JOURNAL OF VIROLOGY, Jan. 2007, p. 202–214
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 1
Repression of Interferon Regulatory Factor 1 by Hepatitis C Virus
Core Protein Results in Inhibition of Antiviral and
Anna R. Ciccaglione,1† Emilia Stellacci,1† Cinzia Marcantonio,1Valentina Muto,1Michele Equestre,2
Giulia Marsili,1Maria Rapicetta,1and Angela Battistini1*
Department of Infectious, Parasitic and Immunomediated Diseases1and Department of Cell Biology and Neurosciences,2
Istituto Superiore di Sanita `, Viale Regina Elena 299, Rome 00161, Italy
Received 17 May 2006/Accepted 9 October 2006
Hepatitis C virus (HCV) proteins are known to interfere at several levels with both innate and adaptive
responses of the host. A key target in these effects is the interferon (IFN) signaling pathway. While the effects
of nonstructural proteins are well established, the role of structural proteins remains controversial. We
investigated the effect of HCV structural proteins on the expression of interferon regulatory factor 1 (IRF-1),
a secondary transcription factor of the IFN system responsible for inducing several key antiviral and immu-
nomodulatory genes. We found substantial inhibition of IRF-1 expression in cells expressing the entire HCV
replicon. Suppression of IRF-1 synthesis was mainly mediated by the core structural protein and occurred at
the transcriptional level. The core protein in turn exerted a transcriptional repression of several interferon-
stimulated genes, targets of IRF-1, including interleukin-15 (IL-15), IL-12, and low-molecular-mass polypep-
tide 2. These data recapitulate in a unifying mechanism, i.e., repression of IRF-1 expression, many previously
described pathogenetic effects of HCV core protein and suggest that HCV core-induced IRF-1 repression may
play a pivotal role in establishing persistent infection by dampening an effective immune response.
Infection with hepatitis C virus (HCV) represents the major
cause of liver disease, affecting more than 170 million individ-
uals worldwide (26). After a subclinical phase, more than 80%
of patients progress to persistent HCV infection, which is the
leading cause of chronic liver disease associated with cirrhosis
and hepatocellular carcinoma (13). The persistence of the virus
in the majority of infected individuals is linked to the ability of
HCV to evade and/or antagonize the host immune response at
both the local and systemic levels. Accordingly, although hepa-
tocytes are a major target of HCV infection, the virus can also
replicate in immune cells, including effector cells (1, 11). In this
respect, resistance to interferon (IFN) therapy is a hallmark of
evolution in persistence, indicating that knocking down the
antiviral and immunomodulating effects of IFN is a successful
strategy for evading the host immune surveillance (21). The
production and secretion of IFN type I is pivotal in inducing a
global antiviral state through paracrine IFN production and
the subsequent activation of interferon-stimulated genes
(ISGs) within the infected cells and in the surrounding tissues
(70). The role of IFN in HCV infection is thus crucial (21).
Functional genomic analyses from cohorts of human subjects
with chronic infection have shown that infection is associated
with a gene expression profile marked by ISGs whose level of
expression is related to different degrees of liver fibrosis and
cirrhosis (67). Similarly, gene expression profiling has demon-
strated that acute resolving infections in chimpanzees are as-
sociated with high levels of hepatic ISG expression (4).
The single-stranded RNA genome of HCV is translated into
a polypeptide precursor of 3,010 amino acids (aa) that is
cleaved by cellular and viral proteases into three structural
proteins (core, E1, and E2), p7, and at least six nonstruc-
tural proteins (NS2 to NS5B) (39). Several HCV proteins
have been shown to interfere with the IFN-induced intra-
cellular signal transduction pathway, thereby inhibiting the
induction of a number of effector proteins. The structural
protein E2 (72) and the nonstructural protein NS5A (22, 23)
modulate interferon responses by inhibiting the interferon-
inducible double-stranded RNA-dependent protein kinase
R (PKR). The NS3/4A protease functions as an antagonist
of virus-induced interferon regulatory factor 3 (IRF-3) and
NF-?B activation and IFN-? expression by blocking the Toll-
like receptor 3 and retinoic acid-inducible gene I signaling
pathways (17, 18, 37). This signaling block not only impairs
IFN production in hepatocytes but also breaks the IFN ampli-
fication loop (8, 18), thereby inhibiting the expression of sev-
eral ISGs, including those involved in the adaptive immune
response (for a review, see reference 21).
HCV core protein (21 kDa) is the first protein to be pro-
duced upon virus infection and possesses multiple functions
affecting both the virus and the host. In addition to forming the
viral nucleocapsids, core protein affects a whole array of host
cell functions, including apoptosis, cell transformation, signal
transduction, and transcriptional regulation (reviewed in ref-
erences 73 and 78). In addition to cytoplasmic and nuclear
localization, the HCV core protein is also secreted from stably
transfected cells and has been found in the bloodstream of
infected individuals, where it may provide an infection-inde-
* Corresponding author. Mailing address: Department of Infectious,
Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita `,
Viale Regina Elena, 299 Rome 00161, Italy. Phone: (3906) 49903266.
Fax: (3906) 49902082. E-mail: firstname.lastname@example.org.
† A.R.C. and E.S. contributed equally to this work.
