(03263)Xiong Wei et al.: Gene Expression in HBV Transfected Cell Line Induced by IFN

Analysis
of Gene Expression in Hepatitis B Virus Transfected Cell Line Induced by
Interferon

XIONG
Wei, WANG Xun, LIU Xiao-Ying, XIANG Li, ZHENG Ling-Jie, LIU Jiang-Xia, YUAN Zheng-Hong*

( Key Laboratory of Medical Molecular
Virology, Ministry of Education and Health, Shanghai Medical College, Fudan
University, Shanghai 200032, China )

Abstract        Infection
of hepatitis B virus (HBV) continues to be a significant health problem. α interferon (IFN-α) and γ
interferon (IFN-γ) have been proven to be
effective in inhibiting HBV replication. To study the global effect of HBV
persistent existence on IFN induced cellular gene expression, cDNA microarrays
dotted with 14 112 human genes were used to examine the transcriptional changes
between an HBV DNA transfected cell line (HepG2.2.15) and its parental cell
line (HepG2) after the treatment of IFN-α
or IFN-γ for 6 h. The results showed
that many genes related to cell cycle, proliferation, apoptosis and new ESTs
were regulated by IFN-α and/or IFN-γ. Many genes involved in kinase and
signal transduction, transcription regulation, antigen presentation and
processing were differentially regulated between these two cell lines post IFN-α or IFN-γ
treatment. Interestingly, several IFN-differentially regulated genes, such as
MyD88 and Diubiquitin, were found to inhibit HBV gene expression, and MyD88 was
proved to inhibit HBV replication. Taken together, our results revealed the
global effects of HBV persistent existence on IFN-induced cellular gene
expression. The novel antiviral genes identified by microarray could be
potentially developed as new anti-HBV drugs or for novel therapies.

(Abstract in Chinese)

Key
words     cDNA microarray;
hepatitis B virus; interferon; cellular gene expression

Infection of hepatitis B virus (HBV)
continues to be a significant health problem. It is estimated that there are
approximately 350 million chronic hepatitis B patients worldwide. These
patients have a high risk of developing liver cirrhosis and hepatocellular
carcinoma with high mortality rate (15%–25%)[1].
In the past two decades, α interferon (IFN-α) has been proven effective in treating
chronic hepatitis B patients[2]. However, when treated with IFN-α alone, only about 30%–40% patients achieved sustained-response.
Recently, extensive researches have been undertaken to study the antiviral
effect of other antiviral cytokines by using HBV transfected cell lines or HBV
transgenic mice. It has been reported that γ
interferon (IFN-γ) secreted by HBV specific cytotoxic
T lymphocytes also effectively inhibited HBV replication. However, the details
of the antiviral mechanisms have not been fully elucidated yet[3].

In this study, cDNA microarrays dotted
with 14 112 human genes were used to analyze transcriptional difference between
an HBV DNA transfected cell line (HepG2.2.15) and its parental cell line
(HepG2) post IFN treatment for 6 h. Many genes were found to be differentially
regulated in expression between these two cell lines post IFN treatment. The
effects of several sensitive genes on HBV replication and expression were
further studied.

1    Materials
and Methods

1.1   Cells, cell culture and RNA
isolation

The HBV DNA transfected cell line
HepG2.2.15 derived from human hepatoblastoma cell line HepG2 has been confirmed
to stably produce infectious HBV particles[4]. HepG2 and HepG2.2.15 cells were
separately seeded into three T-75 flasks at a density of 3×10⁶ cells per flask, and
cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf
serum plus penicillin (105 IU/L) and streptomycin (0.1 g/L ) (Gibco BRL). After
cultured for 24 h, the cells were treated with 1000 IU/mL recombinant human
IFN-α or IFN-γ (R&D Systems), or remained
untreated, respectively. After incubated for an additional 6 h, all cells were
harvested. Total RNA was extracted using Trizol reagent (Gibco BRL), and mRNA
was then isolated from total RNA by Oligotex mRNA midi kit (Qiagen).

