BULLAAND ALEEM SIDDIQUI*

Departmentof Microbiology and Immunology and Programin Molecular Biology, University of ColoradoSchool of

Medicine,

Denver,

Colorado 80262

Received 5 October 1987/Accepted 20December 1987

Wehave usedatransient expressionsystem toexamine the influence of the hepatitis B virusenhanceronthe transcription of the hepatitis B virus surface antigen (HBsAg) gene. This enhancer is located3' to HBsAg

codingsequencesbut is contained withinthematureHBsAgtranscripts. Removal of the enhancer resulted in

asignificant reduction in HBsAggeneexpression in liver cells. By usinganinvitro nuclearrun-on assay,this effectwasdeterminedtobeatthe levelof transcription.We havefurthernoted that HBsAg expressioncanbe restored by thereinsertion of theenhancersequencesinthe enhancerlessvector. However, this reconstitution

ofHBsAg expression appearstodependonlocationofthe enhancer with regardtothe HBsAgpromoters.

Hepatitis B virus (HBV) infection represents a major worldwide human health problem. This viruscauseschronic liver disease and has been linked to hepatocellular

carci-noma(15). Though research has been hindered by the lack of

a suitable model for HBV infection, significant advances have been made through analysis of the cloned virus (37). Four open reading frames have been identified from HBV sequence data andaredesignated S/pre-S, C/e,

pol,

and X.

Major promoter elements and RNA transcripts have been mapped for both the HBV surface antigen (HBsAg)gene(3,

25, 30, 31, 34) and the core/e gene (25, 26). Putative X

gene-specific transcripts have also been detected, and a

separate promoter element has been tentatively identified (30a, 39). In addition, an enhancerelement has been

identi-fied and partially characterized (12, 28, 38). However, there have been few studies involving the role of the enhancer element in HBVgene expression.

Enhancersareeucaryotic control elements which activate

transcription of adjacentgenes inamanner independent of

orientation and position. These control elements have been identified inanumberof viruses and mammaliangenes

(16,

27). The HBV enhancer element, likemanyotherenhancers, exhibits both host- and tissue-specific activity (12, 28). Studies inourlaboratorysuggestaninvolvement of the HBV enhancer incore/e and Xgeneexpression (26; Siddiquietal., in press). The effect of the enhancer on HBsAg gene

expression, however, has notbeendetermined.

The gene encoding HBsAg contains three conserved in-frame translational start codons whichareused to produce HBsAg polypeptides of various lengths, designated

pre-Sl,

pre-S2, and S (Fig. 1). These proteins share common car-boxyltermini. Transcription of the HBsAggeneappears to

be controlledbytwodiscrete promoter elements(23, 30, 34). Thepromoterregion upstreamofthe

pre-Sl

sequence(the

pre-Sl

promoter) contains the canonical TATA sequence

and controls theexpressionof thelarge HBsAg polypeptide (p39/gp42). The secondpromoterelement(pre-S2 promoter) located within the

pre-Sl

region shares homology with the simianvirus40

(SV40)

latepromoterregion (3)and controls expression of pre-S2 (p33/gp36) and major S (p24/gp27) polypeptides.Themajorityoftranscriptsfromthisgenemap tothree sites clustered around thepre-S2translational start

* Correspondingauthor.

site, with barely detectable levels of transcripts originating from the pre-Sl promoter region (23, 30). The HBV en-hancer element has beenmappedtobetween nucleotides (nt) 1073 and 1235(28). This places the enhancer approximately 200base pairs (bp) 3' tothe HBsAg coding sequences but within the HBsAg transcription unit (Fig. 1). In this study,

weshowed that the HBV enhancerservestoelevate expres-sion of the HBsAggeneincells of liver origin.

