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JOURNAL OF VIROLOGY,JUIY1989,p. 2936-2940 0022-538X/89/072936-05$02.00/0

CopyrightC 1989, American Society for Microbiology

Interferon Inhibits Hepatitis

B

Virus

Replication

in

a

Stable

Expression

System of Transfected Viral DNA

YASUYUKIHAYASHIAND KATSURO KOIKE*

Department ofGeneResearch, CancerInstitite, Kami-Ikeblukiuro, Toshima-ku, Tokyo 170,Japan Received 30November 1988/Accepted17 March 1989

Theeffect of interferon(IFN)onhepatitis B virus (HBV)replicationwasinvestigatedinastableexpression system, using HepG2 cells transfected with recombinant HBV DNA. IFN was found to cause a marked reduction in the levels ofboth minus and plus strandsofHBV DNA from core particlesin the cytoplasm.

Neither HBV DNA from virus particles nor the HBV surface antigen in the culture medium primarily

underwentchangeinquantity bytreatmentwithIFN,as wasalso found for HBV mRNAs and the HBVcore

antigen/HBVeantigeninthecytoplasm.IFN exertednoinfluenceonHBV DNAsynthesis by endogenousDNA polymerase in the core particle fraction. From these findings, it would appear that IFN inhibits HBV replication byblocking somestep in thepregenomeRNA-primed assemblyofcoreparticles.

Infection from hepatitis B virus (HBV) causes acuteand chronic hepatitis and frequently leadstohuman livercancer

(20). HBV DNA consists ofa partially single-stranded

cir-cular DNA molecule with a complete minus strand of

approximately 3,200 nucleotides. The replication cycle of HBVpartly resembles that of retroviruses, thoughitappears

that an integration step of the viral genome is unnecessary

forreplication. Thecentralfeature of HBVreplicationis the

use ofanRNA copy of thegenome, designated pregenome

RNA,as anintermediatetemplatein thereplication cycle (9,

10, 17). When pregenome RNA is assembled into core

particles, minus-strandDNA is synthesized byreverse

tran-scriptionand then serves as a template for thesynthesis of plus-strand DNA.Aftercoreparticleshave beencoated with the HBV surface antigen (HBsAg), whether theplus strand is complete or not, the virus particles thus produced are

excreted into the culture medium. Thus, the replication cycle of HBV differs strikingly from that of other DNA

viruses.

Thereareseveralreports(4, 7, 8, 13) indicatingsuccessful therapy ofchronic HBV infection by the use of interferon

(IFN) as anantiviral agent. Though patients vary

consider-ablyintheirresponsetothisdrug, IFNappearstoeffectively decreasevirusparticlesinpatientsera.Suchadecreasemay beexplainedasbeing duetoinhibition of thetranscriptionor

translation of virus genetic information (11). However, the mechanismforthis has remainedunclarified owingtoalack

ofan in vitroculture systemof HBVreplication. Recently,

we (23) and others (2, 14, 18, 21) have found HBV

DNA-transfected human hepatoma cells to stably or transiently produce replicative intermediates and mature virus. The

presentstudywasundertakentoassesstheeffects ofIFNon

HBVreplicationinsuchastable expression systemofHBV and toclarify the mechanism ofIFN action.

MATERIALS ANDMETHODS

DNAs and cell line. A recombinant plasmid, termed pHBV-dimerby Yaginumaetal. and carryingtwo copies of theentire HBVDNA(subtype adr) inahead-to-tail arrange-ment at theBamHI site ofpBR322 asdescribed previously

(23), wasused for DNAtransfection. Another recombinant

plasmid, pSV2-neo-SVgpt (16), contained the region (neo) Correspondingauthor.

essential for the expression of neomycin resistance. A

hu-man hepatoblastoma cell line, HepG2 (6), was used as the

recipient ofDNA transfection.

DNA transfection and establishment oftheHBV-producing cell line. DNA transfection was carried out by the calcium phosphate precipitation method (5) with 10 p.g of pHBV-dimer and 1 p.g of pSV2-neo-SVgpt. After beingincubated

withDNAprecipitatesfor6hat 37°C, the cellswerefurther

cultured in fresh mediumsupplemented with the neomycin analog G418 (400 p.g/ml; GIBCO Laboratories). Colonies resistant to G418 were collected and screened for HBV

particle production.Fivepositivecolonieswereestablished;

one, designated Hep-HB107, was used in this study. Hep-HB107 cells secrete HBV particles and HBsAg into the medium and produce core particles in the cytoplasm as

described previously (23). They each have a

chromosom-ally integrated set of original recombinant HBV DNA and produce two major transcripts, 3.6- and 2.2-kilobase (kb) mRNAs, andone minortranscript, 2.6-kb mRNA. In addi-tion, 0.8-to0.9-kb mRNA has been detected.

