The role of helioxanthin in inhibiting human hepatitis B viral replication and gene expression by interfering with the host transcriptional machinery of viral promoters.

Abstract

A non-nucleosidic compound, Helioxanthin (HE-145), was found to suppress HBV gene expression and replication in HCC cells. To understand the molecular mode of action of HE-145 on HBV gene expression, the effects of HE-145 on four viral promoter activities using luciferase as a reporter were examined. It was found that HE-145 selectively suppresses surface antigen promoter II (SPII) and core promoter (CP) but has no effect on surface antigen promoter I (SPI) or promoter for X gene (Xp). The suppressive effects of HE-145 on either SPII or CP activity is liver-specific, since no suppressive activity of HE-145 was observed when CP or SPII promoter activity was assayed in non-liver cells such as HeLa or 293T. To examine the mode of action of HE-145, EMSA analysis revealed that HE-145 decreased the DNA-binding activity of nuclear extract of HepA2 cells to specific cis element of HBV promoter for core antigen, including peroxisome proliferator-activated receptors (PPARs), PPARs binding site (PPRE), alpha-fetoprotein transcription factor (FTF), and Sp1. Ectopic expression of PPAR gamma or HNF4 alpha partially reversed the HE-145-mediated suppression of HBV RNA. Therefore, HE-145 may represent a novel class of anti-HBV agents which selectively modulate transcriptional machinery of human liver cells to suppress HBV gene expression and replication.

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Available online at www.sciencedirect.com
Antiviral Research 77 (2008) 206–214
The role of helioxanthin in inhibiting human hepatitis B viral replication
and gene expression by interfering with the host transcriptional machinery
of viral promoters
Ya Ping Tsenga, Yueh Hsiung Kuob,c, Cheng-Po Hud,e, King-Song Jengf, Damodar Janmanchia,
Chih Hsiu Ling, Chen Kung Chouh,∗, Sheau Farn Yeha,∗∗
aInstitute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, ROC
bTsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University, Taichung, Taiwan, ROC
cAgricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC
dDepartment of Life Science, Tunghai University, Taichung, Taiwan, ROC
eDepartment of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, ROC
fInstitute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC
gInstitute of Chemistry, Academia Sinica, Taipei, Taiwan, ROC
hDepartment of Life Science, Chang Gung University, Tao-Yuan, Taiwan, ROC
Received 24 August 2007; received in revised form 22 November 2007; accepted 21 December 2007
Abstract
A non-nucleosidic compound, Helioxanthin (HE-145), was found to suppress HBV gene expression and replication in HCC cells. To understand
the molecular mode of action of HE-145 on HBV gene expression, the effects of HE-145 on four viral promoter activities using luciferase as
a reporter were examined. It was found that HE-145 selectively suppresses surface antigen promoter II (SPII) and core promoter (CP) but has
no effect on surface antigen promoter I (SPI) or promoter for X gene (Xp). The suppressive effects of HE-145 on either SPII or CP activity is
liver-specific, since no suppressive activity of HE-145 was observed when CP or SPII promoter activity was assayed in non-liver cells such as
HeLa or 293T. To examine the mode of action of HE-145, EMSA analysis revealed that HE-145 decreased the DNA-binding activity of nuclear
extract of HepA2 cells to specific cis element of HBV promoter for core antigen, including peroxisome proliferator-activated receptors (PPARs),
PPARs binding site (PPRE), ?-fetoprotein transcription factor (FTF), and Sp1. Ectopic expression of PPAR? or HNF4? partially reversed the
HE-145-mediated suppression of HBV RNA. Therefore, HE-145 may represent a novel class of anti-HBV agents which selectively modulate
transcriptional machinery of human liver cells to suppress HBV gene expression and replication.
© 2008 Chen Kung Chou. Published by Elsevier B.V. All rights reserved.
Keywords: Helioxanthin; Hepatitis B virus; Viral promoters; Hepatic nuclear factors; Human hepatocellular carcinoma cells
1. Introduction
Hepatitis B virus (HBV) infection causes acute and chronic
hepatitis, and the affected patients have an increased risk of
Abbreviations: HE-145, helioxanthin; HBV, hepatitis B virus; HBsAg, hep-
atitis B surface antigen; HBeAg, hepatitis B e antigen; HCC, hepatocellular
carcinoma;CP,corepromoter;SPI,surfacepromoterI;SPII,surfacepromoterII;
XP, X promoter; 3TC, Lamivudine, (−)?-l-2?,3?-dideoxy-3?-thiacytidine; EIA,
enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; EMSA,
electrophoretic mobility shift assay.
∗Corresponding author.
∗∗Corresponding author. Tel.: +886 2 2826 7117; fax: +886 2 2826 4843.