?Published ahead of print on 18 October 2006.
by on November 1, 2008
pendent mechanism of immune dysregulation (31, 62). Core-
induced immune dysregulation includes suppression of Th1
polarization, inhibition of IFN-?-mediated cytotoxic T-lym-
phocyte (CTL) activation, and decreased interleukin-2 (IL-2)
and IL-12 production (for a review, see reference 61).
The role of HCV core protein in modulating ISG expression
is more controversial. Data from different groups indicate that
core protein modulates the IFN-induced Jak/STAT signaling
pathway but does not affect the activation of some ISGs. In
contrast, other reports showed a downregulation of interferon-
induced antiviral genes (3, 12, 54).
One of the ISGs, IRF-1, a member of the interferon regu-
latory factor family, was originally identified as a regulator of
the IFN-?/? promoter but later recognized as also able to
regulate several ISGs by binding the IFN-stimulated response
element (ISRE), also known as IRF-E, in the IFN-?/?-stimu-
lated gene promoters and thus amplify the IFN response (20,
60). Intensive functional analyses carried out on this transcrip-
tion factor have revealed a remarkable functional diversity in
the regulation of cellular responses through the modulation of
different sets of genes, depending on the cell type, the state of
the cell, and/or the nature of the stimuli (49, 70). IRF-1 is a
regulator not only of cellular antiviral responses through the
induction of antiviral ISGs, including 2-5A synthetase and
PKR, but also of cellular apoptosis and transformation and
immune responses through the regulation of a number of spe-
cific target genes. Studies in knockout mice have implicated
IRF-1 in the regulation of various immune processes, such as
T-cell selection and maturation, leukemogenic development,
and autoimmunity. Impairment of CD8?cell maturation, de-
fective Th1 responses, exclusive Th2 differentiation, impaired
macrophage production of IL-12, and maturation of NK cells
have all been observed in immune cells from IRF-1?/?mice
(for a review, see reference 70).
Recently, IRF-1 has been implicated in HCV infection as a
negative regulator of HCV subgenomic replicon (30). Using
different experimental approaches, including cells expressing
the entire HCV genomic replicon or cells conditionally or
transiently expressing only the structural proteins, we show
here that the expression of viral proteins, in particular of the
HCV core protein, results in IRF-1 repression, which is re-
versed in IFN-cured cells. IRF-1 expression was inhibited at
the transcriptional level, and this inhibition resulted in the
transcriptional repression of several ISGs, including genes in-
volved in antiviral as well as immunomodulatory activities,
such as 2-5A synthetase, PKR, IL-15, IL-12, and the low-
molecular-mass polypeptide 2 (LMP2). Interestingly, these ef-
fects occurred both in hepatic cells and in T cells. Our data
identify a new mechanism through which the HCV core pro-
tein contributes to HCV-induced persistence and point to
IRF-1 as a unique target to boost a protective innate and
adaptive immune response against HCV.
MATERIALS AND METHODS
Cell culture and treatment. Jurkat cells were grown in RPMI 1640 medium
containing 10% fetal calf serum (FCS) and antibiotics. The Huh-7 cell line
carrying the Sfl HCV full-length replicon (genotype 1b) was obtained from R.
Bartenschlager. The cell clones that stably replicate the HCV replicon were the
21-5, 22-6, and 20-1 clones, passaged as described elsewhere (19, 58). Cured
HCV replicon cells were treated with 100 U IFN-?/ml for 14 days to eliminate
self-replicating full-length HCV replicon RNA (71). Clearance of replicon RNA
was confirmed by reverse transcription-PCR (RT-PCR) and by the loss of resis-
tance to G418. The U2-OS human osteosarcoma-derived, tetracycline-regulated
cell line UTHCNS3-43, inducibly expressing the HCV structural region, was
obtained from D. Moradpour (52). Cells were maintained in complete Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% FCS, antibiotics,
and 1 ?g/ml puromycin, 1 ?g/ml tetracycline, and 500 ?g/ml G418. To induce
HCV protein expression, 1 ? 105/ml cells were seeded in 100-mm dishes. After
24 h, the cells were washed five times over a period of 1 h with DMEM. Finally,
cells were placed in DMEM supplemented with 2% FCS with or without 1 ?g/ml
Where indicated, cells were treated with 100 ng/ml of recombinant IFN-?
(rIFN-?; PeproTech EC Ltd., London, United Kingdom) or 100 ng/ml tumor
necrosis factor alpha (TNF-?; PeproTech) for 16 h.
Expression vectors. Mammalian expression vectors containing the core (aa 1
to 191) and the E1E2p7 (aa 155 to 809) regions of the HCV genome obtained by
RT-PCR from HCV-positive human serum were cloned into the pRc/CMV
mammalian expression vector. The nucleotide sequence of the clones was de-
termined and classified the HCV isolate as type 1a (9). In the E1E2p7 construct,
the signal sequence of E1 protein (aa 155 to 191) was included to allow proteins
to be transported into the endoplasmic reticulum membrane. The sequence of
each recombinant plasmid was confirmed by the modified dideoxynucleotide
method and an ABI 373A automatic sequencer. The IRF-1 expression vector was
a generous gift of J. Hiscott and is described in reference 38.
Transfection experiments and enzymatic assays. Transient-transfection exper-
iments were performed using the FuGENE 6 transfection reagent (Roche Di-
agnostics, Mannheim, Germany) for Jurkat cells and MBS mammalian transfec-
tion kit (Stratagene, La Jolla, CA) for Huh-7 and Vero cells, according to the
manufacturers’ protocols. The construct encoding a portion of the wild-type
LMP2 promoter and a mutated version in the IRF-E consensus sequence (LMP2
mt) cloned upstream of the luciferase reporter gene were a generous gift of K. L.