Cell culture forassayof IFN effects. Toassesstheseeffects

on HBV replication, Hep-HB107 cells were plated at a

density of 5 x 106per 60-mm dish and preincubated for 4 days at 37°C. After being washed with phosphate-buffered saline, theywereculturedat37°Cfor 3daysin fresh medium supplemented with IFN at an appropriate concentration.

The resultingculture medium and cellswere used in subse-quent experiments.The numberof viable cells wascounted

bythetrypan blue exclusionmethod.

ThreetypesofIFNwereused:recombinant humanIFN-ot

(Takeda), natural humanIFN-4 (Toray)derived from human fibroblasts, and natural human IFN-y (Hayashibara/Mo-chida)from humanlymphocytes. Allwerestoredat-40°Cin the culture medium until use.

Preparation and blot analysis of RNA. Total RNA of Hep-HB107 cells with or without IFN treatment was

pre-pared by the guanidium-cesium chloride method (3). After electrophoresis in a formaldehyde-agarose gel, RNA was

transferred to nitrocellulose filterpaper (19). A32P-labeled HBV DNA probe was madeby nick translation (12).

Preparation ofcore or virus particles and assay of HBV

antigens. Preparation ofcore orvirus particles was carried outasdescribedpreviously (23).Acellextractwasprepared

by homogenizing cells with ahypotonic buffer (20mM Tris 2936

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hydrochloride, pH 7.5-50 mM NaCl-5 mM MgCl2-0.1% 2-mercaptoethanol-0.5 mM phenylmethylsulfonyl fluoride). After being assayed for HBVcore antigen (HBcAg)/HBVe

antigen(HBeAg) withanAbbott HBeenzymeimmunoassay

kit, the extract was subjected to 30% sucrose zone centrif-ugation at35,000rpmfor 16 h ina Beckman SW50.1 rotor. The pellet thus obtained was used as the core particle

fraction. Virus particles secreted into the culture medium

were prepared by20% sucrose zonecentrifugation. HBsAg was assayed with anAbbottAUSZYME IT kit.

Preparationandblot analysisof HBV DNA incore orvirus

particles. HBV DNAs in core and virus particles were

prepared from thecore and virus particle fractions,

respec-tively. The particle fraction was treated with 1 mg of

proteinase K (Boehringer Mannheim Biochemicals) per ml

and 1% sodium dodecyl sulfate at 37°C for 2 h and then subjected to 1% agarose gel electrophoresis. DNA was

transferred tothenitrocellulose filterpaper(15), whichwas

then hybridized with a 32P-labeled HBV DNA. 32P-labeled

HBV RNA specific for minus- or plus-strand viral DNA

sequence wasprepared byusingan SP6/T7 transcription kit

(Boehringer Mannheim).

Assay of endogenous DNA polymerase activity. The core

particle fraction (200 p.gofprotein; 20

RI)

was added to a

tube containing 1 pul of the reaction mixture (dATP, dGTP,

and dTTP [1 mM each]-4 FM [32P]dCTP [800 Ci/mmol]-culture medium with or without IFN). In each case, the

reactionmixture wasincubated at 37°Cfor 30min, digested with proteinase K and sodium dodecyl sulfate, extracted with phenol and chloroform (1:1), andthen ethanol precip-itated. The resulting radioactive product was assayed for

[32P]HBV DNAcontent byhybridization withexcess(50ng

each) single-stranded HBVDNA immobilized on filter

pa-per. Radioactivity was detected by exposure to X-ray film followed by scanningwith a densitometer (Zeineh).

RESULTS

Effect of IFNonHBV DNA in core particles. When HBV

DNAs were prepared from the core particle fraction and

subjected to Southern blot analysis, two bands (upper and lower) wereobservedasdescribed in the previous

observa-tionsof thereplicative intermediates ofHBV DNA (23).The

upperbandwashybridized with both minus- and plus-strand

probes, but the lower band was only hybridized with the

plus-strand probe, indicating that the lower band is the single-stranded minus strand (SS in Fig. ld) and the upper

band consists of both minus andplus strands in theform of partially double-stranded circular DNAs (RC in Fig. le). Aftertreatment ofHep-HB107 cells for 3 days with IFN at

various concentrations, the level of HBV DNA from the

coreparticlefractionwasfoundtodecreaseremarkablywith

an increase in IFN concentration (Fig. la through c). The relative values of HBV DNA obtained by densitometer tracing in three separate experiments are given in Table 1.