E-mail addresses: ckchou@mail.cgu.edu.tw (C.K. Chou),
fyeh@ym.edu.tw (S.F. Yeh).
developing liver cancer (Chang et al., 1997). HBV carries a par-
tially double-stranded DNA genome and replicates through an
RNAintermediate.Afterinfectinghostlivercells,therearefour
HBV transcripts transcribed from four different viral promot-
ers (Core, SPI, SPII and X promoter) with 3.5-, 2.4-, 2.1- and
0.7-kb length, respectively. The 3.5-kb transcript is translated to
producethepolymerase,coreandprecoreproteinsandservesas
the pregenomic RNA template. The 2.4- and 2.1-kb transcripts
produce the large, middle and small envelope proteins. The 0.7-
kb transcript is translated to produce the X protein, which can
transactivatebothviralandhostgenepromoters(Chisari,2000).
Although a preventive vaccine for HBV is available, the
therapeutic options for chronically infected patients are still
limited (Zuckerman, 2006). Interferon-alpha (IFN-?), lamivu-
0166-3542/$ – see front matter © 2008 Chen Kung Chou. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.antiviral.2007.12.011

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Y.P. Tseng et al. / Antiviral Research 77 (2008) 206–214
207
dine
[9-(2-phosphonylmethoxyethyl)adenine],
200475) and telbivudine (l-thymidine) have been approved as
the antiviral drugs (Franco and Saeian, 2002; Mohanty et al.,
2006; Yuan and Lee, 2007). However, IFN-? is costly and has a
narrow range of efficacy, safety, and tolerability (Alberti et al.,
1988). 3TC is a nucleoside analogue that inhibits HBV–DNA
reverse transcriptase and decreases HBV replication in patients.
Amajordrawbackof3TCtreatmentisthedevelopmentofHBV
variants with resistance mutations that reduce susceptibility to
3TC and entecavir but remain sensitive to adefovir after a short
periodoftreatment(Delaneyetal.,2001).Othernon-nucleoside
anti-HBV chemicals have been developed. For example, ellagic
acid was reported to block hepatitis B virus e antigen (HBeAg)
secretion (Shin et al., 2005) and the heteroarylhydropyrimidine
Bay41-4109 was shown to inhibit HBV replication by depletion
of nucleocapsids (Deres et al., 2003).
Arylnaphthalene lignan lactone, Helioxanthin (HE-145) and
its analogues were previously reported to inhibit HBV repli-
cation in cultured HepG2.2.15 cells (Li et al., 2005; Yeo et
al., 2005). In this study, we examined molecular mechanism
of anti-HBV activity of HE-145 and revealed that HE-145
could selectively modulate the host transcriptional machinery
inhumanlivercellstosuppressHBVgeneexpressionandrepli-
cation.
[3TC,(−)?-l-2?,3?-dideoxy-3?-thiacytidine],adefovir
(BMS-entecavir
2. Materials and methods
2.1. Materials
Theenzymeimmunoassay(EIA)kitsforhepatitisBvirussur-
face antigen (HBsAg) and e antigen (HBeAg) were purchased
from Bio-Rad (Hercules, CA). Fetal calf serum was obtained
from Hyclone (Logan, UT). Dulbecco’s modified Eagle’s
medium (DMEM) was obtained from Gibco/BRL (Gaithers-
bung, MD). [?-32P] dCTP was obtained from PerkinElmer Life
Sciences(MA).SeaKemLE-agarosewaspurchasedfromFMC
Bioproducts (Rockland, MA). The tetrazolium salt WST-1 for
the quantification of cell viability was purchased from Roche
(Mannheim, Germany). Other chemicals were purchased from
Sigma (St. Louis, MO).
2.2. Cell culture
Thehumanhepatocellularcarcinoma(HCC)HepA2cellline
wasderivedfromHepG2cellsbytransfectingatandemrepeated
full-length HBV DNA and continually secretes HBsAg and
HBeAg into the culture medium (Chang et al., 1987; Yeh et
al., 1993). The Hep3B cell line contains one to two copies of
integratedHBVDNAandcontinuouslysecretesHBsAgintothe
culture medium (Aden et al., 1979). The 1.3ES2 cell line is a
clone derivative of HepG2 cells in which the 1.3 copies of the
entire HBV genome was stably integrated in the genome (Chou
et al., 2005). 1.3ES2 cells therefore continuously produce HBV
viral particles into the culture medium. Stock cultures of HCC
cell lines, human cervix epithelial cells line HeLa (Scherer et
al., 1953) or human kidney epithelial cell line 293T (Graham et
al., 1977) were maintained in DMEM supplemented with 10%
fetalcalfserumandantibiotics(100IU/mleachofpenicillinand
streptomycin) in a humidified atmosphere containing 5% CO2
and 95% air at 37◦C. The cultures were passaged by trypsiniza-
tion every 4 days. For the bioassays, cells were plated either
in 24-well plates at a density of 8×104cells/well or in 100-
mm culture dishes at a density of 1.5×106cells/dish in DMEM
medium containing 10% fetal calf serum.