1.2   Construction of microarray and
probe preparation

The microarrays consisted of 14 112 cDNA
clones representing novel, known and control genes were provided by United Gene
Holdings. The cDNA inserts were amplified by PCR using universal primers
targeting plasmid or vector containing relevant gene fragments and then
purified. All PCR products were examined by gel electrophoresis to ensure the
quality and the identity of the amplified clones. The amplified PCR products
were then spotted onto silylated slides (CEL Associates) using a Cartesian
PixSys 7500 motion control robot (Cartesian Technologies). Glass slides with
spotted cDNA were hydrated for 2 h in 70% humidity, dried for 0.5 h at room
temperature, and UV crosslinked. By further being soaked in 0.2% SDS for 10
min, distilled H₂O for 10 min, and 0.2% sodium borohydride for 10
min, these slides were dried again and ready for use. The fluorescent cDNA
probes were prepared by reverse transcription of the isolated mRNAs and then
purified according to Schena et al.[5]. The RNA samples from IFN untreated
cells were labeled with Cy3-dUTP, and those from IFN treated cells with
Cy5-dUTP.

1.3   Hybridization and washing

Microarrays were pre-hybridized in
solution containing 0.5 g/L denatured salmon sperm DNA at 42 ℃ for 6 h. Fluorescent probe mixtures were
denatured at 95 ℃ for 5 min, and then applied
onto the pre-hybridized chip under a cover glass. Chips were hybridized at 42 ℃ for 16–18
h. The hybridized chips were then washed at 60 ℃
for 10 min each in solutions of 2×SSC
and 0.2% SDS, 0.1×SSC and 0.2% SDS, and 0.1×SSC, then dried at room temperature.

1.4   Detection and analysis

The chips were scanned with a GenePix
4000B array scanner (Axon) at two wavelengths to detect emission from both Cy3
and Cy5. The acquired images were analyzed using GenePix Pro3.0 software
(Axon). The intensity of each spot at the two wavelengths represents the
quantity of Cy3-dUTP or Cy5-dUTP hybridized respectively. Ratios of Cy5 to Cy3
were analyzed for each location on the microarray. Overall intensities were
normalized with a correction coefficient based on the ratios of the
housekeeping genes.

1.5   RNA slot analysis

Total cellular RNA (10 μg) was mixed with 7 μL formaldehyde, 20 μL formamide and 2 μL 20×SSC.
After denatured at 65 ℃ for 10 min, the RNA sample
was applied to the nylon membrane (Roche) by Minifold I (Schleicher &
Schuell). After fixation at 120 ℃ for 30 min, the membrane was
pre-hybridized at 42 ℃ for 6 h in solution
containing 0.1 g/L denatured salmon sperm DNA, and then hybridized with cDNA
probes labeled with [α–³²P]dCTP by
Hexamer random labeling kit (Roche) at 42 ℃
for 16 h. After stringent washing process in 68 ℃,
the membranes were exposed to X-ray film and detected by autoradiography at －70 ℃.
To normalize total RNA quantity, blots were stripped and rehybridized with [α–³²P]dCTP labeled β-actin probes. Quantitative analysis was
undertaken by scanning the intensity of blots. The change of gene expression
between samples could be detected by comparing the ratio of the intensity of
specific gene and β-actin.

1.6   Construction of plasmids

Interferon-inducible 56 kD protein (P56)
(GenBank No: X03557), Diubiquitin (GenBank No: Y12653), MyD88 (GenBank No:
U70451) and DKFZp564A032 (GenBank No. AL050267) encoding genes were amplified
by RT-PCR from the total RNA of HepG2 cells. These amplified segments were
cloned into pET-23a (Novagen) and then transferred into eukaryotic expression
plasmid pcDNA3.1/myc-His (Invitrogen). These plasmids were referred to as
pcDNA-p56, pcDNA-DIU, pcDNA-MyD88, and pcDNA-DKF, respectively. Plasmid pHBV3.8
encoding the whole transcript of HBV DNA (adr subtype) from the core promoter
to the polyA signal region (nucleotides 1403–3215
plus 1 to 1987), was constructed from vector pBS+ (Stratagene) with a 1.2 copy
of the full-length HBV genome between restriction enzyme EcoRI and PstI sites
(kindly provided by Prof. WANG Yuan, Institute of Biochemistry and cell
Biology, the Chinese Academy of Sciences). After transfected into HepG2 cells,
the pHBV3.8 can express both HBV surface antigen (HBsAg) and e antigen (HBeAg),
and the replication of HBV can be initiated in the cells. Plasmid pcDNA-CAT
containing the chloramphenicol acetyltransferase (CAT) gene under control of
the CMV promoter was constructed as described above.