Wright and are described in reference 79. The constructs encoding the IFN-?,
?-casein, and IL-4 promoters upstream of the luciferase reporter gene were
generous gifts of J. Hiscott (McGill University, Montreal, Canada), T. Kitamura
(University of Tokyo, Tokyo, Japan), and M. Li-Weber (Tumorimmunology
Program, German Cancer Research Center, Heidelberg, Germany), respectively,
and are described in references 16, 38, and 55.
The constructs for p3500 (encoding the entire IRF-1 promoter from bp ?3400
to ?168) and the two portions Gas/?B (corresponding to the ?199/?89 region
of the IRF-1 promoter) and NF-?B (corresponding to the ?89/?16 region of the
IRF-1 promoter), cloned upstream of the luciferase reporter gene, were a gen-
erous gift of R. Pine and are described in reference 59. The IL-12 p40 construct
(corresponding to the ?413/?12 region of the IL-12 p40 promoter) cloned
upstream of the luciferase reporter gene was a generous gift of K. Ozato and is
described in reference 44.
Huh-7 (5 ? 105), Huh-21-5 (5 ? 105), Jurkat (2 ? 106), and Vero (1 ? 106)
cells were transfected with 500 ng of luciferase reporter vectors and 500 ng of
expression vectors. The amount of transfected DNA was adjusted with the empty
vector RcCMV. One hundred ng of pAct-Renilla plasmid was cotransfected and
used as a control for transfection efficiency. Reagents from Promega were used
to assay extracts for luciferase activity in a Lumat LB9501 luminometer (E&G
Berthold, Bad Wildbad, Germany).
Western blotting. For NS5B and core analysis, cells were collected by centrif-
ugation at 3,000 ? g for 4 min at 4°C, washed twice in 1? phosphate-buffered
saline, and resuspended in a volume of lysis buffer (12.5 mM Tris-HCl [pH 6.7],
2% glycerol, 0.4% sodium dodecyl sulfate [SDS], 1% mercaptoethanol, 0.1%
bromophenol blue). For IRF-1 analysis, cells were washed twice in ice-cold
phosphate-buffered saline and lysed in cold lysis buffer (20 mM HEPES pH 7.4,
50 mM NaCl, 10 mM EDTA pH 8.0, 2 mM EGTA, 0.5% NP-40, 0.5 mM
dithiothreitol, 10 mM NaMO, 10 mM NaVO3, 100 mM NaF, 0.05 M ?-glycero-
phosphate, 100 ?g/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride) for 20
min at 4°C. Lysates were centrifuged at 10,000 ? g for 10 min at 4°C. Fifty ?g of
cell extracts was separated on 10% or 12% SDS-polyacrylamide gel electro-
phoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Blots were
incubated with anti-IRF-1 (1:200; sc-497; Santa Cruz Biotechnology Inc., Santa
Cruz, CA), anti-IRF-2 (1:200; sc-498; Santa Cruz), anti-IRF-3 (1:200; sc-9082;
Santa Cruz), anti-PKR (1:200; sc-707; Santa Cruz), anti-NS5B (1:200; sc-17532;
Santa Cruz), anti-core (1:1,000; MO-I40015B; Anogen, Mississauga, Ontario,
Canada), anti-E1 and anti-E2 (1:500; C65198M and B65581G; BIODESIGN
Int., Maine), and anti-actin (1:200; sc-1616; Santa Cruz) antibodies and then with
anti-rabbit–, anti-mouse–, or anti-goat–horseradish peroxidase–coupled second-
ary antibody (Calbiochem, San Diego, CA). Immune complexes were identified
VOL. 81, 2007 HCV CORE PROTEIN INHIBITS IRF-1 EXPRESSION 203
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using an enhanced chemiluminescence system (Amersham, Buckinghamshire,
RNA extraction and RT-PCR analysis. Total RNA was extracted from 1 ? 106
cells using RNeasy kits (QIAGEN) as described by the manufacturer and quan-
tified by optical density. One hundred nanograms of total RNA was reverse
transcribed using the high-capacity cDNA archive kit (Applied Biosystems) and
random hexamer primers in an ABI Prism 7000 sequence detector system (Ap-
plied Biosystems) using the following thermal profile: 25°C for 10 min, 42°C for
1 h, and 95°C for 5 min. PCRs were performed in triplicate on the ABI Prism
7000 sequence detector system (Applied Biosystems) using TaqMan chemistry
with primer and probe sets from the Assay-on-Demand list (Applied Biosys-
tems). For each gene, the standard curve was compared with the standard curve
of the reference gene and calculation of the slope of the log(ng RNA) versus ?Ct
was always ?0.1. Fold induction was then calculated by the ??Ct method (39a)
using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level to
normalize values and the mRNA level of the Huh-7 parental cell line or of the
U2-OS (Tet?) cells as a calibrator.
Regulation of IRF-1 expression in cells carrying full-length
HCV replicon. Expression of the full-length HCV poly-protein
in the context of replicating RNA provides a useful system to
evaluate better some aspects of HCV-cell interactions. As it
has been reported that IRF-1 is not directly affected by non-
structural proteins but negatively regulates HCV replication
(30), we investigated IRF-1 regulation in cell lines harboring
the autonomously replicating full-length HCV genome, specif-
ically, the 21-5, 22-6, and 20-1 clones (19, 58).