2.3. Preparation of HE-145
The heartwood of Taiwania cryptomerioides Hayata was
extracted with methanol and subsequently partitioned in n-
hexane and water (1:1, v/v). The n-hexane soluble material
was fractionated by sequential column chromatography using
silica–gel. The active component, HE-145 was eluted using n-
hexane/EtOAc (95:5, v/v) and was further purified by reverse
phasehighperformanceliquidchromatographytohomogeneity.
HE-145-9, a derivative of HE-145, was organically synthe-
sized following standard methods. The structures of HE-145
and HE-145-9 were determined by H-nuclear magnetic reso-
nance, infrared (IR), and mass spectroscopy. For the bioassays,
the compound was dissolved in dimethyl sulfoxide (DMSO)
and filtered through a 0.25?m fluoropore filter (Millipore,
MA).
2.4. Quantification of HBsAg and HBeAg
Cells were seeded in 24-well plates at a density of
8×104cells/well in DMEM containing 10% fetal calf serum.
After 24h of incubation, the cells were washed twice with
PBS, pH 7.0, and treated with various concentrations of drugs
in serum-free DMEM for the time indicated. The HBsAg and
HBeAg in the culture medium were measured by an enzyme
immunoassay (EIA) kit (Bio-Rad, CA). The viability of the
cells was determined by a WST-1 cell proliferation assay. For
the WST-1 assay, WST-1 (Roche Diagnostics, Mannheim, Ger-
many) was added to each well and incubated for 0.5h. The
amountofformazandyeformedcanbecorrelatedtothenumber
of metabolically active cells, which is quantitatively determined
using a scanning multi-well spectrophotometer (ELISA reader)
at the absorbance of 450nm.
2.5. RNA isolation and Northern blot analysis
TotalcellularRNAwasextractedusingthephenolandguani-
dinium isothiocyanate method (Gruffat et al., 1996). The RNA
(20?g) was denatured by 2.2M formaldehyde, separated on
denaturing formaldehyde 1.2% agarose gel, and transferred to
a nylon membrane (Hybond-XL, Amersham, Piscataway, NJ).
The membrane was hybridized with a32P-radiolabelled full-
length HBV probe. The amount of the total RNA applied was
normalized by hybridization of the probe for glyceraldehyde-3-
phosphate dehydrogenase.

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2.6. Endogenous HBV DNA polymerase activity
assay
The culture media of 1.3ES2 cells were clarified by centrifu-
gation at 250,000×g for 1.5h. The pellets were dissolved in
NET buffer (50mM Tris–HCl, pH 7.4, 150mM NaCl, 1mM
EDTA, and 0.5% of Nonidet P-40 at 4◦C) and precipitated with
human antiserum against HBcAg. The endogenous polymerase
activity was measured as described previously (Chang et al.,
1987; Junker et al., 1987). Briefly, the immunoprecipitates were
suspended in HBV polymerase buffer (50mM Tris-HCl, pH
7.4, 40mM NH4Cl, 5mM MgCl2, 0.5% Nonidet P-40, 0.2%
2-mercaptoethanol, 25?M each of dATP, dGTP, dTTP, 100?Ci
[32P]-dCTP) and incubated for 2h at 37◦C. Then unlabelled
dCTP was added at a final concentration of 25?M, and incuba-
tion was continued for 2h. After washing with NET buffer to
remove the free [32P]-dCTP, proteinase K and sodium dodecyl
sulfate (SDS) were added to a final concentration of 200?g/mL
and 0.5%, respectively. The samples were incubated for 2h at
37◦C, followed by phenol/chloroform–isoamyl alcohol extrac-
tion. Then HBV DNA was precipitated with two volumes of
100% ethanol, and the DNA pellet was dissolved in 0.1-fold
TE buffer (10mM Tris, pH 8.0, 1mM EDTA), which contained
50ng/ml RNAase A.
2.7. Quantitative detection of HBV DNA by real-time light
cycler PCR
1.3ES2 cells were seeded in 100mm well plates at a den-
sity of 3×106cells/well in DMEM containing 10% fetal calf
serum.After24hofincubation,thecellswerewashedtwicewith
phosphate-buffered saline (PBS, pH 7.0) and treated with vari-
ous concentrations of drug in serum-free DMEM for 72h. For
quantificationofHBVDNA,viralDNAwasextractedfromcul-
ture media using the High Pure Viral Nucleic Acid Kit (Roche,
Mannheim, Germany) according to the Manufacturer’s instruc-
tions. A series dilution of known amounts of HBV–DNA was
used as a control. The standard curve showed a good linear
range when 102–107copies of plasmid DNA were used as tem-
plates.ThePCRprimersusedwerepurchasedfromTib-Molbiol
(Berlin, Germany). The oligonucleotide sequences of primers
were:HBVForward:5?-CAGGTCTGTGCCAAG-3?(theacces-
sion number of GenBank: AY128092, nt 1168–1182) HBV
Reverse: 5?-TGCGGGATAGGACAA-3?(nt 1359–1345). The
PCR cycling program consisted of an initial denaturing step at
95◦C for 10min, followed by 45 amplification cycles at 95◦C
for 12s, 54◦C for 20s.
