Lack of a Role of the Interferon-stimulated Response Element-like
Region in Interferon
-induced Suppression of Hepatitis B Virus
in Vitro*
Andreas
Rang,
Tilman
Heise, and
Hans
Will
From the Heinrich-Pette-Institut für Experimentelle Virologie
und Immunologie an der Universität Hamburg, Martinistra
e
52, D-20251 Hamburg, Germany
Received for publication, August 28, 2000, and in revised form, November 16, 2000
 |
ABSTRACT |
The antiviral effect of interferon-
(IFN
)
on hepatitis B virus (HBV) is well documented in vitro and
in vivo, but the mechanisms involved are elusive. Recently,
an interferon-stimulated response like element (ISRE) competent for
binding of interferon-stimulated gene factor-3
(p48) has been
identified in the HBV enhancer I region. Mutation of this element was
shown to abrogate IFN
-mediated reduction of HBV X-gene
promoter-driven reporter gene expression. This suggested a role of the
ISRE and of p48 in IFN
-induced antiviral activity against productive
HBV infection. Here, we analyzed the antiviral effect of both IFN
and enhanced p48 expression on complete HBV genomes containing the
wild-type or mutated ISRE. In human hepatoma cells transfected with
both genomes, viral RNA and replicative intermediates were reduced by
IFN
treatment to a similar degree. Enhanced p48 expression increased
IFN
-induced suppression of HBV RNA significantly from 75 ± 22.5% to 46 ± 9.8%, but this was independent of the integrity
of the ISRE-like region. These data imply that p48 neither mediates the
antiviral activity of IFN
against HBV nor down-regulates enhancer I
activity by binding directly to the HBV ISRE-like region, but rather
argue for an indirect role of p48.
 |
INTRODUCTION |
Hepatitis B virus (HBV)1
is a small (3.2 kilobases) enveloped DNA virus, the replication of
which involves reverse transcription of a pregenomic RNA in the
cytoplasm within nucleocapsid, resulting in production of replicative
DNA intermediates (1). From the cytoplasm, the nucleocapsids are either
shuttled to the endoplasmic reticulum and converted to mature virions
for transport out of the cell or to the nucleus for the establishment
of a pool of covalently closed circular DNA (cccDNA) (2). The
cccDNA in the nucleus serves as template for transcription of the
pregenomic/C mRNA and subgenomic RNAs. The designation pregenomic/C
mRNA derives from the fact that it serves both for translation of
the core protein and as template for the generation of the viral DNA
genome by reverse transcription.
Worldwide, more than 300 million people are chronically infected with
HBV (3). Interferon-
(IFN
) treatment is one of the few effective
therapies for chronic HBV carriers. However, it results in efficient
reduction of the viral load only in 10-20% of treated patients and
rarely, if at all, in complete virus elimination. So far, the
mechanisms responsible for the IFN
-mediated reduction of the viremia
in responding patients remain elusive. In vitro, IFN
-induced antiviral mechanisms against HBV have mostly been examined by using hepatoma cell lines expressing viral products from
integrated or transiently transfected HBV DNA, since cell lines
permissive for HBV infection are not available. Antiviral studies with
IFN
in these systems led to intracellular inhibition of one or
several HBV products depending on the type of cell line and system used
(4-14). In the human hepatoma cell line Hep-HB107, which stably
expresses HBV from a chromosomally integrated copy of the viral genome,
IFN
treatment reduces the amount of intracellular replicative DNA
intermediates, but affects neither viral polymerase activity within
nucleocapsids nor the steady state levels of the viral RNAs (8). These
data indicate that a posttranscriptional step of viral replication is
targeted by IFN
-induced mechanisms, which might affect encapsidation
of the pregenomic RNA or the stability of cytoplasmic nucleocapsids. On
the other hand, IFN
-induced reduction of the viral RNA was observed
in studies using a further stably transfected hepatoma cell line
(HepG2.2.15) (5) and the transiently transfected human hepatoma cell
line HuH7 (12). In HuH7 cells viral RNA levels were reduced
posttranscriptionally by IFN
-induced mechanisms. The amount of
replicative intermediates was more effectively reduced, suggesting that
IFN
induces at least two independent antiviral activities acting on
different levels (12). Furthermore, IFN
was reported to suppress the rate of transcription by down-regulating the activity of the HBV core
promoter (13) and of the HBV enhancer I (14), as determined in reporter
gene experiments with subgenomic HBV DNA fragments. Whether this also
occurs in hepatoma cells producing complete HBV genomes has so far not
been analyzed.