IRF-1 mRNA expression was monitored by real time RT-
PCR analysis in naı ¨ve Huh-7 cells and in the clones. This
analysis (Fig. 1A) showed that IRF-1 levels were significantly
and comparably decreased (between 50% and 60% [? 8%,
standard deviation]) in all the clones examined compared with
the Huh-7 parental cell line. For further analyses we chose the
21-5 clone, in which IRF-1 mRNA expression was 46% com-
pared with the Huh-7 parental cell line (Fig. 1B). The IRF-1
mRNA decrease in Huh-21-5 cells resulted in an almost com-
plete reduction in IRF-1 protein expression as assessed by
Western blot analysis (Fig. 1C, lane 2).
To determine whether the inhibition of IRF-1 expression
was specifically mediated by negative regulatory effects of
HCV protein expression, we cured Huh-21-5 cells by culturing
them in the presence of 100 IU/ml of natural IFN-? for 2
weeks, a treatment which has been shown to clear the virus
(71). As shown in Fig. 1B and C, in cured Huh-21-5 cells the
levels of IRF-1 were comparable to those present in the Huh-7
FIG. 1. The expression levels of IRF-1 were decreased in clones harboring the HCV replicon. (A) Total RNA extracted 48 h postseeding from
Huh-7 parental cells and the cell lines 21-5, 22-6, and 20-1 harboring the HCV genomic replicon was subjected to real-time RT-PCR using IRF-1
primers. Data were normalized by the level of GAPDH mRNA expression in each sample and are shown as relative expression units. Levels from
Huh-7 were set as the basis for the comparative results. Shown are the means ? standard deviations of three independent experiments. (B) Total
RNA extracted 48 h postseeding from Huh-7 parental cells, cell line 21-5, and 21-5 IFN-cured cells (21-5c) was subjected to real-time RT-PCR
using IRF-1 primers. Data were normalized by the level of GAPDH mRNA expression in each sample and are shown as relative expression units
as in panel A. (C) Basal level of IRF-1 protein in Huh-7, Huh-21-5, and Huh-21-5c cells was determined by Western blot analysis. Protein extracts
(50 ?g) were separated by 10% SDS-PAGE and analyzed using specific anti-IRF-1 antibody. (D) HCV NS5B expression was analyzed by Western
blotting using anti-NS5B antibody. One representative experiment out of three performed is shown.
204 CICCAGLIONE ET AL.J. VIROL.
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parental cell line. In Fig. 1D the expression of NS5B confirms
the expression of viral proteins in Huh-21-5 cells but not in
cells treated with IFN-?.
These results suggest that HCV replication and viral protein
expression specifically downregulate IRF-1 expression in Huh-
Expression of HCV structural proteins decreases IRF-1 ex-
pression. It has been reported that in cells harboring the sub-
genomic replicon the double-stranded RNA-stimulated IRF-1
is specifically inhibited by NS5A expression (57). In order to
distinguish between the effects of nonstructural and structural
proteins, we first expressed the structural proteins of HCV in
a tetracycline-regulated system (52). As shown in Fig. 2B, when
we performed Western blotting with an anti-core antibody,
only cells cultured in the absence of tetracycline expressed viral
proteins on a kinetic base, whereas in the presence of tetracy-
cline, viral protein synthesis was inhibited. To analyze the
kinetics of gene regulation in cells expressing the HCV struc-
tural proteins, we performed a time course experiment. Cells
that inducibly expressed viral proteins were cultured for 12, 24,
and 48 h in the presence or in the absence of tetracycline, and
IRF-1 expression was monitored by real-time PCR and West-
ern blotting (Fig. 2A and B). Real-time PCR analysis indicated
that at time zero there were no significant differences between
levels of IRF-1 in cells expressing the structural proteins and in
control cells (data not shown). Similarly, no variations in the
IRF-1 mRNA amount were found in cells growing in the pres-
ence of tetracycline and not expressing viral proteins from 12 h
onward (Fig. 2A). Conversely, withdrawal of the antibiotic
from the medium resulted in the expression of HCV proteins
and in a significant decrease in the IRF-1 mRNA levels at all
time points. The decrease was maximum after 48 h of culture,
when IRF-1 mRNA levels were 50% ? 10% of the control
(Fig. 2A). Western blot analysis (Fig. 2B) indicated that the
inhibition of IRF-1 protein expression was even more marked,
and the protein was barely detectable after 48 h. Interestingly,
at this time synthesis of the core protein in this system reached
These results indicate that the downregulation of IRF-1
expression observed in cells expressing the entire HCV repli-
con (Fig. 1) could be due mainly to the expression of HCV
IRF-1 expression is inhibited by HCV core protein. To iden-
tify the HCV-encoded structural protein(s) that influences
IRF-1 gene expression, we transiently expressed individual
fragments from the structural region of the HCV genome
coding for the core or E1 or E2 proteins in Huh-7 hepatoma
cells. We decided to use transient expression, even though the
effects may be underestimated if only a small fraction of the
cell population is transfected, because stable transfection, es-
pecially in the case of the core protein, may be susceptible to
clonal selection. The ability of HCV-expressing plasmids to
code for the core and for the E1-E2 proteins was confirmed by
Western blot analysis (Fig. 3B). After transfection, total
mRNA was isolated, and the amount of IRF-1 mRNA was
evaluated by real-time PCR. As shown in Fig. 3A, the IRF-1
mRNA level was substantially lower (more than 60% ? 10%)
in cells transfected with the plasmid expressing the HCV core
protein than in cells transfected with the empty vector. In
contrast, cells expressing the envelope proteins (E1 and E2)
showed an increase in the amount of IRF-1 mRNA, although
this increase was not statistically significant. Analysis of IRF-1
protein expression by Western blotting (Fig. 3B) indicated that
IRF-1 expression was reduced to 30% in cells expressing the
core protein but was not affected by E1-E2 expression (lane 3).