IFN-a,

IFN-P,

and IFN--y, each at a concentration of104

IU/ml, brought about 79, 89 and 64% reductions in core

particle HBV DNA, respectively. When the plus-strand probe was usedto detectthe amountof HBV minus-strand DNA incoreparticles, theamountofthis DNAwasfoundto

decrease afterIFNtreatmentby essentiallythesameextent asthat ofwhole HBV DNA.

IFN-P,

forinstance, caused a

90% reduction in the minus-strand DNA at104 IU/ml (Fig. ld).

WhenHep-HB107cellsweretreated with 104IUofIFN-ot permlfor 3 daysand then cultured foran additional 3days

a

d

1 2 34 56

1 2 34 56

.

* b

1 2 3 4 5 6

C

1 2 3 4 5 6

e

12 3456

*.X.

*'O

-_-

*

a

*9

to

FIG. 1. Southern blot analysis of HBV DNA fromcoreparticles. Core particles were prepared from Hep-HB107 cells treated with IFN-a (a),IFN-P(b, d, and e),orIFN--y (c). Lanes: 1, 105 IU/ml;2, 104IU/ml; 3,103IU/mI; 4, 102 lU/ml; 5, 101IU/ml; 6,noIFN.The DNA from the core particle fraction was hybridized with a 32p_ labeledprobe specific for the double-stranded (a, b, c), the minus-strand(d),ortheplus-strand (e)HBVDNA.RC and SS indicate the positions of relaxed circular DNA and single-stranded DNA, respec-tively.

in the fresh medium without IFN-ao, HBV DNA from the core particle fraction returned to thecontrol level observed without IFN treatment (data not shown). Thus, reduction in HBV DNA in core particles may be a reversible process induced by IFN.

Effect ofIFN on HBV DNA invirus particles. Since it was quite clear that the amount of HBV DNA in core particles from thecytoplasm decreases at IFN-cx and

IFN-P

concen-trations of 104 IU/ml and at an IFN--y concentration of 105

IU/mI, the level of HBV DNA in virus particles from the culture medium was measured to determine whether IFN suppresses the amount of virus particles. The results of Southern blotanalysisof HBV DNAfrom virusparticlesare shown in Fig. 2. HBV DNA was found not to change at all

TABLE 1. Amounts of HBV DNA incore particlesfrom Hep-HB107 cells treatedseparatelywithIFN-co, IFN-3,orIFN-y IFNconcentration HBV DNA(%)after treatment with:

(lU/ml)

IFN-ox

IFN-,B IFN--y

0 100 100 100

10' 122 104 87

102 101 38 73

103 41 15 52

104 21 11 36

105 11 7 8

.4

110 !.. * It

-0- 0.

44

-*P- 0

-01-- *0

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[image:2.612.312.551.646.733.2]
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2938 HAYASHI AND KOIKE

12 3 4 5 6

RC

FIG. 2. Southern blot analysis of HBV DNA from virus parti-cles. Virus particles were prepared from the culture medium of Hep-HB107 cells treated with IFN-co (104 IU/ml) (lane 1),IFN-3(104 lU/ml)(lane 3), orIFN-y (105IU/ml) (lane 5) or with no IFN (lanes 2, 4, and 6). RC and SS indicate the positions of relaxed circular DNAand single-stranded DNA, respectively.

after a 3-day treatment with IFN, at concentrations of 104 IU/ml for IFN-ot and

IFN-P

and 105 IU/ml for IFN--y.

Accordingly, Hep-HB107cells treated with104 IU of IFN-(x permlfor 3 dayswereculturedagainforanadditional 3days in the fresh medium in the presence of

104

IU of IFN-(x per ml. As aresult, HBV DNA in virus particles in the medium was reduced by 76%. On the other hand, the number of viable cells was not remarkably changed afteranadditional 3 days oftreatment (unpublisheddata).

Production of HBV antigens in IFN-treated Hep-HB107 cells. The effect of IFN on HBV antigen production was assessed from measurements of secreted HBsAg in the culture medium and HBcAg/HBeAg in the cytoplasm. As summarized in Table 2, no HBV antigen reduction was

observed bytreatment with IFNs for 3 days.