Taken together, it is well established that IFN
can induce an
antiviral state against HBV in vitro, but neither the
cellular proteins nor the IFN
-sensitive HBV targets involved are
clear. In general, induction of an antiviral state in a cell by IFN
starts with the activation of the cytoplasmic protein tyrosine kinases
Tyk2 and Jak1, and phosphorylation of latent cytoplasmic transcription
factor subunits called STATs (signal transducer and activator of
transcription) (15, 16). The STATs then assemble with interferon
stimulated gene factor 3
(p48) to form functional IFN-stimulated
gene factor 3 (ISGF3), which translocates into the nucleus and binds to
IFN-stimulated response elements (ISRE) located within the promoter
region of IFN
-inducible genes.
Recently, in HuH7 cells treated with IFN
, a protein complex
containing the transcription factor p48 was reported to interact with
an ISRE-like region located within the enhancer I region of the HBV
genome (11). Functional inactivation of the ISRE-like element that
abrogated p48 binding in gel shift experiments abolished IFN
-induced
suppression of HBV enhancer I activity in reporter gene expression
experiments performed with subgenomic HBV DNA fragments. These results
suggested that IFN
triggers direct binding of p48 to HBV DNA in
concert with other proteins and thereby can modulate enhancer I
activity. Since promoter and enhancer activities of HBV in part differ
dramatically when full-length genomes or subgenomic fragments are
tested (17), the relevance of these findings for IFN
-induced
suppression of HBV propagation remains to be determined. This should be
possible in hepatoma cells producing infectious virus when transfected
with full-length HBV DNA. If IFN
treatment reduces the enhancer I
activity also in this system and not only in reporter gene expression
experiments, one should observe reduced levels of viral RNA and of
other viral gene products (18). Since the HBV enhancer I is known to
support synthesis of all viral transcripts in these cell lines (19),
its modulation should affect all viral transcripts and subsequently all
products synthesized from them.
In the present study, we investigated whether the ISRE-like region and
p48 contribute to the antiviral activity induced by IFN
on HBV when
propagated in human hepatoma cells. We demonstrate that inactivation of
the ISRE-like region has no detectable effect on the IFN
-induced
response in this system. Furthermore, overexpression of p48 enhanced
IFN
-induced suppression of the viral RNA levels irrespectively of
the mutation introduced. These data indicate that neither the ISRE-like
region of the HBV nor direct binding of p48 to this element, if
occurring at all in the full-length genome context, play a detectable
role in the IFN
-induced antiviral state against HBV in hepatoma cells.
 |
EXPERIMENTAL PROCEDURES |
Plasmids--
Plasmid pHBV(wt) contains the HBV-DNA of subtype
ayw (20) as head to tail linked dimer cloned via the
EcoRI site (12). Plasmid pHBV ISRE(M) was produced by
polymerase chain reaction-mediated oligonucleotide-directed
mutagenesis. The mutation was introduced into pHBV-1 (plasmid
containing the HBV-DNA as monomer, cloned via the EcoRI
site) by amplification with Pwo DNA polymerase (Roche Deutschland Holding GmbH, Grenzach-Wyhlen, Germany) and the
complementary primer pairs HBV ISRE(M) (5'-GCA GGC ccT CAC cTT CTC
GC-3', only sense strand is shown, mutations are indicated by lowercase
letters). Correct mutagenesis was verified by sequencing. The mutated
HBV full-length fragment was isolated after EcoRI digestion
by elution from an agarose gel. Thereafter, the fragment was inserted
as head to tail dimer into the EcoRI site of pUC19. Plasmid
pCMV/p48, which codes for human p48 under the control of a CMV
promoter, was kindly provided by David E. Levy.