1.7   Cell transfection and analysis of
HBV proteins

HepG2 cells were seeded onto a 12-well
plate at the density of 1×10⁵ cells per well.
Plasmid pcDNA-p56, pcDNA-DIU, pcDNA-MyD88, pcDNA-DKF, or pcDNA3.1 (each 2.0 μg) was transiently co-transfected with
pHBV3.8 (1 μg) and pcDNA-CAT (0.3 μg) into HepG2 cells using the calcium
phosphate precipitation method. After 16 h post-transfection, the culture
medium was replaced with fresh medium with or without 1000 IU/mL recombinant
human IFN-α. After transfected for 48 h,
culture supernatants were collected and analyzed for the expression of HBsAg
and HBeAg by standard enzyme linked immunosorbent assay (ELISA) (Sino-American
Biotech.). Transfection efficiency was normalized by detecting the activity of
CAT in cell lyses using CAT ELISA (Roche). Results were representative of three
independent experiments performed in duplicate.

1.8   Southern blot analysis of viral
replicative intermediates

HepG2 cells were seeded onto four 60-mm
dishes at the density of 1×10⁶ cells per dish.
Plasmids pHBV3.8 (6 μg) and pcDNA-CAT (1 μg) were transiently co-transfected with
pcDNA-MyD88, pcDNA-DKF, or pcDNA3.1 (each 12 μg)
into HepG2 cells. At 16 h post-transfection, medium was replaced with fresh
medium or medium supplemented with 1000 IU/mL recombinant human IFN-α. After transfected for 48 h, cells were
washed twice with chilled PBS and lysed in 650 μL
of lysing buffer (10 mmol/L Tris-HCl, pH 7.9, 1 mmol/L EDTA, 1% NP-40, 80 g/L
sucrose). After centrifugation at 12 000 r/min for 2 min at 4 ℃, nuclei and debris were removed and
supernatant was collected. 50 μL supernatant was kept to
detect the activity of CAT to normalize transfection efficiency. The
intracellular core particles were purified from the rest of the supernatant and
the HBV replicative intermediate DNA was extracted from core particles as
described by Lin et al.[6]. The normalized viral replicative intermediate DNA
were electrophoresed onto 1% agarose gel and blotted onto a positive nylon
membrane (Roche). Hybridization was undertaken as described above using [α–³²P]dCTP labeled full-length
HBV DNA probes. After stringent washing process, the signals were detected by
autoradiography. The experiments have been performed twice and blots were
quantified by densitometry.

2    Results

2.1   Quality control of microarray
hybridization

To assess the specificity of microarray
hybridization, reference genes including Arabidopsis gene (16 spots),
the rice COP II gene (16 spots), the hepatitis C virus coat protein gene
(8 spots) and blank solution were applied as negative control. All those spots were
undetectable after hybridization. To assess the reproducibility of the
microarrays, 82 housekeeping genes were applied to normalize the differences of
signal intensity between microarrays.

2.2   Global characteristics of gene
expression in HepG2 and HepG2.2.15 cell lines post IFN-α or IFN-γ treatment

In HepG2 cells, the number of genes whose
expression changed more than 3.0-, 2.0- or 1.8-fold was 18, 104 and 278 for
IFN-α; 30, 134 and 241 for IFN-γ. In HepG2.2.15 cells, the number of
changed genes was 23, 129 and 293 for IFN-α;
8, 101 and 218 for IFN-γ. To better characterize the
gene expression post IFN-α or IFN-γ treatment, the genes changed more than
1.8-fold in expression of HepG2 and HepG2.2.15 cells were classified in
functional categories (Table 1). Results showed that many genes
including those for cytoskeletal and extracellular matrix, interferon
inducible, ligands and receptors, kinases and signal transduction, protease and
proteasome components and transcription factors were significantly changed in
HepG2 and HepG2.2.15 cells. It is interesting to note that about 60% to 80% of
the changed genes belong to new ESTs or unknown genes.