Figure 1 shows the recombinant plasmidconstructs used in this study. Plasmid pML18 contains the 2.8-kilobase (kb) BglII restriction fragment of HBV, including the entire HBsAg geneplus flanking sequences. Transientexpression

of the HBsAg gene (pML18) in a variety of cell lines

produced levels of HBsAg that were virtually undetectable (Table 1). However, in the HepG2 human hepatoma cell line (17), the levels of HBsAg expressionwereverypronounced

(Table 1). We estimate that levels of HBsAg in the HepG2 line were approximately 100-fold higher than levels in the other cell lines tested. These differentlevels of expression

arenotlikelytobe duetotheefficiency of transfection, since uptake of other plasmid vectors appears to be efficient in these cell lines(datanotshown). Although replication of the plasmidvectorin the HepG2 cells could accountfor these results, analysis of extrachromosomal DNA from

trans-fected cells indicated thatreplication of pML18 didnotoccur

(S. Jameel, unpublished results).Thispromptedusto inves-tigate the roles of HBVsequencesthatwereresponsiblefor

thisapparenttissue-specific expression of the HBsAggene.

Inaprevious study,wefound that bothHBsAgpromoter regions

(pre-Sl

and

pre-S2)

of theHBsAggenelack host-or tissue-specific activity when linkedto the chloramphenicol acetyltransferasegene(30).Recent identification of the HBV enhancerelement and its tissuespecificity (12, 28) prompted us to examine the role of this enhancer in HBsAg gene

expression. It is noteworthy that this enhancer element is

locatedinthe 3'untranslatedregionof theHBsAggene(Fig. 1).

To address the question of enhancer involvement in

HBsAg expression, we transfected HepG2 cells with

en-hancer deletion plasmidspMLAE and pMLAE344 (Fig. 1). Figure 2A compares levels of HBsAg RNA produced in

HepG2 cells transfectedwith native (pML18)or

enhancer-less (pMLAE) HBsAg plasmids. A 32P-labeled antisense

riboprobe (21) wasused to detect HBsAggenetranscripts.

1437

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TATA P. Int.P.

TATA pre-S EN

pM

118

jy Bg

!

V

.

p

Ap Ba I )(b NpCSPHP,

I Ic

89

TATAAA,

I,

pMI.AE

pM1,AE334

pENAP

pENBS

pENAE

1

i^< ~~~~~~~~-273

--273

-.as

-2

i _.-273z

FIG. 1. Construction of recombinantplasmids. The 2.8-kb BgIII restriction fragment of HBVwasinserted into theBamHI site ofpML (19) toproduce plasmid pML18.PlasmidspMLAE and pMLAE344areenhancer deletionplasmids derived from pML18 bydeletingthe273-bp HpaI-SphI (nt 964 to 1235) or 344-bp HpaI-HpaII (nt 964 to 1310) restriction fragment, respectively. Enhancer reconstitution plasmids pENAP andpENBS contain the344-bp HpaI-HpaII enhancerregion inserted into the ApaI (nt 2603)orBstEII (nt2824) site ofpMLAE. PlasmidpENAE contains the 273-bp enhancer fragment inserted 257bp downstream of thepolyadenylation signalof theHBsAg gene. In each case, enhancerreconstitution plasmids contain the enhancer with the putative Xgene promoter directed away from the HBsAg gene transcription unit. Open boxes represent the openreading frames of the HBV genome. Allconstructsweremadebyusingstandard methods (20).Pre-S, S, C, X, andPrefer to the openreading frames ofHBV.Thelocations of the HBsAg genepre-Sl promoter (TATA P.), internal promoter(Int.P.), and polyadenylationsignal areindicated. Thearrow indicates the polarity of the enhancer fragments in relationto the HBsAg gene. Abbreviations: Ap,ApaI; Bg, BglII; Bs, BstEII; Hp, HpaI; HpII, HpaII; RI, EcoRI; Sp, SphI; Xb,XbaI; Ac, AccI; EN, enhancer.

Hybridization signals

were

quantitated by measuring

peak areas

from densitometer

tracings of

autoradiographs

and

normalized

to

signals obtained by probing

a

replicate

filter

with

a

rabbit

P-globin

gene probe

(data

not shown) which was used as an

internal

control in the

transfections.