Thelevel of HBV mRNA in IFN-treated Hep-HB107cells. The effect of IFN treatment on the HBV mRNAs was examined. No decrease occurred in the two major

tran-scriptsof 3.6- and 2.2-kb mRNAs in IFN-treated cells (Fig.

3), as was also found for the amount of 2.0-kb ,B-actin

transcript used as the control. Theamountof HBVDNAin core particles, however, apparently decreased, indicating

thatIFN-mediated reduction in HBV DNA of core particles isunlikely to be correlated with the amount ofHBV mRNAs (Fig. 1 and Table 1).

[image:3.612.133.241.80.223.2]

Viabilityof IFN-treatedHep-HB107cells.Aftercultivation for 3daysinthe presence or absence of IFN, the number of

TABLE 2. Effectof IFN on HBVantigen production

Treatment HBV antigen(%)

andconcentration

(lU/ml) HBcAg/HBeAg HBsAg

IFN-a

0 100 100

104 94 108

IFN-p

0 100 100

104 93 111

IFN-y

0 100 100

105 104 96

TABLE 3. Effect of lFN on cellviability

IFNconcentration Viable cells(%)after treatment with:

(IU/ml) IFN-o. IFN-f3 IFN--y

0 100 100 100

101 93 93 100

102 86 79 100

lo, 79 71 100

104

79 64 93

105 64 64 86

viable cells was counted. Table 3 shows that there were 21, 36, and 7% decreases in viable cells at 104 IU of IFN-ot, IFN-1, and

IFN--y

per ml, respectively. IFN at the same concentration, however, strongly inhibited HBV DNA pro-duction in the core particles (Fig. 1 and Table 1). Growth inhibition was frequently observed in IFN-treated tumor cells (1). In separateexperiments, 5 x 105 Hep-HB107 cells per60-mmdishwereplated andexposedto 104IU ofIFN-ot,

IFN-P,

or

IFN-y

per ml for 3 days, and cell growth was inhibited by 35, 45, and 19%, respectively (unpublished data). It is evident that IFN has a weak antiproliferative effectonHep-HB107 cells.Therefore,the decreased growth ratecoupled with the increase of nonviable cells may beat least partially responsible for the effects of IFNs upon the amount of minus-strand DNA in the core particles.

In vitro effect of IFN on endogenous DNA polymerase activityinthe coreparticlefraction. Since nochangein 3.6-kb mRNA occurred with reduction in HBV DNA of core particles in IFN-treated Hep-HB107 cells (Fig. 1 and 3 and Table 1), wesoughttodetermine whether reductionin HBV DNAresults from inhibitionof HBV DNA synthesisin core particles. Endogenous DNApolymerase activitywas exam-ined by an in vitro assay system using the core particle fractionin the presence orabsence ofIFN, asdescribed in Materials and Methods. Relative activity in three indepen-dentexperiments is shown inTable4. The incorporationof

[32P]dCTP depended on the presence ofdATP, dGTP and dTTP, since incorporation was inhibited by 91% in the absence ofall three unlabeled triphosphates. Treatment of the core particle fraction with DNase I or RNase A before and duringthe polymerase reaction had negligible effecton

1 2 3 4

HBV

a m

3;

kb

,3-actin

W

U

o

"

-2.Okb

FIG. 3. Northern (RNA) blot analysis of HBV mRNAs from Hep-HB107 cells. Total cellular RNA was prepared from Hep-HB107 cells treatedwith 104 IU ofIFN-ctper ml(lane 1), 104IUof IFN-,Bper ml (lane2) and105 IUofIFN-yper ml(lane 3) and from untreated cells (lane4). 32P-labeled HBV DNA and ,B-actin cDNA were usedasprobes.

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[image:4.612.50.291.98.198.2]

TABLE 4. Relative activity of HBV DNA synthesis in the core particlefraction inthe presenceofIFN

[32PIdCTP

Reactionconditions incorporation

Complete"... 100

±LFN-ot(104IU/ml) ... 92

+IFN-, (104 IU/ml) ... 95

+IFN-y(105 lU/ml) ... 97

+DNase I"... 97

+RNase Al... 99

NodATP,dGTP, or dTTP... 9

"Thereaction mixture contained 10 mg ofcor-eparticlefractionperml.50

,uM each dATP. dGTP anddTTP. and 0.2 p.M [32PJdCTP (800 Ci/mmol).