2.8. Plasmids construction
AllplasmidswereconstructedbystandardDNAcloningpro-
cedures (Proudfoot and Baralle, 1979) and polymerase chain
reaction (PCR) methods (Mullis et al., 1986). To generate
pSPI-Luc,pSPII-Luc,pCP-Luc,andpXP-Luc,theXbal-HindIII
fragments containing the surface promoter I (SPI), surface
promoter II (SPII), or core promoter (CP) from pA3SPICAT,
pA3SPIICAT (Kuo and Ting, 1997), pA3CPCAT, pXPCAT
respectively, were inserted into the NheI-HindIII site of the
pGL3-Basic vector (Promega, Madison, WI). The pSPII-Luc
plasmid contains the entire major surface gene corresponding
to map positions at nucleotides 2869–3180 of HBV (the acces-
sionnumberofGenBank:AM282986,EcoRIsiteasnucleotide
1). The pCP-Luc plasmid contains core promoter of the HBV
pregenomic promoter (nt 1636–1851) into the pGL3-Basic vec-
tor. The pSPI-Luc plasmid was from nucleotides 2710 to 2826
and the pXP-Luc was from nucleotides 1177 to 1376 of HBV.
Plasmid pHBV1.3, containing a 1.3-fold-HBV genome (ayw
subtype) (Chou et al., 2005; Galibert et al., 1979), was used for
transient transfection experiments.
2.9. Transient transfections and luciferase
assay
HepA2 cells were transfected with various plasmids, using
the calcium phosphate precipitation method (Graham and van
der Eb, 1973). The cells were transfected in DMEM supple-
mented with 10% FCS for 16h. After 8–10h transfection,
the cells were transferred into a fresh medium to recover for
16–18h. The transfected cells were then incubated with serum-
free DMEM and treated with HE-145 for 2 days. To prepare
the total cell lysate from transfected cells for luciferase activity
measurements, the medium was aspirated from the cell culture
and the cells were gently rinsed with PBS. Cells were scraped
from the plates and collected by centrifugation. The supernatant
was collected for protein and luciferase activity measurements
immediately following lysate preparation. The protein concen-
trations of the resultant cell lysates were measured by the
Bradfordmethod(Bradford,1976).Usingaluminometerandthe
PromegaLuciferaseAssaySystemasdescribedbytheManufac-
turer(Promega,Madison,WI),lysatespreparedfromtransfected
cellswereanalyzedforluciferaseactivity.Foralltransienttrans-
fections with promoter-luciferase reporter constructs, the level
of luciferase activity was determined without drug treatment.
The transfection efficiency was normalized using the activity of
?-galactosidase as an internal control.
2.10. Gel electrophoretic mobility shift assay
Double-stranded oligonucleotide probes were obtained by
annealing equal moles of single-stranded complementary
oligonucleotides.TheprobescorrespondingtothedifferentSp1,
FTF, HNF3 or HNF4 binding sites, identified in the core pro-
moter of HBV, were labeled with [?-32P]ATP using T4 polynu-
cleotidekinase(Promega).Thesequencesoftheoligonucleotide
probes used for the EMSA are as follows: FTF: 5?-CAC CAA
ATA TTG CCC AAG GTC TTA-3?(nt 1631–1654). Sp1 (1): 5?-
CCG TGA ACG CCC ACC AAA TAT TGC-3?(nt 1620–1643).
Sp1 (2) and (3): 5?-TGG GAG GAG TTG GGG GAG GAG
ATT-3?(nt 1733–1756). HNF4 (1): 5?-AAG AGG ACT CTT
GGA CTC TCA GCA-3?(nt 1658–1681). HNF3 (1): 5?-GAC
TCT CAG CAA TGT CAA CGA ACC G-3?(nt 1671–1694).
HNF3 (2): 5?-TAC TTC AAA GAC TGT TTG TTT AAA-3?(nt
1706–1729). PPAR: 5?-GAG ATT AGG TTA AAG GTC TTT
GTA CT-3?(nt 1751–1780). HNF4 (2): 5?-GAG ATT AGG TTA

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AtG aTC TTT GTA CT-3?(nt 1751–1780). Non-specific: 5?-
TTG AGG CAT ACT TCA AAG ACT GTT-3?(nt 1698–1721).