Table 1   Functional categories of over 1.8-fold regulated genes
in HepG2 and HepG2.2.15 cell lines post IFN-α or IFN-γ treatment

The
number of gene expression up-regulated (↑)
or down-regulated (↓)
over 1.8-fold in HepG2 cells (HepG2) and HepG2.2.15 cells (2215) post IFN-α
or IFN-γ
treatment was presented.

2.3   Genes differentially regulated over
2.0-fold in HepG2 and HepG2.2.15 cell lines post IFN-α or IFN-γ treatment

To compare the changes of gene expression
in both cell lines after IFN-α or IFN-γ treatment, partial genes regulated over
2.0-fold were selected (Table 2). The data show the ratios of partial
2.0-fold regulated genes in HepG2 cells (HepG2) and HepG2.2.15 cells (2215)
post IFN-α or IFN-γ treatment.As data shown, some genes,
such as MxA, p56 and IFP35, were only regulated by IFN-α, but not IFN-γ, while some genes, such as γ2 protein and Diubiquitin, were only
regulated by IFN-γ, but not IFN-α. However, some genes, such as PBEF,
p9-27 and 1-8D, were regulated by both IFN-α
and IFN-γ. Interestingly, it was
observed that, for some genes, the changes of gene expression between HepG2 and
HepG2.2.15 cells were greatly different even after IFN treatment. For example,
after treated by IFN-α or IFN-γ, the transcript levels of some genes,
such as Weel hu, BTG1, BTG2, PNAS-2, MCP-3 and SMIF, were higher in HepG2 cells
than in HepG2.2.15 cells, while the transcript levels of other genes, such as
Grb14, PRKY and carboxypeptidase D, were lower in HepG2 cells than in
HepG2.2.15 cells.

Table 2   Differentially expressed genes in HepG2 and HepG2.2.15
cell lines post IFN-α or IFN-γ treatment

2.4   Validation of microarray results

To confirm the results of microarray, RNA
slot analysis was carried out to analyze the expression of p56, Diubiquitin,
MyD88 and DKFZp564A032 in HepG2 and HepG2.2.15 cells after treated with IFN-α or IFN-γ
for 6 h (Fig.1). The ratios of the signal intensity of specific genes in
HepG2 and HepG2.2.15 cells before or after treated with IFN-α or IFN-γ
for 6 h were listed in Table 3. It is observed that the result of RNA
slot analysis was aligned with the result from the microarray analysis.

Fig.1       RNA slot analysis of gene
expression after IFN treatment in HepG2 and HepG2.2.15 cells

The mRNA level of P56 (A), Diubiquitin
(B), MyD88 (C), and DKFZp564A032 (D) in HepG2 cells (HepG2) and HepG2.2.15
cells (2215) after IFN-α
or IFN-γ
treated for 6 h was analyzed by RNA slot (upper panel) and β-actin
gene was used as control to normalize the total RNA (lower panel).

Table 3   Comparison of microarray and RNA slot analyses on
selected genes

The
values represent the ratio of gene expression in HepG2 cells (HepG2) and
HepG2.2.15 cells (2215) post IFN-α
or IFN-γ
treatment compared to that of IFN-untreated cells.

2.5   Identification of novel cellular
genes that inhibit HBV proteins synthesis and gene replication

To study the role of novel cellular genes
induced by IFN in antiviral activity, p56, Diubiquitin, MyD88 and DKFZp564A032,
were cloned into eukaryotic expression plasmid and transiently co-transfected
with HBV replicative competent plasmid into HepG2 cells. The antiviral activity
of those genes against HBV proteins synthesis was investigated by detection of
HBsAg and HBeAg in the culture supernatants. Results showed that the expression
of Diubiquitin and MyD88 could effectively decrease the secretion of HBsAg and
HBeAg in transfected cells, while the expression of p56 and DKFZp564A032 had no
apparent effect (Fig.2). Southern blot analysis was further carried out
to detect effect of MyD88 and DKFZp564A032 on HBV replication. Compared to IFN-α treatment, the expression of MyD88 could
also inhibit HBV replication by dramatically reducing the synthesis of HBV
replicative intermediates, while DKFZp564A032 had no apparent effect on HBV
replication (Fig.3).

