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Vol. 66, No.2 JOURNALOFVIROLOGY, Feb. 1992, p. 1223-1227

0022-538X/92/021223-05$02.00/0

Copyright © 1992,American SocietyforMicrobiology

Hepatitis

B

Virus

X

Protein

Is Not

Central

to

the Viral Life Cycle

In

Vitro

HUBERT E. BLUM,t* ZHEN-SHENG ZHANG, EITHAN GALUN, FRITZ VON WEIZSACKER, BILLGARNER, T. JAKE LIANG, AND JACK R. WANDS

MolecularHepatology Laboratory, MGH Cancer Center, Massachusetts General Hospital, HarvardMedical School, Charlestown, Massachusetts02129

Received 18September1991/Accepted11November 1991

Thehepatitis B x (HBx) gene is the smallest open reading frame of the hepatitis B virus (HBV) genome. It is conserved among all mammalian hepadnavirusesand is expressed during viral infection. While the HBx protein (pX) has been shown totrans-activatethetranscriptionof awide range of viral and cellular genes and to induce livercancer in transgenic mice, thesignificance of pX forthe life cycle of HBV itself has not been elucidated. To assess thefunction of pX in viral replication and virion export, we designed an X-minus mutant byintroduction of a stop codon at the beginning of the HBx gene withoutaffectingthe viral polymerase gene product. Transient transfection analyses usingdifferentcell linesrevealed that this X-minus mutant directs the synthesis of wild-type levels of viral proteins, replicative intermediates,and virionexport. These data suggest that theexpression of the highly conserved HBx gene is not central for the life cycle of HBV in vitro but may beinvolved in thepathogenicity ofhepadnavirusinfection, including liver cancer development.

Human hepatitis B virus (HBV) belongs to a group of hepatotropic DNA viruses (hepadnaviruses) that includes thehepatitis viruses ofthewoodchuck, ground squirrel, tree squirrel, Pekin duck,and heron. Thehepadnavirusgenomes have similar sizes and structures and replicate via reverse transcription ofanRNAintermediate,areplication strategy characteristic for RNA-containing retroviruses (25). Three of the four open reading frames code for proteins with essential functions for the viral life cycle, i.e., DNA poly-merase-reverse transcriptase, hepatitis B surface antigen (HBsAg), and hepatitis B core/e antigen (HBc/eAg). The fourth open reading frame, termedthe hepatitis B x (HBx) gene, codesfora17-kDaprotein which is expressed during the natural course of HBV infection (8, 26) and which appears tobea novelprotein kinase(27). TheHBxprotein (pX) has been showntotrans-activatethetranscription ofa widerangeof viral and cellular genes (2, 5, 9, 22-24, 29, 30) and to induce liver cancer in transgenic mice (11). While

most

previous

studiesanalyzed the trans-activatingpotential

of pX

by

cotransfection of pX expression vectors with differenttargetconstructs,theendogenousfunction ofpXin the lifecycle ofHBVitself has notbeenelucidated.

To assessthecontribution ofpX to viral geneexpression,

replication,

and virion export in vitro, we designed an

X-minus mutantby site-directedmutagenesis ofawild-type HBV genome via introduction of a stop codon at the

beginning

ofthe HBx gene ina

replication-competent

HBV

construct. Site-directed

mutagenesis

was carried out

by

polymerase chain reaction (PCR)

amplification

ofthe

repli-cation-competent,

incomplete

HBV DNA dimer adwR9

(3)

with a

primer spanning

the genome

region

consisting

of

nucleotides (nt) 821 to 842(EcoRIsite startposition 1)anda primer spanningtheregion

consisting

ofnt1415 to1388 and

carrying

aC-to-T mutationat nt1397. This mutation results

inastopcodon mutationin the HBx gene(codon 8)without

* Correspondingauthor.

tPresentaddress: DepartmentofMedicine, University

Hospital

Zurich, Raemistrasse 100, CH-8091 Zurich,Switzerland.

affecting the polymerase gene product (Fig. 1). The PCR productwasgelpurifiedand cloned into pCR 1000 (Invitro-gen, San Diego, Calif.). The EcoRV-BamHI fragment was isolated to replace thecorresponding fragment in the repli-cation-competent, incompleteHBVDNAdimer adwR9 (nt 1044 to 1403),yieldingthe mutant clone HBX-21. Fromthis clone a complete head-to-tail dimer (HTD), designated HBX-21HTD,wasconstructed in theEcoRI site of pGEM-7Zf(+) (Promega, Madison, Wis.). The functional compe-tence of wild-type and mutant clones was assessed by transfection ofhuman hepatoma cell lines and primary rat hepatocytes followed by detection and characterization of viral proteins, RNA, and replicating DNA species in cell

lysates

and culturemedia (3).