Nuclear extracts were incubated with or without added unla-
beled competitor oligonucleotides in a total volume of 20?l
containing 10mM Hepes, pH 7.8, 50mM KCl, 2.5mM MgCl2,
20% glycerol, 0.5mM DTT, and 2.5mg of poly (dI-dC). After
15min of incubation on ice, the radiolabeled oligonucleotide
was added, and the reaction mixture was further incubated for
15minoniceandthen20minat30◦C.Protein–DNAcomplexes
were separated with a 5% polyacrylamide gel.
3. Results
3.1. Antiviral activity of HE-145 in stably HBV-transfected
HepG2 cells
We have shown previously that HE-145 and its analogues
have been reported to inhibit both HBV replicative intermedi-
ates and mRNA in stably HBV-replicated HepG2 2.2.15 cells
(Li et al., 2005). Since two other HBV genome stably trans-
fected HepG2 cell lines have been established, we intended to
examinemolecularmechanismofHE-145inthesecells.HepA2
cells contain a head to tail dimer HBV genome which continu-
ally secretes HBsAg and HBeAg into the culture medium (Yeh
et al., 1993). As shown in Fig. 1B, HE-145 suppressed both
HBsAg and HBeAg production with an IC50about 0.17?M
and 0.13?M, respectively, in HepA2 cells.
Fig.1. (A)ChemicalstructuresofHE-145andHE-145-9.(B)EffectofHE-145
on the production of HBsAg and HBeAg in HepA2 cells. HepA2 cells were
seeded on 24-well plates at a density of 8×104cells/cm2in DMEM with 10%
fetal calf serum and allowed to attach overnight. The cells were then washed
twice with phosphate-buffered saline (pH 7.0) and treated with various concen-
trations of HE-145 in serum-free DMEM for 48h. The amount of HBsAg and
HBeAgproductionintheculturemediumwasdeterminedbyenzymeimmunoas-
say. Viable cells in each well were determined by the WST-1 assay. Data are
expressed as mean±S.D. (n=3).
Fig. 2. Antiviral activity of HE-145 in 1.3ES2 cells. (A) 1.3ES2 cells were
seededon100-mmculturedishesandtreatedwithvariousconcentrationsofHE-
145 in serum-free DMEM for 48h. Total RNA was extracted from serum free
(SF)andHE-145-treatedcellsandanalyzedbyNorthernhybridizationwithHBV
DNA probe as described in Section 2. GAPDH was used as control. (B) 1.3ES2
cells were seeded. Different doses of HE-145 were added into the complete
medium (CM), and the expression level of secreted viral particles was analyzed.
HE-145wassupplementedat2daysafterplating(day0)andthetotalviralDNA
ofmediumwereharvestedevery2daysfor6days.DNAwereelectrophoresedin
1.2%agarosegel.Amountsofsecretedrelaxedcircular(RC)andlinear(L)forms
ofHBVwerereducedintheHE-145-treatedcultures.(C)Quantitativereal-time
PCR was used to detect HBV titer in the media of 1.3ES2 cells. Cultured cells
were treated with various concentrations of HE-145 (0, 1.5 and 5.0?M) and
HE-145-9 (5.0?M) in serum-free (SF) DMEM for 72h and media collected
for real-time PCR analysis using primer pair HBV DNA as template. Data were
expressedasmean±S.D.(n=3).ThedifferenceinHE-145(1.5?MinAandB,
1?M in C)-treated vs. untreated serum free (SF) cells is statistically significant
by t test (*p<0.05).
1.3ES2 cell line was also derived from HepG2 cells by sta-
ble integration of a 1.3-fold HBV genome (Chou et al., 2005).
Northern blot analysis revealed two major HBV specific RNA
species with molecular sizes of 3.5-kb (HBeAg mRNA and
pregenomic RNA) and 2.4/2.1-kb (large HBsAg mRNA and
middle/major HBsAg mRNA) in 1.3ES2 cells. As shown in
Fig. 2A, all three viral transcripts in 1.3ES2 cells were sig-
nificantly decreased in a dose-dependent manner after 48h of
HE-145 treatment. HE-145 also decreased the viral particles
released from 1.3ES2 cells into the culture medium measured
by Endogenous HBV DNA polymerase activity assay (Fig. 2B)
and Quantitative real-time PCR (Fig. 2C). Anti-HBV activ-
ity of HE-145 is structure-specific, since a synthetic analog
HE-145-9, shares similar chemical structure and hydropho-
bicity as HE-145 but showed no anti-HBV activity at all
(Fig. 2C).