To assess the specificity of the inhibitory effect of the core
protein on IRF-1 expression, dose-response experiments were
performed. As shown in Fig. 3C, increasing doses of HCV core
protein resulted in a correspondingly dose-dependent inhibi-
tion of IRF-1 protein expression, whereas expression of other
FIG. 2. Tetracycline-regulated expression of HCV structural proteins represses IRF-1 expression. (A). Total RNA was extracted at the
indicated time points from U2-OS cells expressing the structural region of HCV and cultured in medium containing 1 ?g/ml tetracycline where
indicated (Tet?). The IRF-1 RNA level was determined using real-time RT-PCR as for Fig. 1. The GAPDH mRNA level from control cells
(Tet?) was set as the basis for the comparative results. Shown are the means ? standard deviations of three independent experiments. (B) Protein
extracts (50 ?g) were separated by 10% or 12% SDS-PAGE and analyzed by Western blotting using specific anti-IRF-1, anti-core, and anti-actin
antibodies. The intensity of the IRF-1-specific bands was measured by densitometry and is reported as the percentage of IRF-1 expression in HCV
protein-expressing cells compared with control cells after normalization with actin levels.
VOL. 81, 2007 HCV CORE PROTEIN INHIBITS IRF-1 EXPRESSION205
by on November 1, 2008
IRFs, e.g., IRF-2 and IRF-3, was not affected. The core protein
was detectable by Western blot analysis starting from a dose of
1 to 5 ?g, although doses as low as 0.2 ?g were equally able to
decrease IRF-1 expression.
Taken together, these results indicate that among structural
HCV proteins only the core is able to down-regulate signifi-
cantly IRF-1 expression in HCV-infected cells.
HCV core protein inhibits transcriptional activity of the
IRF-1 promoter. IRF-1 expression is mainly regulated at the
transcriptional level by several inducers (70). To determine
whether the HCV core protein was able to inhibit IRF-1 pro-
moter activity, Huh-7 cells were transiently transfected with a
3,500-bp IRF-1 promoter construct upstream of the luciferase
reporter gene in the presence of an expression vector coding
for the HCV core protein. As shown in Fig. 4A, basal IRF-1
promoter activity was substantially lower (80% ? 5%) in cells
expressing the core protein than in cells expressing the empty
vector (lane 2 versus lane 1). Moreover, since it is known that
inflammatory cytokines such as IFN-? and TNF-? substantially
induce the expression of IRF-1 (59), we checked the ability of
HCV core protein to affect cytokine-induced IRF-1 transcrip-
tion. Interestingly, as shown in Fig. 4A (lanes 3 to 6), the
treatment with IFN-? and TNF-? did not counteract the in-
hibitory effect exerted by the core protein, suggesting that cells
expressing the core protein do not respond to inflammatory
stimuli by inducing IRF-1. As shown in Fig. 4B, HCV core
protein expression also impacted the basal and cytokine-in-
duced IRF-1 transcriptional activity in cells of the immune
system, specifically Jurkat T cells. This may be of physiological
importance, since cells of the immune system not only repre-
sent another target of HCV infection (11) but can also be
affected in a bystander manner by core protein circulating in
the bloodstream (31).
To determine which sequences on the IRF-1 promoter were
FIG. 3. Inhibition of IRF-1 expression by HCV core protein. Huh-7 cells were transfected with 5 ?g pRc/CMV parental plasmid or vector
expressing the core or the E1-E2, respectively. (A) Forty-eight hours after transfection, cells were harvested, and total RNA was used to assess
the IRF-1 RNA level using real-time RT-PCR as in Fig. 1. Levels were normalized to cellular GAPDH mRNA abundance, and concentrations
from cells transfected with the parental vector pRc/CMV were set as the basis for the comparative results. Shown are the means ? standard
deviations of three independent experiments. (B) Total cell extracts (50 ?g) were separated by 10% or 12% SDS-PAGE and analyzed by Western
blotting using anti-IRF-1, anti-core, anti-E1, anti-E2, and anti-actin antibodies. The intensity of specific IRF-1 bands was measured by densitometry
and is reported as the percentage of IRF-1 expression in HCV protein-expressing cells compared with control cells after normalization with actin
levels. (C) Huh-7 cells were transfected with increasing doses of an HCV core-expressing vector. After 48 h total cell extracts (50 ?g) were analyzed
by Western blotting using anti-IRF-1, anti-IRF-2, anti-IRF-3, anti-core, and anti-actin antibodies. Percentages of IRF-1 expression were calculated
as for panel B.
206CICCAGLIONE ET AL. J. VIROL.