An

approximately

sixfold

decrease in the levels of

HI3sAg

gene

steady-state

RNA was

observed when

pMLAE

was used

(Fig. 2A). This decrease of HBsAg steady-state

RNA

levels

correlates

well with

HBsAg

protein

data obtainedfromthese

transfections (Fig.

2A).

We found

that the extended

en-hancer

region deletion plasmid (pMLAE344) produced

HB-sAg levels

similar

to

those of

pMLAE

(data

not

shown).

We

next

characterized

the

HBsAg

gene

transcripts from

HepG2 cells transfected with either pML18

or pMLAE. Northern

(RNA) blot

analysis of poly(A)+ purified

RNA

(Fig. 3A) revealed

a

2.1-kb HBsAg transcript from

pML18-transfected cells

and a

smaller transcript

from

pMLAE-transfected

cells,

the latter

being consistent with

the extent

of

the enhancer

deletion.

The

0.8-kb band (Fig.

3A, lane 1) is

probably

an X

gene-related transcript

(30). To determine whether enhancer removal altered the specificity of

tran-script

initiation,

Si

nuclease mapping

was carried out(Fig. 3B). The

transcriptional

initiation sites for both native (30)

TABLE 1. Transient expression of HBsAg gene in variouscelllinesa

Cell line Source HBsAgS/Nb

APB Ratfibroblast 1.3

3T3 Mouse fibroblast 1.1

HeLa Human cervical carcinoma 1.2

T47D Human breast carcinoma 1.2

MCF-7 Human breast carcinoma 1.1

HepG2 Human hepatoma 33.3

a Cellsweretransfected with20 ,ug ofplasmidpML18byusing theCaPO4 procedure (9), and culturesupernatantswereassayed forHBsAg48 h laterby theAusriaIIradioimmunoassay(AbbottLaboratories).

bS/N, Ratio ofcounts per minute of sample to counts per minute of negativecontrol serum, asdeterminedbyradioimmunoassay.

A.

[HBsAg]

ng/mi MLIB 16.5

RN A

em,

MLAE 4.0 ENAP 2.1

ENBS 145 V

ENhE 105 _

B.

SHIS

3-4

4U_

[image:2.612.64.551.68.225.2]

SHAE 2.0

am

FIG. 2. HBsAg gene expression from recombinant plasmids in HepG2 and COS cells. Unconcentrated cell supernatants were

assayed by acommercially available radioimmunoassay (Abbott), and levels were determined from a standard curve. For mRNA quantitation, 10 F±g of total cytoplasmic RNA isolated from

trans-fected cellsby the method of Chirgwin etal. (4)wasdenaturedat

65°Cfor 10min in 6.15 M formaldehyde-lOx SSC (1x SSC is1.5 M

NaClplus 0.15 M sodium citrate) and immobilized onto nitrocellu-lose membranes (14) by usingaslot blotapparatus (Schleicher& Schuell, Inc.). BoundRNAwasprobed with a32P-labeled antisense RNA generated from a 744-bp HincIl (nt 222 to 966) restriction fragment. This probe includes HBsAg gene coding sequences but

excludes the enhancer region. As an internal transfection control, a duplicate filterwasusedto hybridize with a

P-galactosidase

nick-translated probe. Filters were exposed to XAR5 film (Eastman

KodakCo.). Relativelevels of HBsAg expression were determined by densitometer scanning (Quickscan R & D) of the autoradio-graphs, and signals were normalized to

P-galactosidase

signals. HBsAgproteinandRNA levelsfrom transfected HepG2 cells (A)

andfrom COS cells transfected with pSH18 and pSHAE (B) were

used.

I

2.A

I

I

I I

I

I I

I Ii

i

I

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A.

pML18t

I1

.2.IS pMLAE344t t 5l7--T---I---[( 344)-- -- --

----BgAp ~~~BgSi

lgI tprobes

= =SV40polyA

ML18t

Sense a-Sense

I v

,1

U:

'....