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Fig. 3. HE-145 suppressed the promoter activity of HBV in HepA2 or Hep3B cells but not in HeLa or 293T cells. (A) Different cells were transfected with SPII
of HBV promoter region with luciferase reported gene, using the calcium phosphate precipitation method. Left panel: Hep3B and HepA2. Right panels: 293T and
HeLa. (B) Different cells were transfected with core of HBV promoter region with luciferase reporter gene. Left panel: Hep3B and HepA2. Right panels: 293T and
HeLa. (C) HepA2 and Hep3B cells were transfected with SPI of HBV promoter region with luciferase reporter gene. (D) HepA2 and Hep3B cells were transfected
with XP of HBV promoter region with luciferase reporter gene. After transfection, the cells were treated with serum free (SF) media (lane1), 0, 0.3, 1.5, 3.0?M
(lanes 2–5) of HE-145 for 48h. The transfection efficiency was corrected by co-transfecting ?-gal expression vector and assayed ?-gal activity simultaneously. The
normalized luciferase activities (CPS/OD) of core promoter were 2×106, 1.5×106, 7×105and 2.5×106in HepA2, Hep3B, HeLa and 293T cells, respectively.
The similar expression level of SPII was also observed in both liver and non-liver cells. Data were expressed as mean±S.D. (n=3).
3.2. HE-145 selectively suppressed viral promoter activity
for HBV major surface antigen and core antigen only in the
liver cells
Toinvestigatethemolecularmechanismofanti-HBVactivity
ofHE-145,weexaminedfourHBVpromoteractivitiesforlarge
viral surface antigen (SPI), major viral surface promoter (SPII),
viral core protein (CP) and viral X protein (XP), respectively,
upon HE-145 treatment, using luciferase as a reporter assay. We
foundthatHE-145selectivelysuppressedSPIIandCPpromoter
activities but has no effect on SPI and XP activities (Fig. 3A–D)
in both HepA2 and Hep3B cells. Interestingly, the suppressive
activity of HE-145 against HBV promoters is cell type-specific.
Neither SPII nor CP promoter activity was suppressed by HE-
145 in non-human liver cell HeLa or 293T cells (Fig. 3A and
B).
3.3. Effect of HE-145 on the DNA-binding activity of
nuclear extract of HepA2 cells to the cis element of Sp1,
FTF, HNF4, HNF3 and PPAR in HBV core promoter
HBV preC/C promoter/Enh II region contains many tran-
scriptional factor binding sites and many of these cis elements
have been shown to play critical role in viral gene expression
(Gilbert et al., 2000; Li and Ou, 2001; Yu and Mertz, 2003). We
designed twelve primer pairs to cover all the putative cis ele-
ments in this region to perform electrophoretic mobility shift
assay (EMSA) to evaluate which transcriptional factor bind-
ing site is responsible for anti-HBV activity of HE-145. As
shown in Fig. 4, synthetic DNA probe containing the Sp1, FTF,
HNF4, HNF3 and PPAR binding site could be bound by the
nuclearextractsofHepA2cellstoformacomplexwithaslower
electrophoretic mobility on the EMSA gel. The binding activ-
ity of nuclear extract prepared from HE-145-treated (1.5 or
3.0?M) HepA2 cells to Sp1, FTF, HNF4 and PPAR probes
was significantly decreased. However, the binding activity of
the same nuclear extract to HNF3 DNA probe did not yield any
significant change. The specificity of DNA-protein complexes
formation in EMSA assay was established by showing that the
complex band could only be competed by unlabeled specific
probe but not by the non-specific probe. Another control exper-
iment showed that treatment of HepA2 cells with the inactive
analog HE-145-9 has no effect on the DNA binding activity
examined.
3.4. Ectopic expression of PPARγ and HNF4α could
relieve the suppression of HE-145 to core promoter of HBV
In order to examine the involvement of PPAR? and HNF4?
in HE-145-mediated suppression of HBV genes expression, we
compared the anti-HBV activity of HE-145 in control and in
PPAR? or HNF4? ectopically expressed in HepG2 cells. We
co-transfected pHBV1.3 with control vector or expression vec-
tor for PPAR? or HNF4? and then examined HBV transcripts

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Fig. 4. HE-145 decreased the DNA-binding activity of FTF, Sp1, HNF4 and PPAR but not HNF3 in HepA2 cells. Gel electrophoretic mobility shift assays were
carried out as described in Section 2. Nuclear extracts of HepA2 cells were incubated with double-stranded32P-end-labeled FTF, Sp1, HNF4, HNF3 or PPAR
oligonucleotide, and treated with various concentrations of HE-145 (lanes 3, 4 and 5) or HE-145-9 (lane 6 in HNF4 and PPAR oligonucleotides) and untreated (SF,
lane 2) in serum-free DMEM for 48h. Probe incubated only without nuclear extracts is shown in lane 1. Excess unlabeled self-competitor oligonucleotide was added
to confirm specific binding (cold). Non-specific element (nt 1698–1721) showed no effect on the complexes.
in these cells after HE-145 treatment by Northern blot analysis.