Relativeincorporation wasdeterminedby usingradioactivityof the reaction in the absenceofIFN as100%.

"Coreparticle fractionwaspreincubated at37TCfor 15 min with 1 igof DNase I or RNase A perml.

this incorporation. If the 32P-labeled product of the DNA polymerase reaction in the presence or absence of IFN is

equivalent to the amount of HBV DNA synthesized, this would mean that none of the IFNs has any in vitro effect at all on the endogenous DNA polymeraseactivity in the core

particles.

DISCUSSION

An in vitro system for assessing the effect of antiviral

drugsonHBVreplicationwasestablished, andexamination of IFN by this system indicated that it apparently reduced theamountof HBV DNA in core particles (Fig. 1and Table 1)but had only a weak effect on cell viability (Table 3). This drug thus exerts a selectively inhibitory effect on the core

particles. Unexpectedly, the level of HBV DNA in virus particles from the culture medium was found not to change atallfor 3 days of treatment with IFNs. The amount of HBV DNA in virus particles secreted into the culture medium decreased after Hep-HB107 cells were treated for an

addi-tional 3 days with IFN in the fresh medium, and this was preceded by a decrease in HBV DNA of core particles in the cytoplasm. It is clear from this that IFN does not directly affect the secretion of HBV particles from the cells. Hep-HB107cells may function to preserve a large number of core particles.

IFN treatment remarkably decreased HBV DNA in core

particlesin the stableexpressionsystem(Fig. 1and Table 1), while the amount of HBV mRNAs was found not to change

by IFN treatment(Fig. 3). However, preliminary data indi-cated that treatment with 104 IU of IFN-ox per ml reduced 3.6- and 2.2-kb mRNAs by 88%in the transient expression

system, in which HBV particles were transiently produced

by transfected HBV DNA (pHBV-dimer) as described pre-viously (23). In the simian virus 40 system, expression of chromosomally integrated simian virus40genomes is resis-tant to IFN, while that of replicating simian virus 40 ge-nomes is sensitive to this drug (11). The primary effect of IFNs on HBV replication may appear different accordingto the different assay system used.Thus, it would appear from presentfindingsthatIFN mayinhibitsomestepparticipating

in the pregenome RNA-primed assembly ofcore particles. IFN-ox and

IFN-1

(type I IFN) appeared to be more effective for inhibiting HBV replication than IFN-y (type II IFN) on the basis of measurements of their reduction of HBV DNA in core particles (Table 1). Also, a low concen-tration of type I IFNs appeared to slightly increase HBV

DNAincore

particles

(Table 1). However,wecouldfind no

significant

difference in the

inhibitory

effects of type I and TI

IFNs,despitetheirdifferentmechanisms foreliciting cellular

responses (22). It is evident from the present data that all three IFNs examined inhibitHBVDNA

replication

in

HBV-producing cells stably transformed by recombinant HBV DNA. A stable expression system such as Hep-HB107cells should help pave the way for

producing

new antiviral drugs

for treating HBV-related chronic liver diseases.

ACKNOWLEDGMENT

This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategyfor Cancer Control. Japan, to K.K.

LITERATURE CITED

1. Borden, E.C. 1983. Interferons andcancer: how thepromise is being kept. Interferon 5:43-83.

2. Chang,C., K.-S. Jeng, C.-P. Hu, S.J.Lo, T.-S.Su,L.-P.Ting, C.-K. Chou, S.-H. Han, E. Pfaff, J. Salfeld, and H. Schaller. 1987. Production of hepatitis B virus in itro by transient expressionofclonedHBV DNAin ahepatomacell line. EMBO J.6:675-680.

3. Chirgwin, J. M., A. E. Przybyla, R.J. MacDonald, and W.J.

Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299.

4. Dusheiko, G.,A.Dibisceglie,S.Bowyer,E.Sachs,M.Ritchie,B. Schoub, andM. Kew. 1985. Recombinant leukocyte interferon

treatmentof chronic hepatitis B. Hepatology 5:556-560. 5. Graham,F.L.,andA.J.vander Eb.1973. Anewtechniquefor

the assayofinfectivity of humanadenovirus5 DNA. Virology 52:456-467.