Cell Culture and Transfection Procedure--
The human hepatoma
cell line HuH7 was grown as monolayer in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum. Plasmid DNAs used for
transfection were purified by ion exchange chromatography and were
transfected using FuGENE 6 Transfection Reagent (Roche
Deutschland Holding GmbH, Grenzach-Wyhlen, Germany) according to the
protocol supplied. Cells (1.3 × 106 cells/5-cm plate)
were transfected with 2 µg of the corresponding HBV expression
plasmids and 0.25 µg of pCMV/SEAP, which codes for a secreted
alkaline phosphatase (SEAP). Plasmid pCMV/SEAP was cotransfected as
control to monitor transfection efficiency and potential
IFN
-mediated cytotoxic effects. IFN
-2b (1,000 IU/ml,
Intron A, Essex Pharma, Munich, Germany) was added to the medium
16 h after transfection, and cells were harvested for analysis 48 h later. The amount of SEAP activity secreted into the medium of transfected cells was determined as described previously (12). For
overexpression of p48 the pCMV/p48 expression plasmid was used,
containing the p48 cDNA under control of a CMV promoter (21).
Purification of HBV-DNA from Intracellular Core Particles and of
HBV RNA--
Isolation of intracellular HBV-DNA was performed as
described recently (22). Total RNA was prepared by use of the High Pure RNA Isolation Kit (Roche Deutschland Holding GmbH,
Grenzach-Wyhlen, Germany) according to the protocol supplied.
Southern and Northern Blot Analysis--
DNA isolated from
cytoplasmic core particles were separated on an 1.2% agarose gel. Ten
µg of total RNA were separated on an 1.2% agarose-formaldehyde gel.
DNAs and RNAs were blotted onto "Hybond N" nylon membranes (Nycomed
Amersham, Buckinghamshire, United Kingdom) and hybridized with a
32P-labeled gel-purified full-length HBV DNA fragment. To
standardize the loading, Northern blots were rehybridized with
32P-labeled glycerolaldehyde-3-phosphate
dehydrogenase-specific or actin-specific probes. HBV- and
actin-specific probes were generated using a random-primed labeling kit
(Nycomed Amersham, Buckinghamshire, United Kingdom).
Glycerolaldehyde-3-phosphate dehydrogenase-specific probes were
generated by in vitro transcription using T7 RNA polymerase
according to the manual supplied (Promega, Madison, WI). Blots were
exposed to Fuji imaging screens, and signals were quantified by a Fujix
BAS 2000 bio-imaging analyzer (Fuji, Tokyo, Japan) and by TINA
software (Raytest, Straubenhardt, Germany).
Protein Extracts and EMSA--
HuH7 cells were treated with
1,000 IU/ml IFN
for 3 h or left untreated. In addition, HuH7
cells were pretreated with 100 IU/ml IFN
for 16 h and,
thereafter, with 1,000 IU/ml IFN
for 30 min to increase the amount
of activated ISGF3, as described previously (23, 24). Nuclear proteins
were extracted as described previously (22). For EMSA, 15 µg of
nuclear protein was preincubated for 5 min at room temperature with or
without competitor in 25 µl of 20 mM HEPES (pH 7.9),
0.1% Nonidet P-40, 1 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol, 8% glycerol, 2 µg of poly[d(I-C)], and 0.1 ng of an unrelated single-strand
oligonucleotide (5'-ATGGTGAGCAAGGGCGAGGAGC-3'). For the supershift
experiments 2 µg of the indicated antibodies were added and incubated
for 1 h at room temperature before addition of the labeled
double stranded oligonucleotide probes. Polyclonal rabbit antisera
against STAT 1 (p84/p91), STAT 2, ISGF-3
(p48), IRF1, and IRF2 were
obtained from Santa Cruz Biotechnology, Inc. After addition of the
indicated 32P-labeled double-stranded oligonucleotide
probes and incubation for 30 min at room temperature, DNA-protein
complexes were analyzed on 4% polyacrylamide gels (29:1) containing 25 mM Tris borate and 0.25 mM EDTA. For
competition unlabeled double-stranded oligonucleotides were used in
equimolar amounts, 10-fold, and 100-fold molar excess. The sequence of
double-stranded oligonucleotides (only plus strand sequence indicated)
used as probes and/or as competitors in EMSA were: HBV ISRE(wt),
5'-GCAGGCTTTCACTTTCTCGC-3'; ISG15, 5'-GGGAAAGGGAAACCGAAACTGAAGCC-3'; Sie m67, 5'-GTCGACATTTCCCGTAAATC-3'; control,
5'-GATATAGATTCTGATTTTGGAGAAGAGTCTCTCTTTGATCTGTTCCTCTCAGA-3'.