As shown in Fig. 5, both 3.5-kb and 2.4/2.1-kb HBV transcript
transcribed from pHBV1.3 were enhanced by ectopic expres-
sion of HNF4? but not by ectopic expression of PPAR?. As
we expected, HE-145 suppressed both 3.5-kb and 2.4/2.1-kb
HBV transcript in the control cells. However, ectopic expres-
sionofHNF4?partiallyrelievedHE-145-mediatedsuppression
of both 3.5-kb and 2.4/2.1-kb HBV transcript. Surprisingly,
ectopic expression of PPAR? only marginally affected HE-
145-mediated suppression of 3.5-kb HBV RNA but completely
abolished HE-145-mediated suppression of 2.4/2.1-kb HBV
RNA.
4. Discussion
To develop an antiviral drug against HBV infection with a
novel mode of action to overcome the problems associate with
current approved drugs is still a major challenge. Previously, we
have shown that a new class of lignans, Helioxanthin (HE-145),
isolated from Taiwania cryptomerioides, has potent inhibitory
activity on HBV gene expression and replication in cultured
human hepatoma cells (Li et al., 2005). In this study, the mode
of action of HE-145 on HBV transcription and the involvement
of several nuclear receptors such as HNF3, HNF4, and PPAR
were examined.
In this study, all three HBV viral transcripts in 1.3ES2 cells
were significantly decreased under HE-145 treatment (Fig. 2A).
1.3ES2 cells have been shown to carry both chromosomal inte-
grated1.3copyofHBVgenomeandthecccformofHBVDNA
(Chou et al., 2005, 2007). Currently, we cannot distinguish the
viral mRNA transcribed from integrated HBV genome from the
viral mRNA transcribed from ccc DNA of HBV genome. How-
ever, HE-145 suppressed all viral transcripts expressed from
the transient transfected episomal form of HBV DNA (Fig. 5).
We speculate that HE-145 may suppress viral gene expression
from both chromosomal integrated HBV genome and episomal
cccDNA of HBV.
HBV has a very compact genome size and all four transcrip-
tional units extensively overlap. It is difficult to dissect or to
identify the cis element in HBV genome that is responsible for
the inhibitory activity of HE-145. We therefore examined the
effect of HE-145 on individual HBV promoter activity using
luciferase as reporter gene.
Our studies first revealed that HE-145 is a potent transcrip-
tional inhibitor of viral promoter for core antigen (CP) and
major surface antigen (SPII), but has no effect on other two

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Fig. 5. PPAR? and HNF4? involved in the HE-145 suppression of HBV gene
expression. (A) HepG2 cells were co-transfected pHBV1.3 with pCDNA3.1
or pPPAR? and pHNF4?, respectively. The transfected cells were cultured
in serum-free (SF) medium or treated with HE-145 3?M, and Northern blot
analysis was performed as described in Fig. 2A. (B) HE-145-mediated suppres-
sion was relieved by overexpression pPPAR? and pHNF4?. The intensity of
the 3.5-kb and 2.4/2.1-kb RNA was normalized with the GAPDH transcript.
The suppression activity of HE-145 to the HBV viral mRNA in the control
cells was assigned as 100%. The difference in HE-145-mediated suppression of
HBV mRNA between pCDNA- and PPAR? or HNF4?-overexpressed cells are
statistically significant (*p<0.05).
viral promoters for pre-S antigen and X protein in human hep-
atoma cells. This observation suggests that the reduction of
3.5kb HBV pregenomic RNA and 2.1kb major surface antigen
RNA introduced by HE-145 treatment may be mainly situ-
ated at the transcriptional level. Even though HE-145 has no
effect on viral promoter for pre-S antigen (Fig. 3), we con-
sistently observed the reduction of 2.4kb pre-S RNA after
HE-145 treatment (Fig. 2). One possibility is that the regula-
tion of the pre-S promoter activity in the intact HBV genome
is different from that in the cloned HBV DNA fragment. Pre-S
promoter activity may be influenced by several regulatory cis
elements such as viral enhancer I (Doitsh and Shaul, 2004)
or II (Fu et al., 1996; Su and Yee, 1992) which are located
thousand base pairs downstream of the Pre-S promoter. Sev-
eral studies have reported that the Pre-S promoter activity is
indeed influenced by viral enhancer I or II (Faktor et al., 1988).
Our results also showed that HE-145 reduced the activities
of HBV enhancer I when viral enhancer I is linked to the
viral promoter for X gene (data not shown). Therefore, HE-
145 reduced 2.4kb pre-S RNA and may act indirectly through
other control elements such as enhancer I or II in the HBV
genome. This explanation deserves further investigation in the
future.
Another interesting finding is that the inhibitory activity of
HE-145 against CP or SPII appears to be liver cell-specific. HE-
145 selectively inhibited CP and SPII activity in liver derived
HepG2orHep3Bcellsbutnotinnon-liverderivedHeLaor293T,
even though both CP and SPII exhibited significant activity in
either HeLa or 293T cells (Fig. 3A and B). The liver tropism
of HE-145-mediated activity suggests that the target of HE-145
may be the liver-specific transcriptional machinery that is also
responsible for liver tropism of HBV infection and replication.