6. Knowles, B. B., C. C. Howe, and D. P. Aden. 1980. Human hepatocellular carcinoma cell lines secrete the major plasma proteins andhepatitisB surface antigen. Science 209:497-499. 7. Lai, C.-L.,A. S.-F.Lok,H.-J. Lin, P.-C.Wu,E.-K. Yeoh,and

C.-Y. Yeung. 1987. Placebo-controlled trial of recombinant cx.-interferon in Chinese HBsAg-carrier children. Lancet ii: 877-880.

8. Lok, A. S. F., D. M. Novick, P. Karayiannis, A. A. Dunk, S. Sherlock,and H. C. Thomas. 1985. Arandomized study ofthe effectsof adenine arabinoside 5'-monophosphate (shortorlong courses) and lymphoblastoid interferon on hepatitis B virus replication. Hepatology5:1132-1138.

9. Miller,R.H.,P. L.Marion,and W.S.Robinson.1984.Hepatitis B viral DNA-RNA hybrid molecules in particlesfrom infected liverare converted toviral DNAmolecules during an endoge-nous DNApolymerasereaction. Virology 139:64-72.

10. Miller, R. H.,C.-T.Tran, andW.S. Robinson. 1984. Hepatitis B virus particles ofplasma and livercontain viral DNA-RNA hybrid molecules. Virology 139:53-63.

11. Revel,M. 1979. Molecularmechanismsinvolved in theantiviral effects ofinterferon. Interferon 1:101-163.

12. Rigby, P. W. J., M. A. Dieckmann, C. Rhodes, and P. Berg. 1977. Labelingdeoxyribonucleicacidtohighspecific activityin

lvitro

bynick translation with DNApolymerase 1. J. Mol. Biol. 113:237-251.

13. Scullard, G. H., L. L. Andres, H.B. Greenberg, J. L. Smith,

V. K. Sawhney, E. A. Neal, A. S. Mahal, H. Popper, T.C. Merigan, W.S. Robinson, and P.B. Gregory. 1981. Antiviral treatmentofchronichepatitisBvirusinfection:improvementin liver disease withinterferon and adeninearabinoside. Hepatol-ogy 1:228-232.

14. Sells, M. A., M.-L. Chen, and G. Acs. 1987. Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA 84:1005-1009.

15. Southern, E. M. 1975. Detection ofspecific sequences among DNAfragmentsseparated by gel electrophoresis.J. Mol. Biol. 98:503-517.

on November 10, 2019 by guest

http://jvi.asm.org/

(5)

2940 HAYASHI AND KOIKE

16. Southern,P. J., and P. Berg. 1982.Transformation of

mamma-lian cells to antibiotic resistance with a bacterial gene under control of theSV40early regionpromoter.J.Mol.Appl. Genet. 1:327-341.

17. Summers,J., andW. S.Mason.1982. Replication of thegenome ofahepatitis B-like virus byreversetranscription ofan RNA intermediate. Cell 29:403-415.

18. Sureau, C.,J.-L. Romet-Lemonne, J. I. Mullins,and M.Essex. 1986.Production of hepatitisBvirus byadifferentiated human

hepatoma cell line after transfection with cloned circular HBV DNA.Cell 47:37-47.

19. Thomas,P.S. 1980.Hybridization ofdenatured RNA and small DNAfragments transferredtonitrocellulose. Proc. Natl. Acad.

Sci. USA 77:5201-5205.

20. Tiollais, P., C. Pourcel, and A. Dejean. 1985. The hepatitis B virus.Nature(London) 317:489-495.

21. Tsurimoto, T., A. Fujiyama, and K. Matsubara. 1987. Stable expression and replication ofhepatitis B virus genome in an

integrated stateinahumanhepatomacell line transfected with the cloned viral DNA. Proc. Natl. Acad. Sci. USA 84:444 448. 22. Vilcek, J. 1982. The importance ofhavinggamma. Interferon

4:129-154.

23. Yaginuma,K., Y. Shirakata, M.Kobayashi,and K.Koike. 1987. HepatitisBvirus(HBV) particlesareproducedinacell culture

systemby transient expression of transfected HBV DNA. Proc. Natl. Acad. Sci. USA 84:2678-2682.

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Figure

FIG.1.CorelabeledIFN-aDNAstrandpositions104 Southern blot analysis of HBV DNA from core particles
FIG.3.wereHep-HB107untreatedHB107IFN-,B Northern (RNA) blot analysis of HBV mRNAs from cells
TABLE 4. Relative activity of HBV DNA synthesis in the coreparticle fraction in the presence of IFN

References

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