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targets of the inhibitory effect of the HCV core protein, Huh-7
and Jurkat cells were transfected with portions of the IRF-1
promoter containing the consensus binding sites for STAT1
(GAS/NF-?B) or NF-?B transcription factors together with an
expression vector coding for the core protein. Eight hours after
transfection, cells were mock treated or stimulated with IFN-?
or TNF-? for a further 16 h, after which the promoter tran-
scriptional activity was measured. As shown in Fig. 5A to D,
both basal and cytokine-induced activation of constructs bear-
ing the GAS or the NF-?B elements, respectively, was signif-
icantly reduced in Huh-7 and Jurkat cells expressing the HCV
Finally, IRF-1 expression following core expression was de-
termined in IFN-deficient Vero cells to exclude an involvement
of IFN blockade in the core-mediated IRF-1 repression. As
shown in Fig. 5E, the amount of IRF-1 mRNA, evaluated by
real-time PCR, was reduced in core-expressing Vero cells at
levels comparable to those observed in core-expressing Huh-7
cells (45% ? 8%). Accordingly, basal IRF-1 promoter activity
was significantly repressed in Vero cells expressing the core
protein compared to cells transfected with the empty vector
Regulation of IRF-1-dependent gene expression during
HCV RNA replication. To determine whether the regulation of
IRF-1 by HCV affected host cell gene expression during HCV
RNA replication, we examined the expression of different
ISGs, targets of IRF-1 and endowed with both antiviral and
immunomodulatory activities, in cells either expressing the en-
tire replicon (Fig. 6A) or conditionally expressing only the
structural proteins (Fig. 6B). The ISGs examined were PKR
and 2-5A synthetase, IL-15, IL-12, and IFN-?. Real-time PCR
analysis indicated that expression of the entire replicon (Fig.
6A) resulted in a significant decrease in mRNA accumulation
in all the genes examined. The expression of structural proteins
(Fig. 6B) determined a comparable inhibition of PKR, IL-15,
and IL-12 but not of IFN-?. Interestingly, data in the literature
report that the signaling pathway leading to IFN type I pro-
duction is mainly affected by the HCV nonstructural protein
(21). We further determined the levels of PKR protein expres-
sion by Western blotting (Fig. 6C), confirming the data ob-
tained by RT-PCR analysis.
Inhibition of IRF-E/ISRE activity in cells harboring the
replicon or expressing the core protein. The downstream effect
of IRF-1 inhibition in cells harboring the entire replicon or
transiently expressing increasing amounts of the HCV core
protein was also demonstrated by determining the transcrip-
tional activity of reporter constructs bearing the IRF-E/ISRE
sequences present on the LMP2 and IL-12 gene promoters,
both of which are dependent on IRF-1 for transcription. As
shown in Fig. 7A, the relative IRF-E-regulated luciferase ac-
tivity was significantly repressed (80% ? 10%) in Huh-21-5
cells harboring the HCV replicon compared with naı ¨ve Huh-7
cells. Notably, increasing doses of the core protein (Fig. 7B and
C) cotransfected with the same construct caused a dramatic
dose-dependent inhibition of the wild-type IRF-E luciferase
reporter construct activity in both T and hepatoma cells. In-
deed, doses of 1 ?g of the HCV core protein-expressing vector
practically abolished the promoter transcriptional activity.
Conversely, basal expression of the LMP2 promoter construct
mutated in the IRF-E element (Fig. 7B, mt) was lower than in
the wild-type construct, but its transcriptional activity was not
affected by the expression of the HCV core protein. The in-
hibitory effect of the core protein was directly mediated by
IRF-1 repression, as demonstrated by restoring IRF-1 expres-
sion in Huh-7 cells expressing the HCV core protein. As shown
in Fig. 7D, repression of the IRF-E-regulated luciferase activ-
FIG. 4. HCV core protein inhibits transcriptional activity of the IRF-1 gene promoter. The p3500-luc construct containing a 3,500-bp fragment
of the IRF-1 promoter linked to the luciferase reporter gene was transfected alone or in combination with 0.5 ?g expressing vector coding for the
HCV core protein in Huh-7 (A) and Jurkat (B) cells. After 8 h, where indicated, cells were mock treated or stimulated with 100 ng/ml of rIFN-?
or TNF-? for 16 h, and then total cell extracts were processed for luciferase activity. Means ? standard deviations from three separate experiments
were calculated after normalization with the pAct Renilla activity.
VOL. 81, 2007HCV CORE PROTEIN INHIBITS IRF-1 EXPRESSION 207
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ity was completely reversed by overexpression of IRF-1 in a
dose-dependent fashion. To demonstrate further the specificity
of the core inhibition, the activity of other promoter constructs
was determined in Huh-7 cells transfected with 1 ?g of the core
protein. As shown in Fig. 7E, neither the transcriptional activ-
ity of ?-casein nor that of the IL-4 gene promoter was affected
by the core protein expression. Similarly, and in accord with
the RT-PCR data shown in Fig. 6B, the IFN-? proximal pro-
moter basal activity was maintained irrespective of the expres-
sion of the HCV core protein.