 |
RESULTS AND DISCUSSION |
HBV enhancer I activity was shown to affect expression of all
viral products (19), and its IFN
-mediated suppression was reported
to depend on binding of p48 to an ISRE-like region (11). If p48 binding
plays a major role in the antiviral activity of IFN
, as speculated
previously, inactivation of the corresponding binding site should
reduce IFN
-mediated suppression of HBV on the level of viral RNA and
replicative intermediates in cells propagating HBV genomes (18). To
test this, we first wanted to confirm that specific mutations in the
HBV ISRE-like region (Fig. 1A)
inactivate p48 binding in an EMSA, as described recently for the
corresponding oligonucleotides (11). We used nuclear extracts from
IFN
-treated HuH7 cells or untreated cells and
32P-labeled double-stranded oligonucleotides of the HBV
ISRE-like region (HBV-ISRE wt), the mutated HBV ISRE-like region
(HBV-ISRE(M)), the ISRE region of the IFN-stimulated gene 15 (ISG15),
or the high affinity serum-inducible element sequence (Sie m67). As
reported recently (11), IFN
treatment induced a pronounced increase in the amount of DNA-protein complexes (Fig. 1B, lanes
2, 3, 14, and 15). Increasing
amounts of the unlabeled HBV-ISRE(wt) oligonucleotide reduced formation
of this complex (Fig. 1B, lanes 4-6). In
contrast, addition of the same amounts of the unlabeled HBV ISRE
oligonucleotide with mutations (HBV-ISRE(M)) or of a control
oligonucleotide unrelated to the ISRE had no effect (Fig.
1B, lanes 7-10). When using the mutated HBV
ISRE-like element (HBV-ISRE(M)) as labeled probe for EMSA, no
IFN-activated DNA-protein complexes were detected (Fig. 1B,
lanes 16 and 17). As expected IFN
-activated
DNA-protein complexes were detected using the ISRE of gene ISG15 (Fig.
1B, lanes 11 and 12) and the Sie m67
(Fig. 1B, lanes 18 and 19)
oligonucleotide probes as positive controls. To further characterize
the DNA-protein complexes bound to the HBV ISRE-like region, supershift
experiments were performed with antisera against STAT 1, STAT 2, and
p48. In these experiments a supershift of DNA-protein complexes was detected only with the Sie m67 oligonucleotide probe after addition of
antiserum against STAT 1 (data not shown). Since neither binding of p48
to the HBV ISRE-like region nor binding of the ISGF3 complex (STAT 1, STAT 2, and p48) to the ISG15 ISRE were detected when using these
extracts, we performed analogous experiments with nuclear extracts from
HuH7 cells pretreated with IFN
for 16 h before addition of
IFN
. This treatment is known to increase the activation of ISGF3
(23, 24) and to induce binding of p48 to the HBV ISRE-like region (11).
As expected, EMSA with these nuclear extracts showed supershifted
signals with antibodies against STAT 1, STAT 2, p48, IRF1, and
IRF2 when using the ISG15 (Fig. 1C, lanes 12-16)
or the Sie m67 probes (Fig. 1C, lane 19), similar as published previously (23-25). When the mutated HBV-ISRE probe (HBV-ISRE(M)) was used in these experiments, neither the same nor other
DNA-protein complexes were observed (Fig. 1C, lanes 8 and 9). This rules out the possibility that binding
of other transcription factors to the mutant oligonucleotide
compensated for the IFN responsiveness of wt HBV ISRE. The IFN-induced
protein-DNA complexes seen with the HBV-ISRE(wt) probe in our
experiments did not contain p48 (Fig. 1C, lane 5)
but did contain IRF 1 (Fig. 1C, lane 6).