MLfE344t

Sense a-Sense

FIG. 3. RNA analyses of pML18- and pMLAE-directed

tran-scriptsinHepG2cells. Poly(A)+ RNAwaspurified by chromatog-raphy through an oligo(dT)-cellulose column (1). For Northern

analysis(36), 5 ,ug ofpoly(A)' RNAwasfractionatedon a

formal-dehyde-agarose gel. RNA was transferred to nitrocellulose and

probed with 32P-labeled HBV DNA. ForS1nuclease analysis (41), 20,ugof totalcytoplasmicRNAwasincubated witha5'-end-labeled BglII-XbaI restriction fragment(nt2432to250)(30). Hybridswere

digestedwith Si nuclease and analyzed on an 8%

urea-polyacryl-amidegel. (A) Northern (RNA) blot analysis.Lane 1, pML18 RNA; lane2, pMLA&ERNA.Molecular sizesareindicated in kilobases.(B) Si nuclease analysis.A20-,ug sample of cytoplasmicRNAwasused

for each lane. Lane 1, pML18 RNA; lane 2, pMLAE RNA.

Molecular size of protected fragments is indicated inbasepairs. and enhancerless HBsAg genes werefoundto be identical. These results indicate that thedeletion of the enhancer does

not influence the specificity of initiation of HBsAg gene transcription.

Theobserved decrease in HBsAg expressionas aresult of enhancer deletion could be due to either a decrease in transcription rate of the HBsAg gene or a post transcrip-tional event (e.g., altered mRNA transport or stability).

Because analysis of steady-state mRNA cannotdistinguish between theseprocesses,weusedtwoindependent methods

to account for the enhancer deletion effect. Our first

ap-proachwas to useacell line in which measurable levels of HBsAg synthesis are produced but in which the HBV

enhancer has been foundto be relatively inactive. For this

purpose, we used plasmids pSH18 (29) and enhancerless pSHAEin which the native orenhancerless HBsAggene is clonedinto theplasmidvectorpSVO10. pSVO10contains the SV40origin ofreplication and willreplicate toa high copy

number whenintroduced into COS cells(18).Transfection of COS cells with pSH18 has been previously shown to

pro-duce detectable levels ofHBsAg (29).Levels ofHBsAggene

mRNA and HBsAg proteinfrom both pSH18- and pSHAE-transfected COS cells wereapproximately equal (Fig. 2B). Althoughdifferential RNAprocessingandstabilitybetween the COS andHepG2cellscannotbe ruledout,these results

suggest that enhancerdeletion does notsubstantially affect

HBsAg expressionin the nonliver cell line.

Wenextcarriedoutnucleartranscriptionrun-onassaysto

determine if the observedeffect of enhancer deletionwasat

the level oftranscription. For these experiments, the

liver-derived Huh-7 (22) andHepG2 cell lines were used. While

both cell linesgavesimilarresults, the results obtainedwith

the Huh-7 cell line arepresentedhere. Nucleiwereisolated

Vector

*

Gal

Act

.,z. .Alb

;;)i: SP65

C

a-Amant.

HBV pBR

FIG. 4. Effect ofenhanceron nuclear transcription ofHBsAg

gene. Nuclear run-on assays weredone by amodification of the

method ofClaytonetal.(5).(A) Huh-7cellsweretransfectedwith

pML18torpMLAE344t,which contain theHBsAggeneconstructs

flankedby SV40 polyadenylationsequences. PlasmidpCH110 (11),

encoding the 0-galactosidase gene, was used as a transfection

control. At 48 hposttransfection, nucleiwereisolated and used in

the nuclearrun-onprocedure (5). (B)Effectof enhancer deletionon

HBsAggenetranscription. Run-on-generatedRNA from transfected cellswashybridizedtoeithersingle-stranded HBsAg gene-specific

DNA fragments (fragments I and U indicated in panel A) or

double-stranded restrictionfragmentsof controlgenesandexposed

tofilm. Abbreviations: Act, Actin; Bg, BgIII; Gal,

0-galactosidase;

S1,Sall; Alb, albumin; SP65, plasmidDNAcontrol; Vector,

single-strandedpTZ18RDNA(Pharmacia, Inc.). (C) Transcriptionrun-on

ofpML18-transfected cells in the presenceorabsence of 3 ,ugof

a-amanitinperml. Each laneorslot contains 3to5 ,ugof DNA.

