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 atube 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 of104IU/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 a90% 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
.
* b1 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 105IU/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--y0 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]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 93105 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,
orIFN-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 m3;
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:3.612.322.561.92.183.2] [image:3.612.368.516.531.671.2] [image:3.612.66.305.598.732.2]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 HBVDNAincore
particles
(Table 1). However,wecouldfind nosignificant
difference in theinhibitory
effects of type I and TIIFNs,despitetheirdifferentmechanisms foreliciting cellular
responses (22). It is evident from the present data that all three IFNs examined inhibitHBVDNA
replication
inHBV-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 drugsfor 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.
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