The latter result is consistent with a perfect IRF element (IRF-E)
consensus sequence, known to bind IRF 1 and IRF 2, within the HBV
ISRE-like region (Fig. 1A). Taken together, in contrast to a
previous publication (11), in which binding of p48 to HBV ISRE wt
sequence was claimed without including a specificity control for the
anti-p48 antibodies used, our data indicate that p48 (although present
and active in nuclear extracts from the same type of cells treated with
IFN
or IFN
plus IFN
) neither binds to the HBV-ISRE(wt) nor to
the corresponding mutant oligonucleotide, while IRF 1 binds to
the HBV-ISRE(wt), but not to the mutant oligonucleotide.

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Fig. 1.
Binding of IFN-activated proteins to the HBV
ISRE-like region is abrogated by mutations. A, scheme
depicting the location of the ISRE-like region, viral open reading
frames, and enhancer I within the HBV genome. Framed sequence
regions indicate perfect match between the HBV ISRE(wt) and the
consensus sequences of the ISRE (26) and of IRF-E (IRF element) (27)
(N, any nucleotide; Y, purimidine; R,
purine; S, G or C). Mutations introduced in HBV ISRE(M) to
inactivate the previously reported binding of p48 are marked
(bold/underlined). B, EMSA performed with nuclear
extracts from HuH7 cells treated with 1,000 IU/ml IFN . For
competition, increasing concentrations (equimolar amounts, 10-fold, or
100-fold molar excess) of unlabeled double-stranded HBV ISRE(wt)
(lanes 4-6), or HBV ISRE(M) (lanes 7-9), or an
100-fold molar excess of an oligonucleotide with an unrelated sequence
(lane 10) were used. C, treatment of HuH7 cells
with IFN and IFN reveals binding of IRF 1 to the HBV ISRE-like
region. Nuclear extracts used for supershift analysis were prepared
from HuH7 cells pretreated with IFN for 16 h (100 IU/ml) and
thereafter with IFN for 30 min (1,000 IU/ml). Antisera
(AB) and probes used for supershift experiments are
indicated.
|
|
For functional analysis of the ISRE-like region in the complete HBV
sequence context, we used a plasmid-integrated head to tail dimer of a
fully functional HBV genome (pHBV wt) and in parallel the analogous
construct containing mutations in the ISRE-like region (see above) of
HBV (pHBV ISRE(M)), which abrogated binding of IFN-activated proteins
in EMSA. These constructs were transfected into HuH-7 cells together
with a plasmid pCMV/SEAP, which codes for a SEAP. Expression of SEAP
was measured to control transfection efficiency and to exclude
potential cytotoxic effects of IFN
. Sixteen hours after
transfection, cells were treated with 1,000 IU/ml IFN
or left
untreated. Two days after addition of IFN
, the cell culture medium
and the cells were harvested. Both DNA and RNA were extracted and
analyzed by Southern and Northern blots. In untreated cells, Northern
blots revealed only a minor difference in the levels of the viral RNA
produced by HBV wt and the mutant genome, indicating that the mutations
had no significant effect on the enhancer activity (Fig.
2B). The slight increase of
viral RNA in cells transfected with ISRE mutant virus DNA visible on the Northern blot reflects the corresponding higher transfection efficiency obtained with the mutant genome DNA compared with HBV wt
DNA, as evident from the higher levels of SEAP (Fig. 2A).
The amount of replicative DNA intermediates in pHBV ISRE(M)-transfected cells was about 5-fold lower than in cells producing wild-type HBV DNA,
as determined by Southern blot analysis (Fig. 2C). This reduced replication efficacy is probably due to one of the three mutations introduced into the ISRE-like region causing an amino acid
mutation (F656L) in the C-terminal end of the viral P-protein and the
corresponding defect.