Fig.2       Effect of the expression of
selected cellular genes on the syntheses of HBV s and e antigens

Plasmid pHBV3.8 was transiently
co-transfected to HepG2 cells with pcDNA3.1 (V), pcDNA3.1 (V+IFN), pcDNA-p56
(V+p56), pcDNA-DIU (V+DIU), pcDNA-MyD88 (V+MyD), or pcDNA-DKF (V+DKF).
pcDNA-CAT was included as the internal control of transfection efficiency. At
16 h post-transfection, 1000 IU/mL IFN-α
was added in IFN treated group (V+IFN). After transfected for 48 h,
supernatants were collected to detect the expression of HBV s antigen (HBsAg)
and HBV e antigen (HBeAg) by the standard immunoassay. Bars show the standard
errors of three independent experiments performed in duplicate.

Fig.3       Effect of the expression of selected
cellular genes on HBV gene replication

Plasmid pHBV3.8 was transiently
co-transfected with pcDNA3.1(V), pcDNA3.1 (V+IFN), pcDNA-MyD88 (V+MyD),
pcDNA-DKF (V+DKF) into HepG2 cells. pcDNA-CAT was included as the internal
control of transfection efficiency. At 16 h post-transfection, 1000 IU/mL IFN-α
was added in IFN treated group (V+IFN). Cells were harvested 48 h after
transfection and HBV replicative intermediates DNA extracted from core
particles was loaded onto 1% agarose gel according to the transfection
efficiency detected by CAT ELISA and then transferred to nylon membranes. For
hybridization, full-length HBV DNA probes labeled with [α–³²P]dCTP
were used. The arrows indicated relaxed circular (RC), double stranded linear (DS),
and single stranded linear (SS) HBV DNA forms. The values of scanned bands were
shown in histograms.

3    Discussions

Persistent infection of HBV is the
outcome of the interaction between HBV and host cells, which ultimately leads
to the development of liver cirrhosis and hepatocellular carcinoma[7]. As the
interaction between HBV and host cells is complex and involved in many cellular
signaling pathways, only parts of them have been defined. Development of
microarray technology has provided an opportunity to analyze the complex
interaction at the global level.

In this study, HepG2.2.15 cell line,
which was derived from HepG2 cell line by integrating HBV genomes and could
persistently secrete HBV virions, was used as HBV infected cells. The HepG2
cell line was used as control of uninfected cells. The gene expression profiles
of these two cell lines post IFN-α
or IFN-γ treatment for 6 h were
examined by microarrays. Our previous studies showed that the mRNA levels of
many cellular genes between HepG2 and HepG2.2.15 cells prior to IFN-α or IFN-γ
treatment were different (data not shown). In addition to HBV replication, this
difference may also be related with cellular genes’ integration and
rearrangement. To observe the effects of HBV gene expression and replication on
IFN induced gene transcription, the genes, which did not show apparent
difference of expression (below 2.0-fold) between these two cell lines pre IFN
treatment but was significantly regulated (over 2.0-fold) post IFN treatment,
were selected for further study (Table 2).

IFN-α
and IFN-γ are pleiotropic cytokines
that mediate antiviral, antiproliferative, antitumor responses and modulate the
immune system. Upon binding to their specific cell surface receptors, IFN-α and IFN-γ
activate the expression of a number of cellular genes, through the distinct,
yet not completely understood pathways of signaling cascade in a rapid and
direct manner[8]. Our results showed that the expression of many genes with
known biological function was regulated by IFN-α and/or IFN-γ. For example, MxA and 2-5 OAS
that have been proved to be IFN-induced antiviral genes were all up-regulated
in HepG2 and HepG2.2.15 cells post IFN-α
treatment[8]; Weel hu, BTG1 and BTG2, which were associated with cell cycle and
proliferation, were also up-regulated in HepG2 cells post IFN-α or IFN-γ
treatment; likewise, many apoptosis related genes including those for apoptosis
related protein (PNAS-2), kinase and signaling transduction proteins (S6K,
DRAK1, MAP4K3), and transcription factors (PCAF, SMIF) were significantly
up-regulated in HepG2 after the treatment of IFN-α
or IFN-γ. Increasing expression of
these genes would improve the sensitivity of cells to apoptosis[9－14]. Besides, the expression of many
mitochondrial respiratory chain related genes was down-regulated after IFN-α or IFN-γ
treatment, which could affect the mitochondrial metabolisms and lead to
apoptosis[15,16]. By regulating cellular gene transcription, IFN not only
induces direct antiviral response, but also regulates cell cycle, proliferation
and apoptosis to indirectly interfere with viral replication.