The liver-specific inhibitory activity of HE-145 towards CP
mayprovideamechanisticexplanationonhowHE-145reduced
3.5kbHBVpregenomicRNAtranscribedeitherfromintegrated
HBV DNAs in the host genome (Fig. 2) or from transiently
transfected HBV DNA (Fig. 5). The 3.5kb HBV pregenomic
RNA not only encoded the core and polymerase protein, but
also served as template for reverse transcription. Therefore,
HE-145 reduced the 3.5kb HBV pregenomic RNA production
through blocking viral CP activity, which is consistent with
our observations that HE-145 decreased intracellular replicative
intermediates of HBV DNA (data not shown) and HBV viral
particle production by 1.3ES2 cells (Fig. 2).
Recent studies by Ying et al. on the anti-HBV activity of one
analogue of HE-145 in human hepatoma cell line, HepG2.2.15
have reached similar conclusions as ours (Ying et al., 2007).
However, two discrepancies between their conclusions and ours
have been noticed. First, they suggested that HE-145 inhibits
CP activity may be due to the decrease of HNF4? and HNF3?
protein. In our study, we did not observe any changes in the pro-
tein levels of HNF4?, PPAR?, C/EBP?, and C/EBP? in HepG2
cells after HE-145 treatment (data not shown). Secondly, Ying
et al. reported that HE-145 suppresses all four viral promoter
activities, whereas we found that HE-145 selectively suppresses
CP and SPII without affecting the SPI and XP promoters in the
HepG2 and Hep3B cells. One possible explanation is that we
examined protein level of HNF4? and viral promoter activity in
cells treated with HE-145 only for 48h in serum-free medium.
In contrast, Ying et al. treated cells with HE-145 in the medium
with 10% FCS for 6 days. We observed no changes of the abun-
dance of HNF4? protein, while both CP activity and binding
activity of nuclear extract to HNF4 response element were sig-
nificantly reduced in HE-145-treated HepG2 cells. Therefore,
the primary effect of HE-145 may be the direct interference of
specific transcriptional complex formation in vivo. The reduc-
tion of HNF4? protein and the inhibition of SPI or XP promoter
activity may be the consequence of the long-term treatment of
HE-145.
How exactly HE-145 inhibits HBV viral CP activity is still
not clear. The CP consists of a basal core promoter (BCP) and
a core upstream regulatory sequence (CURS) that is overlapped
with the enhancer II and contains several binding sequences of
differentnuclearreceptors.EMSAanalysisrevealedthattheHE-
145 treatment reduced binding activity of nuclear extract to the
response elements of Sp1, HNF4, FTF and PPAR, respectively.
However, HE-145 has no effect on the binding of HNF3 to its
response element in the CP. We proposed that HE-145 may not
reduce protein level of transcriptional factors but interfere with
the transcriptional complex formation in viral core promoter in

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213
vivo. Alternatively, HE-145 may directly associate with the CP
specific transcriptional machinery in vivo and reduce its affinity
to the cis element in vitro. Our current study cannot distinguish
betweenthesetwopossibilities.Aquantitativecomparisonofthe
abundanceandaffinityofthespecifictranscriptionalcomplexto
CP in the control and HE-145-treated cells may solve this criti-
calquestion.HE-145treatmenthasnoeffectontheproteinlevel
of PPAR? and HNF4?. Then, why can overexpression of either
PPAR? or HNF4? partially reduce the suppression activity of
HE-145 towards the production of both 3.5 and 2.4/2.1kb HBV
transcripts in HepG2 cells? All attempts to show specific physi-
cal interaction between HE-145 and different nuclear receptors
failed(datanotshown).Onepossibilityisthatoverexpressionof
PPAR? or HNF4? may shift the less active transcriptional com-
plex induced by HE-145 to a more active state. We are currently
investigating this possibility by a quantitative CHIP assay.
WehaveshownthatHE-145suppressedtranscriptionalcom-
plexformationinHBVcorepromoterinvivo.However,HE-145
neither reduces binding of transcriptional complex to HBV core
promoter in vitro, nor binds to naked DNA directly (data not
shown). We could not rule out the possibility that HE-145 may
be modified or degraded by hepatic CYP450 enzymes in vivo
and that the metabolites of HE-145 possess anti-HBV activity.
In conclusion, our results strongly suggest that HE-145 is an
anti-HBV agent with a novel mode of action. It may act through
liver-specific transcriptional machinery for viral CP to inhibit
HBV viral gene expression and viral particle production.
Acknowledgments
The authors thank Dr. Ling-Pai Ting for providing pA3SPI-
CAT, pA3CP-CAT and pA3SPII-CAT plasmids, and Dr. Chung
Ming Chang for providing the pXp-CAT plasmid. We are
grateful that this work was supported by grants NSC 96-3112-
B-010-005 and CMRPD140103.
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