Thesynthesis andexportoftheviral proteinsHBsAgand HBc/eAg intotheculture mediumwasfirst assessed with the humanhepatoma cell lineHuH-7(16).Thecellsweregrown to nearconfluence inEagle minimal essential medium sup-plemented with

10%

fetal bovine serum. Cells were trans-fectedasdescribed by Chen andOkayama (4);20 ,ugofDNA wasroutinely usedper100-mm-diameter plate.Analiquot of culture mediumwasremoved

daily

and

analyzed

for

HBsAg

by a radioimmunoassay (Centocor, Malvern, Pa.) and for

HBc/eAg

by an enzyme immunoassay

(Abbott,

North

Chi-cago,

Ill.).

As shown in

Fig.

2

(left panel),

mutant HBX-21 directsthe

synthesis

of bothHBsAg and

HBc/eAg

atlevels similarto those of

wild-type

adw R9.

For the

analysis

ofviral RNAandDNA,HuH-7cellswere

grown andtransfectedas described above. RNAandDNA

analyses

were

performed

asdescribed earlier

(3).

For

North-ern (RNA) blot

analysis,

total cellular RNA was isolated from HuH-7 cells 2 days after transfection with either wild-type(adwR9) or mutant(HBX-21)HBV DNA.

Hybrid-ization was carried out with recombinant

full-length

HBV DNA labeled to

high

specific

activity (2

x

108

to 4 x 108 cpm/,lg) with a

multiprime

labeling

system

(Amersham,

Arlington

Heights,

Ill.).

For Southern blot

analysis,

particle-associated viral DNA was isolated as

previously

described (3)from celllysate ofone 100-mm-diameter

plate

and from 10mlof culturemedium5

days

aftertransfection with either

1223

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(2)