and incubated in thepresenceof

[IP]rUTP

to allow elonga-tion of RNAtranscripts by previouslybound RNA polymer-aseIImolecules. Extracted32P-labeled RNAwashybridized

tosingle-strandedHBVfragmentsand double-stranded

con-trol genes immobilized on nitrocellulose filters. Relative

HBsAg gene transcription was normalized to signals

ob-tained with both 3-galactosidaseand actin DNAprobes.The

,B-galactosidase signal serves as a control for transfection

efficiency. This method allows foranestimationof the RNA

A.

NORTHERN

B.

1 2

Si

1 2

4probe

B.

.4c

235-270

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polymerase loading on

a gene (10) and

therefore indicates

whether an enhancer affects the initiation

of

transcription.

Initial experiments

indicated

that plasmid vector pBR322

sequences

were being

efficiently

transcribed,

making HBV

transcription

results difficult to interpret. To

limit

transcrip-tion from possible cryptic

pBR322

promoters

interfering

with HBsAg gene

transcriptional

analyses, we

flanked

the

native (pML18)

and enhancerless (pMLAE344)

HBsAg

genes with the

SV40 polyadenylation

sequences

contained in

a

BamHI-BclI fragment

(Fig.4A). Run-on analyses

of nuclei

from Huh-7 cells transfected with these HBsAg gene

plas-mids showed readily

detectable

HBV-

and

pBR322-specific

RNA being produced. In addition,

levels of

transcription of

both HBV and plasmid sequences appear to

be increased

by

the presence of the linked

HBV

enhancer.

Transcription of

vector

sequences

does not appear to influence

the signal

obtained

from the

internal S

gene sequences

(designated I),

however,

since the upstream S gene sequences

(designated

U) are

transcribed

only to a

small

degree.

Therefore,

the

transcriptional

activity

detected for the HBsAg

gene appears to be initiated within the HBV sequences. As an

internal

control,

signals

detected

by

using

the

P-galactosidase-co-transfected

control gene were

approximately equal. This

indicates

that the observed increase in HBsAg gene

tran-scription

is enhancer

dependent.

Densitometry of

autoradiographs

and direct

counting of

filters show an

approximate

fivefold increase in sense-strand

HBsAg transcription

in the presence of the enhancer

(Fig.

4B).

This

correlates well

with both steady-state

and

protein

data described

previously. Transcription

occurring from the

pre-Sl promoter

was near the background level

(Fig.

4B, U

fragment).

We also noted a significant level

of antisense

HBsAg

gene RNA being produced by the HBsAg gene. The

significance

of this

antisense

RNA

production

is

not

known.

It

is

unlikely

that this represents

transcription

originating from

vector

sequences,

since the SV40

polyadenylation

signal is located

3' to the HBsAg gene

fragment.

It has been suggested that the antisense message from this

region is produced in

murine

cells containing

multiple

HBV

integrates

(42). In

addition,

an RNA

polymerase

III

transcript

derived from the opposite strand has been previously reported from the 3' region of the HBsAg

gene

region (33).

As afinal control, the run-on analysis was carried out in the presence of 3

p,g

of

cx-amanitin

per ml. Results indicate that

transcription

of both HBsAg gene and plasmid se-quences was reduced by

approximately

80%, indicating that the majority of

transcription

isdirected by RNA

polymerase

II molecules (Fig.

4C).

We therefore conclude that the majority of

enhancement

ofHBsAg gene

expression

by the HBV enhancer is at the level of

transcription.

If the enhancer region is

functionally

able to

increase

transcription

of the HBsAg gene,

placement

of these se-quences outside of the

enhancer-deletion

HBsAg transcrip-tion unit should recover expression. To test this, we used plasmids with the enhancer region inserted 5' or 3' to the HBsAg gene in

pMLAE

(Fig. 1). Insertion of the

enhancer

element

(HpaI-HpaII)

5' to the

pre-Sl

promoter

element

(plasmid pENAP) resulted in no increase in HBsAg gene

expression

(Fig. 2).