It has been inferred that persistent HBV
infection exerts great impact on the expression of IFN-induced cellular genes.
In accordance with this hypothesis, compared to HepG2 cells, many genes, such
as those for cell cycle and proliferation (e.g. Weel hu, BTG1, BTG2), kinases
and signaling transduction (e.g. S6K, DRAK1, MAP4K3, MyD88), transcription
factor (e.g. PCAF, SMIF), antigen presentation and processing (e.g. RING4, LAP,
Diubiquitin, LMP7), were differentially regulated in HepG2.2.15 cells post IFN-α or IFN-γ
treatment. The transcription of those genes was significantly up-regulated
(over 2.0-fold) by IFN-α and/or IFN-γ in HepG2 cells, but were only mildly
increased (below 1.5-fold) or reduced in HepG2.2.15 cells (Table 2). The
differential expression of MyD88 and Diubiquitin between these two cell lines
was confirmed by RNA slot analysis (Fig.1, Table 3). The
reduction of those genes transcripts might decrease the IFN-induced antiviral
response and host immune defense. For example, LAP, RING4 and LMP7 are
associated with the presentation and processing of antigen and the generation
of epitopes[17–19]; Diubiquitin containing
two ubiquitin-like domains in head-to-tail arrangement was found to be
generally and synergistically induced by IFN-γ
and TNF-α, indicating its functions in
antigen presentation or other cellular processes controlled by these two
cytokines[20,21]; MyD88 protein is a critical component of signaling cascade,
which is mediated by Toll-like receptors as well as IL-1 receptor and IL-18
receptor[22,23] and can lead to liberation of nuclear factor-kappa B (NF-κB) into the nucleus to activate gene
transcription[22,23]. The reduction of the transcription of these genes in HBV
existing cells may protect HBV from host cells’ eradication, while the
increasing expression of these genes may inhibit HBV replication. Further
analysis of the antiviral activity of some IFN-induced genes against HBV by
transient transfection experiment could support the above hypothesis. For
example, over-expression of Diubiquitin and MyD88 could effectively inhibit the
synthesis of HBV proteins (Fig.2). Furthermore, MyD88 was found to
inhibit HBV replication by reducing the synthesis of HBV replicative
intermediates (Fig.3). Thus, HBV may decrease the expression of some IFN
induced genes to escape the antiviral effect of the IFN system, which may
partially explain the mechanisms of the persistent infection of HBV.

Besides, it is interesting to note that,
many new ESTs or yet uncharacterized genes were significantly regulated in
HepG2 and HepG2.2.15 cells after IFN treatment. Presumably, some of these genes
may play an important role in regulating HBV replication and participating in
the antiviral response of host cells.

Taken together, our results revealed the
global effects of HBV persistent existence and IFN treatment on the expression
of cellular genes. By examining differential gene expression between HepG2 and
HepG2.2.15 cells post IFN treatment, this study provided a unique opportunity
to investigate the complex mechanisms responsible for the antiviral activity of
IFN and the persistent existence of HBV in the cells. Work is in progress to
further investigate the hypothesis suggested in this study by manipulating the
genes, which are differentially expressed between HepG2 and HepG2.2.15 cells as
well as those markedly down- or up-regulated by IFN treatment. The novel
antiviral genes identified by microarrays could be potentially developed as new
anti-HBV drug or for novel therapies.

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_____________________________

Received:
July 10, 2003        Accepted:
September 12, 2003

This
work was supported by the grants from the Major State Basic Research
Development Program of China (973 Program)(No. 1999054105), Med-X Foundation of
Fudan University and Graduate Innovation Foundation of Fudan University

*
Corresponding author: Tel, 86-21-64161928; Fax, 86-21-64227201; e-mail, [email protected]

Updated
at: 2003-12-18