1224 NOTES

A. B.

am

m

12

~~~~~~~~~~~~~~~~

A;d.'^

X W 76-,

f~~~~~~~~~~~~~v7z~

CL ME

X 2 1 2

Kb 36

-2 1 /-2.4- tI"

PCR PRODUCTS

Culture Medium

odwR9 '-'

1,

_ ,,

FIG. 1. (Upper panel) Map of the hepadnavirus polymerase (P-ORF) and X (X-ORF)openreadingframes and DNAsequences

of HBVwild-type(WT)andmutant(MT) X and Pgenes.AA,amino acid. (Lower panel) DNA sequences of HBV wild-type (adw R9) and X-minus mutant (HBX-21) clones and of PCR amplification products of viralDNAisolatedfrom culture medium after transfec-tion of the human hepatoma cellline HuH-7 with either wild-type (adw R9)orX-minus(HBX-21) mutantDNA.

wild-type (adw R9)ormutant(HBX-21) HBV DNA. Hybrid-ization was carried out as described above. Northern blot

analysis of total RNA (Fig. 3A) identified virtually identical levels of the twomajor transcripts of 2.1 or 2.4 kb (subge-nomic) and 3.6 kb (prege(subge-nomic) in mutant- and wild-type-transfected cells. Southern blot analysis (Fig. 3B) revealed that the replicating viral DNA species in cell lysate and

HuH-7 cells

FIG. 3. ViraltranscriptsandreplicatingDNAspeciesafter

trans-fection with either wild-type (lanes 1) or mutant (lanes 2) HBV

DNA.(A) Northern blotanalysis of total cellularRNA(10 ,ug per lane).Theautoradiographicexposuretimewas2daysat-80°C. (B) Southern blotanalysisof viral DNAisolated from celllysate (CL) (equivalent to cells fromone 100-mm-diameter plate) and culture

medium (ME) (equivalent to medium from one 100-mm-diameter

plate). The autoradiographicexposuretimewas1dayat-80°C;the

markerwas5pgof cloned HBV DNA.

culture medium were qualitatively and quantitatively

indis-tinguishableaftertransfectionwith mutantHBX-21or wild-typeadw R9HBV DNA.Identical results(not shown)were

obtained after transfection of HuH-7 cells with a complete

HTD of wild-type HBV DNA (adw HTD) or a complete

HTD of the X-minus mutantHBV DNA(HBX-21 HTD). Tofurther characterize the viralantigens synthesizedand toanalyze thecompetenceofHBX-21todirect the export of

Primary Rat Hepatocytes

HMsAg hs&Ag

SQOOD-iodwR9 -dw R9I MHBX-24 M~~~~~~~iOoHBX-24

BoO, -

ll~~~5=

oX l

-~~~~~~~~~~~SO

123 4 5 ~~~~~~~ ~ 2 3 4 5

DAYSPosr rRANsFrTooN DaYS Posr rRAAsFECr/Oa

HBcleAg H6c/eAg

FIG. 2. Viral proteins in culture medium after transfection with either wild-type (adw R9) or mutant (HBX-21) HBV DNA of HuH-7 human hepatoma cells and primary rat hepatocytes. The graphs indicate detection of HBsAg (HuH-7 cells, 1:5 dilution; primary rat

hepatocytes, undiluted) and HBc/eAg(HuH-7cells, 1:10dilution;primary rathepatocytes, undiluted).

CLON ES

.II

"..,VPq 1:...X

Kbp 3.2

-;iT:. :,

IRR ,.::

:4:.f:;,

040

f.;,.

J. VIROL.

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(3)

NOTES 1225

0-0

I-

ci-0-0

1-.. >.-1 * c

'_

0T

FRACTION

:... im

....m

__ :!

~44

I'l

o.

I.-,

.j :;!

[image:3.612.136.478.67.294.2]

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

FIG. 4. Export of virions from HuH-7cells into culture medium. Cesium chloride equilibrium gradientanalysis of viral proteins and

replicative DNA intermediates from culture medium (equivalent to one 100-mm-diameter plate) was performed after transfection with

wild-type (adwR9)ormutant(HBX-21) HBV DNA.(Upperpanel)Densityand HBsAg andHBc/eAgcontentof gradient fractions. (Lower

panel) Southern blotanalysis of viralDNA extractedfrom gradient fractions 14and15 (lanes 1), 16and 17 (lanes 2), 18 and 19 (lanes 3), 20

and 21(lanes4), 22and23(lanes5), 24and 25 (lanes 6), 26 and 27 (lanes7),28 and 29 (lanes 8) and 30 and 31 (lanes 9). The autoradiographic

exposuretimewas 1dayat -80°C; the markerwas20pgofcloned HBV DNA.

virions, culture media from adw R9- and

HBX-21-trans-fected cells were subjected to cesium chloride equilibrium centrifugation, and then viralantigens and replicating DNA species were analyzed. HuH-7 cells were grownand

trans-fected as described above. Culture medium (10 ml) was

harvested 5 days aftertransfection. Solid cesium chloride (1.5 g)wasaddedto4.5 ml of medium, and the mixturewas

centrifugedat235,000 x gfor 60 hat4°C (21). The gradients

were fractionated into 350-pdl samples. The density of the

fractions was determined by weight. HBsAg and HBc/eAg weremeasuredasdescribed above aftera1:10dilution of the

fractions withphosphate-buffered saline. For DNA extrac-tion, two gradient fractions each (700

[lI)

were pooled,

diluted to5ml with10 mMTris-HCl (pH7.4)-i mM EDTA and centrifuged for 5 h as described above. DNA was

isolated from thepellet and analyzedasdescribedpreviously (3). As shown in Fig. 4, HBsAg and HBc/eAg in culture mediafrom wild-type adw R9- and mutant HBX-21-trans-fected cells bandatdensities of 1.15 and 1.25g/ml,

respec-tively. Furthermore, the replicating DNA species are

iden-tical for wild-type- and mutant-transfected cells and are

associated withHBsAg(fractions 18 through 23),