However,

insertion of the

enhancer

between the

pre-Sl

promoter and the

pre-S2

promoter at nt 2824 resulted in levels of HBsAg expression similar to levels

produced

with the native HBsAg gene (Fig. 2).

Enhancer

insertion 3' to the HBsAg gene (pENAE) led to a similar increase in HBsAg

expression.

These results indicate that

enhancer-mediated activation

of HBsAg gene expression

can be

restored by insertion

of the

enhancer

element

but is

partially

location

specific.

The

inability of the enhancer insertion

5' tothe

pre-Sl

promoter

to reconstitute

expression

is not

entirely unexpected

be-cause

of

the apparent

negative regulatory

constraints

placed

on

the

pre-Sl

promoter

during

normal

HBsAg

expression

(3,

23, 30).

These constraints

may be

interfering

with direct

activation of the transcription

control elements. Preferential actionof enhancers on proximal rather than distal promoters (6, 13, 40) may

also

contributeto this effect.

In

this study,

we

have

addressed the question of the involvement

of the

HBV

enhancer

element in expression of the HBsAg gene. Using a transient expression system, we demonstrated that

(i) the

HBVenhancer functions to elevate HBsAg gene

expression

atthe levelof transcription, (ii) the

deletion of

enhancer sequences neither alters the specificity

of

initiation of stable HBsAg transcripts

nor

appreciably

affects mRNA stability, and (iii) enhancer reconstitution restores theoriginal level of HBsAg expression.

The HBV enhancer is strategically located within the 3' untranslated

region of the

HBsAg gene and 5' to the X and

core/e

genes as well as within the coding region of the

polymerase

gene. It isthe

only

enhancer, to our

knowledge,

whose sequences are partof a mature transcript, in this case the HBsAg geneand

polymerase

gene

transcripts.

Theonly other

example of

an

internal

enhanceris the

immunoglobulin

enhancer. However, with the

immunoglobulin enhancer,

the enhancersequences are

subsequently spliced

out anddo not appear in the stable

transcript (2,

8, 24).

The HBV enhancer has been suggested as dramatically increasing expression from the HBV core promoter as well as from a heterologous promoter gene in the human hepa-toma cell line

PLC/PRF/5

(28).

However,

we have consis-tently found the HBV enhancer to

increase

expression

ata more modest level (8- to 10-fold) both in the context of

homologous

(core and HBsAg) and

heterologous (SV40)

promoters fused to a reporterchloramphenicol acetyltrans-ferase gene (12; unpublished results). That the enhancer alone cannot account for the large difference between HB-sAg gene expression in liver versus nonliver cell lines suggeststhe involvement of multifactorial mechanisms in the

regulation

of this gene.

However,

theenhancer may

contrib-ute to the abundant synthesis of HBsAg

during

HBV infec-tion. Similarly, theenhancer

probably

also

plays

asignificant role in the generation of pregenomic RNA, thus regulating replication of the viral genome.

If the major role of the HBV enhancer is to potentiate

transcription

of HBV

transcription

units located 3' to the enhancer (core, X, andpot), then the activation of HBsAg gene expression from this strategic

location

of the HBV enhancer 3' totheHBsAg open reading frame is perhaps due to the constraints of the small size (3.2 kb) of the HBV genome. Also, the spatial

arrangement

of this enhancer between HBsAg and

X/core promoters

may ensure proper

proportions

of

pre-Sl,

pre-S2, and major S polypeptides. The apparent liver

cell-specific

regulation of HBV gene expression by this enhancer further suggests a pivotal role of the enhancer in overall viral gene

regulation

during HBV infection. Such a role for an enhancer in viral pathogenesis has been described with other viruses (7).

Thisinvestigationwassupported by Public Health Service grants A121182 and CA33135 from the National Institutes of Health and by agrant from the MilheimFoundation forCancer Research to A.S. We thank Scott Stockert for technical help and J.E. Mapoles for

criticalreading of this manuscript.

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