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Fig. 2.
Mutational inactivation of the ISRE-like
region does not alter IFN -mediated suppression
of HBV transcription or replication. HuH7 cells transfected with
pHBV(wt)- or pHBV ISRE(M)-DNA were treated with or without 1,000 IU/ml
IFN for 48 h. To monitor transfection efficiency and potential
IFN -mediated cytotoxic effects cells were cotransfected with
pCMV/SEAP. The amount of SEAP secreted into the medium (A)
was determined by a SEAP enzyme activity assay. The levels of HBV
transcripts (B) and of viral intracellular replicative DNA
intermediates (C) were determined by Northern or Southern
blotting, respectively. Quantitative evaluation of the data is shown in
the adjacent panels. The SEAP activities are given as mOD(405
nm)/min and the signal intensities of the blots as arbitrary
units ± S.D. (n = 4).
|
|
In cells treated with IFN
, the levels of viral RNA produced by HBV
wt and mutant HBV were both decreased (Fig. 2B), indicating that inactivation of ISRE-like region did not abrogate the reduction of
viral RNA levels by IFN
. Similarly, the amount of replicative DNA
intermediates was also reduced by IFN
treatment both in the HBV wt
and the HBV mutant genome transfected cells (Fig. 2C). This
was not due to a cytotoxic effect of IFN
, as SEAP expression levels
were very similar in treated and untreated cells (Fig. 2A).
Independent of the functionality of the ISRE, the antiviral effect of
IFN
was reproducibly weaker for viral RNA than for viral DNA
(reduction to roughly 70% compared with 15%). This is consistent with
our previously reported data, which suggested the existence of at least
two antiviral mechanisms activated by IFN
, one acting on the RNA the
other on the DNA level (12). Taken together, these data argue against a
significant role of the ISRE-like element in IFN
-induced reduction
of viral RNA and replication in HBV producing hepatoma cells. One
reason for the lack of a role of the ISRE-like region on replicating
virus may be that the ISRE-like region is covered by proteins, which
bind to adjacent regions not present in the reporter gene constructs used in previous studies (11, 14). Another reason may be that in the
full-length genome context efficient transcription of the HBV DNA from
promoters located upstream of the ISRE-like region prevents binding of
IFN-activated proteins to the ISRE-like region, while in the reporter
gene constructs this region is not transcribed. The major differences
in promoter and enhancer activities observed in many reported studies
with subgenomic HBV fragments compared with full-length HBV DNA (17) is
consistent with these interpretations.
Suppression of enhancer I activity by IFN
-induced p48 binding to the
ISRE-like element in previous reporter gene experiments was enhanced by
overexpression of p48 in HuH7 cells (11). Therefore, we tested whether
overexpression of p48 can increase IFN
-induced suppression of viral
RNA and replicative DNA intermediates in the full-length genome
context. HuH7 cells were cotransfected with pHBV(wt) and 0.5 or 2 µg
of pCMV/p48, treated with IFN
or left untreated, and analyzed as
described above. As determined by Northern blotting, IFN
treatment
reduced the signal intensities for the viral RNA to about 75% compared
with untreated cells (Fig. 3).
Cotransfection of 0.5 or 2 µg of pCMV/p48 increased IFN
-induced suppression of the viral RNAs to about 59 and 46%, respectively (Fig.
3), as evident from quantitative evaluation of the signals using a
bioimager. The results indicate that enhanced expression of p48
increases the antiviral effect of IFN
on the RNA levels in human
hepatoma cells. On the level of replicative DNA intermediates, an
increase of IFN
-induced suppression was not observed with 0.5 µg
of pCMV/p48 expression plasmid and only a modest, but significant, increase with 2 µg of pCMV/p48 compared with the antiviral activity induced by IFN
in the samples from cells not cotransfected with p48-DNA (Fig. 4). Taken together, these
data suggest that p48 is present in limiting amounts in human hepatoma
cells and contributes to the IFN
-induced antiviral response against
HBV on the RNA and DNA level. To examine this further, and to find out
whether this contribution depends on the ISRE-like region on HBV, we
compared the IFN
-induced suppression on the RNA level of HBV wt and
ISRE mutant HBV under conditions of enhanced p48 expression. HuH7 cells were cotransfected with 2 µg of pCMV/p48 and pHBV wt or pHBV ISRE(M), treated with IFN
or left untreated, and analyzed as described above.