represent-ing Dane particles, as well as with HBc/eAg (fractions 26

through 31), representingcoreparticles (28).Takentogether, these data demonstrate that in HuH-7 cells, the X-minus mutant HBX-21 isfunctionallyindistinguishable from wild-typeHBV DNA.

The trans-activating potential of wild-type and mutant viral DNA was analyzed by cotransfection of viral DNAs

with pSV2CAT, which contains the simian virus 40 early

promoterandenhancer fusedtothechloramphenicol acetyl-transferase (CAT) gene. HuH-7 cells were cotransfected

withpSV2CAT and different hepadnavirusDNAconstructs. Theseanalyses revealed four-tofivefoldtransactivationby

anHTDofHBV andwoodchuck hepatitisvirus(WHV)but

notby the X-minus mutant of HBV (HBX-21 HTD) or an HTD of duck HBV (DHBV HTD) which lacks an x open reading frame (Table1).These findingssuggestthat the stop codonmutation in codon 8ofthe HBx geneleadsto alossof the trans-activating potential of these clones without af-fecting viral gene expression, replication, orexport of

viri-ons. In

addition,

the absence of trans-activating activityof

DHBVHTDindicates that the HBxgene-relatedsequences identified in the core gene of DHBV (6) have no trans-activating potential in the cell culture systemused.

Transfection efficiency was assessed by the detection of hepatitis B core antigen (HBcAg) in cells transfected with wild-type or X-minus mutant HBV DNA. HuH-7cellswere grownand transfected asdescribed above. Five days after

TABLE 1. CAT trans-activation by differentconstructs

CATtrans P/N ratio forHBsAg

Construct' activation

[fold(cpm)]b in culturemediumC

pSV2CAT 1.0(910)

pSV2CAT + pJ30mega 0.8 (720)

pSV2CAT + HBV HTD 5.2(4,780) 95

pSV2CAT+ HBX-21 HTD 1.0(880) 114 pSV2CAT +WHV HTD 4.5(4,140)

pSV2CAT + DHBV HTD 0.9(850)

aAllhepadnavirus constructs werereplication-competent HTDsinserted into theEcoRIsiteofpGEM-7Zf(+). WHVHTDwaspreparedfromWH81

(7), and DHBV HTDwaspreparedfrom DHBVFl-6 (14).The

nonhepadna-virus controlwas pJ30mega DNA(15). Transfection of HuH-7 cells was

carriedoutwith1.0,ugofpSV2CAT(obtainedfromAmericanTypeCulture Collection[ATCC37155])and 3

pg

of theadditionalconstructindicated.Two

days aftertransfection,cellswereharvested.

bCelllysateswereanalyzedfor CATactivityasdescribedelsewhere(31),

with10 p.gofprotein per assay.

cApositive-to-negative

(P/N)

ratioof -2.2 indicatespositivityforHBsAg.

VOL.66, 1992

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transfection,

the cells were stained for

HBcAg by

the

peroxidase-antiperoxidase technique using polyclonal

rabbit

anti-HBc antibodies (Accurate Chemical & Scientific

Cor-poration, Westbury, N.Y.).

The transfection efficiencies

were identical for

wild-type (adw

R9 or adw HTD) and

mutant

(HBX-21

orHBX-21

HTD) DNA,

with about 15% of HuH-7cells

showing

nuclearor

cytoplasmic HBcAg

expres-sion orboth.

Analysis

of the cell culture media for

HBsAg

and

HBc/eAg

from the same

experiment

confirmed that

wild-type

and X-minusmutantHBV DNAdirect the

synthe-sis and export of

virtually

identical amounts ofthese viral

proteins.

To exclude a

pX-like

contribution of HuH-7

hepatoma

cellstoviralgene

expression, replication,

and virionexport, the human

hepatoblastoma

cell line

HepG2 (1)

and

primary

adultrat

hepatocytes (17)

weretransfected witha

wild-type

HTDofthe HBVgenome

(3)

orwithmutantHBX-21HTD. As in the case ofHuH-7

cells,

viral

replication

and virion exportinto the culture mediumwereidentical for

wild-type-and X-minus mutant-transfected

HepG2

cells

(data

not

shown). HBsAg synthesis

and

HBc/eAg synthesis,

on the

other

hand,

were

reproducibly

lowerin mutant-transfected

HepG2

cells

(40

to

70%).

Considering

the

complex, partially

cell

line-dependent

interplay

of at least six

transcription

factors which activateandonewhichrepresses

transcription

ofthe

major

Sgene

(19),

more studies areneededto assess

the moleculareffect ofpXonthe

regulation

ofviral genes. In view ofthe

wild-type

levels of viral

replication

and virion exportdirected

by

the X-minus mutant, thedifferent

antigen

levels observed in

HepG2

cells do not affect the viral life

cycle,

however.

Furthermore,

in

primary

adult rat

hepatocytes,

the

wild-typeandX-minus mutant constructsresulted in

synthesis

of identicalamounts of viral

transcripts

of2.1and3.6 kb

(data

not

shown)

as well as secretion of identical amounts of

HBsAg

and

HBc/eAg

intothe culture medium

(Fig.

2,

right

panel).

Thelevels of viral

replication

in these cellsweretoo

lowtobe

assessed,

however.Taken

together,

these

findings

demonstrate

that,

not

only

in HuH-7 human

hepatoma

cells but also in

HepG2

human

hepatoblastoma

cells and in

primary

rat

hepatocytes,

pX is not

required

for the viral

functions

analyzed.

A

significant

reversion ofHBX-21 tothewildtype, while