It is known that IRF-1 binds not only to IRF-E sequences but
also to the overlapping ISRE, regulating the expression of several
ISGs (69, 70). We therefore investigated whether the promoter
activity of an ISRE-containing promoter was similarly affected by
HCV core protein expression. To this end we cotransfected in
Huh-7 and Jurkat cells a luciferase reporter construct under the
control of the ISRE present on the IL-12 p40 gene promoter. As
shown in Fig. 8A and B, a dose of 0.5 ?g of a core-expressing
vector significantly reduced IL-12 promoter activity in both cell
types (50 to 70% [? 10%] compared with control cells).
FIG. 5. HCV core protein inhibits transcriptional activity of the NF-?B and STAT consensus sequences containing portions of the IRF-1 gene
promoter. Portions of the IRF-1 gene promoter containing the NF-?B and STAT consensus sequences (GAS) linked to the luciferase reporter
gene were transfected in Huh-7 (A and B) and Jurkat (C and D) cells alone or in combination with 0.5 ?g HCV core protein-expressing vector.
Where indicated cells were mock treated or stimulated with 100 ng/ml of rIFN-? or TNF-? for 16 h. Cell extracts were processed for luciferase
activity. Means ? standard deviations from three separate experiments were calculated after normalization with the pAct Renilla activity. (E) Vero
cells were transfected with 5 ?g pRc/CMV parental plasmid or vector expressing the core, and total RNA was used to assess IRF-1 mRNA level
using real time RT-PCR as in Fig. 1. Shown are the means ? standard deviations of three independent experiments. (F) The p3500-luc construct
containing a 3,500-bp fragment of the IRF-1 promoter linked to the luciferase reporter gene was transfected alone or in combination with 0.5 ?g
expressing vector coding for the HCV core protein in Vero cells and the IRF-1 promoter activity was evaluated as for Fig. 4.
208 CICCAGLIONE ET AL.J. VIROL.
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Together, these results suggest that the block of IRF-1 ex-
pression in HCV genomic replicon-bearing cells was mainly
mediated by the core protein and was sufficient to repress the
expression of IRF-1-dependent genes during HCV replication.
Most HCV-infected individuals are unable to clear the virus
and develop a chronic infection with inauspicious outcome.
The mechanisms by which HCV resists the host antiviral de-
fenses and induces liver injury remain poorly defined. Never-
theless, it is well established that HCV induces IFN, and the
ability to circumvent the antiviral effects of this cytokine seems
to be mainly responsible for the establishment of persistence.
Accordingly, only 10% to 20% of patients with chronic infec-
tion respond to IFN-? therapy (51). Thus, clarification of the
mechanisms involved in impaired anti-HCV actions by IFN-?
is an important goal for the definition of effective therapies.
In the present study we have demonstrated that HCV core
gene expression, either alone or in the context of HCV repli-
cation, inhibits IRF-1, a transcription factor involved in IFN
signaling. Furthermore, IRF-1 repression attenuates ISG re-
sponses mediated by the IRF-E/ISRE. Specifically, we showed
that IRF-1 target genes, endowed with both antiviral and im-
munomodulatory functions, are repressed and IRF-1 suppres-
sion occurs at the transcriptional level through inhibition of
both basal and cytokine-induced IRF-1 promoter activity.
IRF-1 is a pleiotropic transcription factor, critical for cell
defense against viral infections but also crucial for the de-
velopment of both the innate and adaptive responses. In-
deed, the targets of IRF-1 during the antiviral response are
genes directly involved not only in virus elimination but also
in differentiation, proliferation activity, and apoptosis of
cells, including those of the immune system (42, 70). Ac-
cordingly, dysregulation of IRF-1 expression can lead to
defective antigen presentation, NK and T-cell activity dis-
orders, and tumorigenesis.
The different levels of IRF-1 expression seem to be one of
the factors that dictate how it functions. Expression of the
IRF-1 gene is rapidly induced following virus infection, and
IFNs, proinflammatory cytokines, and high IRF-1 expression
levels are required for the induction of a set of ISGs necessary
for an efficient antiviral response (24, 33). Here we have shown
that HCV RNA replication affects IRF-1 expression, down-
modulating both mRNA and protein at levels no more suffi-
cient to induce the expression of ISGs direct targets of IRF-1.
The inhibition of IRF-1 expression and IRF-E/ISRE activity
in cells expressing a subgenomic replicon of HCV was reported
by Kanazawa and colleagues (30). However, in the system
analyzed, no direct correlation was found between the viral
RNA replication and IRF-1 repression.
The development of in vitro culture systems for expression
of the entire replicon of HCV greatly improved our under-
standing of the complex virus-host interactions and recapitu-
lated most of the previous observations of the impact of single
HCV proteins on cell physiology. In contrast to observations in
FIG. 6. Regulation of IRF-1-dependent gene expression during HCV RNA replication. Total RNA was purified from the parental Huh-7 and
the Huh-21-5 cells harboring the HCV genomic replicon harvested 48 h postseeding (A) and the U2-OS cells (B) expressing the structural region
of HCV and cultured in medium supplemented, where indicated, with 1 ?g/ml of tetracycline (Tet?) and expression of the indicated genes was
monitored by real-time RT-PCR. Data were normalized by the level of GAPDH expression in each sample and are shown as relative expression
units. Concentrations from Huh-7 (A) and U2-OS Tet? (B) cells were set as the basis for the comparative results. Shown are the means ? standard
deviations of three independent experiments. (C) The basal level of PKR protein in Huh-7 and Huh-21-5 cells was determined by Western blot
analysis. Protein extracts (50 ?g) were separated by 10% SDS-PAGE and analyzed using specific anti-PKR antibody.