Independent of the type of HBV genome transfected, RNA levels were
reduced to very similar levels by IFN
treatment (Fig. 5). These data imply that even under
nonlimiting concentrations of p48, the binding of proteins activated by
IFN
to the ISRE-like region does not significantly contribute to
IFN
-induced reduction of the levels of HBV transcripts in human
hepatoma cells. However, our findings do not exclude a function of the
ISRE in infected hepatocytes in which the template of transcription is
cccDNA, which might allow binding of IRF-1 and/or ISGF3 to the
ISRE-like region. A suitable animal system is required to investigate
this further.

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Fig. 3.
Effect of enhanced p48 expression on
IFN -induced reduction of HBV RNA levels.
HuH7 cells cotransfected with pHBV(wt)- and pCMV/p48-DNA were treated
with or without 1,000 IU/ml IFN for 48 h. The amount of HBV
transcripts was determined by Northern blotting. The relative
intensities of the hybridization signals for the viral RNA from
IFN -treated and untreated cells are shown below the blot and are
expressed as mean ± S.D. (n = 4). IFN -induced
antiviral activity is enhanced significantly by cotransfection of 2 µg of p48-DNA compared with samples from cells not cotransfected with
p48-DNA (p = 0.018, as determined by Student's
t test).
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Fig. 4.
Effect of enhanced p48 expression on
IFN -induced reduction of HBV replication.
HuH7 cells were treated as described in legend to Fig. 3. The amount of
HBV replicative intermediates was determined by Southern blotting, and
the relative intensities of the hybridization signals for the viral DNA
from the IFN -treated and untreated cells are shown below the blot,
expressed as mean ± S.D. (n = 4). IFN -induced
antiviral activity is enhanced significantly by cotransfection of 2 µg of p48-DNA compared with samples from cells not cotransfected with
p48-DNA (p = 0.035).
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Fig. 5.
Enhanced
IFN -mediated suppression of HBV transcripts by
overexpression of p48 is independent of a functional ISRE-like
region. HuH7 cells were cotransfected with 2 µg of pCMV/p48 and
pHBV(wt)- or pHBV ISRE(M)-DNA and treated thereafter as described in
legend to Fig. 3. The relative intensities of the hybridization signals
for the viral RNA from IFN -treated and untreated cells are shown
below the blot expressed as mean ± S.D. (n = 4).
|
|
 |
ACKNOWLEDGEMENTS |
We thank David E. Levy for generously
providing the expression plasmid for p48. We appreciate critical
reading of the manuscript by Martin Forster.
 |
FOOTNOTES |
*
This work was supported by a grant from the
Bundesministerium für Forschung, Bildung und Technologie. The
Heinrich-Pette-Institut für Experimentelle Virologie und
Immunologie is supported by the Bundesministerium für Gesundheit
und the Freie und Hansestadt Hamburg.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed:
Heinrich-Pette-Institut für Experimentelle Virologie und
Immunologie an der Universität Hamburg, Martinistra
e 52, D-20251 Hamburg, Germany. Tel.: 49-40-48051-221; Fax:
49-40-48051-222; E-mail: will@hpi.uni- hamburg.de.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.C000584200
 |
ABBREVIATIONS |
The abbreviations used are:
HBV, hepatitis B
virus;
cccDNA, covalently closed circular DNA;
IFN
, interferon-
;
STAT, signal transducer and activator of transcription;
p48, interferon-stimulated gene factor 3
;
ISGF3, IFN-stimulated gene
factor 3;
ISRE, IFN-stimulated response element;
SEAP, secreted
alkaline phosphatase;
EMSA, electrophoretic mobility shift assay;
ISG15, interferon-stimulated gene 15;
Sie, serum-inducible element;
IRF, IFN regulatory factor;
IRF-E, IRF element.
 |
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