highly

unlikely

to occurin a transienttransfection system,

wasexcluded

by

directsequence

analysis

ofthePCR ampli-fication

product

of viral DNA isolated fromculture media 5

days

aftertransfection. Culture mediawere

processed

(with

DNase

digestion performed

as part ofthe

processing),

and

DNA was

prepared

as described

previously

(3). Two PCR

primers

spanning

the HBV genome

regions consisting

ofnt

1080 to 1108

(primer 1)

and 2437 to 2410

(primer

2) were

selected. On the basis of the

incomplete

dimerstructure of

the

wild-type

adw R9(3)and itsX-minusderivativeHBX-21,

PCR

amplification

of cloned

input

DNA should result in a

5.25-kbp

PCR product

[including

3.0 kbp from

pGEM-7Zf(+)

vector DNA]; PCR amplification of circular virion

DNA,

on the other

hand,

should result in a 1.35-kbp PCR

product.

When DNA isolated from culture media from

wild-type-

andX-minusmutant-transfected HuH-7 cells was

amplified

by PCR,

only a1.35-kbpPCR product was

gener-ated, indicating

that the template was indeed virion rather

than cloned

input

DNA. The 1.35-kbp PCR products were

gel purified

and

sequenced directly.

As shown in Fig. 1

(lower right

panel), these analyses demonstrated the

pres-ence of

wild-type

CAA in culture medium from adw

R9-transfected

cellsandofmutant

TAA

inculture medium from

HBX-21-transfected cells. In addition, an aliquot of each PCR product was cloned into pCR 1000. DNA sequence analyses of several randomly selected TA clones derived from each PCR product confirmed the wild-type CAA or

mutant TAA codon with downstreamvirion-specific,generic HBVsequences not present in cloned input DNA (3). Taken together, these PCR product sequence analyses demonstrate that the majority, if not all, of the exported virion DNA molecules carry the expected wild-type or mutant base at nt 1397 and exclude a significant revertant of HBX-21 to

wild-type HBV DNA.

Koike and collaborators (12, 28) studied an HBV mutant carrying a frameshift mutation in the 3' region of the HBx gene. Their results differed from our results in that in their study, in HepG2 cells this frameshift mutant was less effi-ciently transcribed and showed lower levels ofreplication thanwild-type HBVDNA. This apparent conflict in results isdifficult toresolve, however, without further data demon-stratingtransfectionefficiency, mRNA stability, and frame-shift mutation-inducedablation of HBxgene-mediated trans-activating activity.

Inconclusion, on the basis of ourdata,theexpression of thefull-lengthHBxproteinis notrequiredfor viral transcrip-tion, replicatranscrip-tion, or synthesis of structural proteins and therefore appears not to be central to the life cycle of HBV invitro. The biological significanceofcarboxy-terminalpX peptides potentially translated from transcripts initiated at downstream AUGs is unclear at present. Recent findings with simian immunodeficiency virus (SIVmac 239) demon-strate that the nefgene, which is highly conserved among lentivirusesandnonessential for viralreplicationin vitro,is requiredinvivoformaintainingahighvirusloadand for full pathogenic potential (10). To assessthe potential contribu-tion of pXtohepadnavirus propagation and pathogenicityin vivo,we have therefore initiated long-term studies withthe woodchuckmodel (20), using WHV-positive culture media from HuH-7 cells transfected with a replication-competent HTDofwild-type WHV andanX-minusmutantoftheWHV genome(2a).Mostimportantly,these invivo studies should determinethecontribution ofpX to thedevelopmentofliver cancer,extendingthe recentfindings with transgenic animals (11, 13) ina natural model ofhepadnavirus hepatocarcino-genesis (18).

We thank R. Carlson for expert technical assistance, D. B. Rhoadsforproviding primary adultrathepatocytes,J. L.Gerin for making WHV clone WH81 available, and J.-I. Lee for preparingan HTDof WH81.

H.E.B. wassupported byaHermannandLilly Schilling

profes-sorship from the Stifterverband fur die Deutsche Wissenschaft,

Z.-S.Z.wassupported byaWHOfellowship, F.v.W.wassupported

byafellowshipfrom the Deutsche Forschungsgemeinschaft, T.J.L. wassupported byaNIDDK clinical investigatoraward, and J.R.W.

wassupported by a research scientist award. This work was funded in partbyPublicHealthServicegrantsfromthe National Institutes ofHealth.

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Mapping the transcriptional transactivation function of simian virus 40 largeTantigen. J. Virol. 65:2778-2790.

VOL.66, 1992

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Figure

FIG.1.(P-ORF)ofandacid.tionproducts(adw HBV (Upper panel) Map of the hepadnavirus polymerase and X (X-ORF) open reading frames and DNA sequences wild-type (WT) and mutant (MT) X and P genes
TABLE 1. CAT trans-activation by different constructs

References

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