VOL. 81, 2007 HCV CORE PROTEIN INHIBITS IRF-1 EXPRESSION209
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HCV subgenome-expressing cells, we found that in cured
Huh-7 cells from which the entire replicon has been elimi-
nated, IRF-1 expression is restored (Fig. 1), and we postulate
that the decrease in IRF-1 expression could be due to active
suppression by viral proteins. Indeed, using cells that tran-
siently or conditionally express HCV structural proteins, we
have demonstrated a specific repression of IRF-1 by the core
Several in vitro studies have already defined the role of some
viral structural and nonstructural proteins in inhibiting the
expression and/or activity of key factors in IFN synthesis and in
the IFN-induced signal transduction pathway (21). Conversely,
FIG. 7. Inhibition of IRF-E/ISRE activity in cells harboring the replicon or expressing the core protein. (A) Naive Huh-7 and Huh-21-5 cells
harboring the HCV genomic replicon were transfected with a 200-bp fragment of the proximal LMP2 promoter linked to the luciferase reporter
gene. After 24 h, total cell extracts were processed for luciferase activity. Means ? standard deviations from three separate experiments were
calculated after normalization with the pAct Renilla activity. (B and C) Jurkat and naive Huh-7 cells were cotransfected with the LMP2 luciferase
construct, wild type (wt) or mutated in the IRF-E consensus sequence (mt), and increasing doses of an HCV core-expressing vector. After 24 h
cells were processed as for panel A. (D) Naive Huh-7 cells were cotransfected with the proximal LMP2 promoter, 1 ?g of an HCV core-expressing
vector, and increasing doses of IRF-1-expressing vector. After 24 h cells were processed as for panel A. (E) Naive Huh-7 cells were cotransfected
with 1 ?g of an HCV core-expressing vector and the LMP2, IFN-?, ?-casein, and IL-4 proximal promoters. After 24 h cells were processed as
described for panel A.
210 CICCAGLIONE ET AL.J. VIROL.
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to date conflicting results have been reported on the role of
HCV core protein. Whereas recent papers agree on the inter-
ference with the Jak/STAT pathway by inhibition of ISGF3 and
STAT-1 activities, there is no agreement on whether this in-
terference results in a modification of transcription of ISGs (3,
6, 7, 12, 47, 48, 50, 54). Variations in the experimental system
used, structural or genotypic differences in the protein and,
possibly, the amount of protein used may all be implicated in
the reported discrepancies. Using conditional and transient
expression of HCV structural proteins, we have shown that the
core protein was mainly responsible for the inhibition of IRF-1
expression in cells bearing the entire HCV replicon. Con-
versely, and in agreement with previous reports (57), other
structural proteins, E1 and E2, did not impact IRF-1 expres-
sion (Fig. 2).
We have also shown that the inhibition of IRF-1 expression
occurred at the transcriptional level through both the STAT-1
and NF-?B consensus sequences on the IRF-1 promoter. In
agreement with these data it has recently been reported that
core protein inhibits both the activation and nuclear translo-
cation of STAT-1 in IFN-treated cells (7, 47). In addition, in
core-expressing cells NF-?B nuclear translocation and activity
is also suppressed (29). Data obtained in Vero cells, which are
deficient in IFN production, seem also to exclude the possibil-
ity that the core-inhibited IRF-1 expression resulted from the
blockade of the endogenous IFN in core-expressing cells.
Given that no reports have, as yet, clarified the mechanism
through which the core regulates gene transcription, our data
support the conclusion that the core regulates gene transcrip-
tion by means of indirect effects eventually leading to modu-
lation of signal transduction and transcription factors.
Notably, we have also shown that the down-modulation of
IRF-1 expression by the HCV replicon and by the core protein
in turn affects host gene expression mediated by IRF-E/ISRE.
Among the repressed genes, we found genes with antiviral
functions, including 2-5A synthetase and PKR, but also immu-
nomodulatory cytokines, such as IL-12 and IL-15, and genes
that are important for antigen presentation, such as LMP2.
These results suggest that inhibition of IRF-1 by the core in the
context of HCV replication may trigger a cascade of events
that affect a wide range of immune responses via direct or
Numerous studies have shown that the HCV core protein is
a major cause of most of the pathogenic features associated
with HCV infection, and in this respect its role in the suppres-
sion of immunological functions has been established. In par-
ticular, impairment of the activation and functions of dendritic
cells and of the proliferation of T cells has been reported (25,
34). In accordance with our observations, a selective suppres-
sion of IL-12 in stimulated human macrophages mediated by
the suppression of AP1 has also recently been described (15,
36). Our data also indicate that the HCV core protein affects
IL-12 p40 gene promoter activity in both hepatoma and T cells.
In this respect it has been demonstrated that induction of the
gene encoding both the p40 and p35 subunits of IL-12 is totally
dependent on IRF-1 (41, 43, 68). Therefore, our results imply
that in addition to the reported mechanism of IL-12 repression
in macrophages mediated by AP-1 (15), the inhibition of IRF-1
is probably most responsible for IL-12 suppression by the HCV
IL-12 is known to play a pivotal role in the generation of Th1
immune responses and provides a crucial link between innate
and adaptive immunity (74). IRF-1 has been defined as a
“super” Th1-cell transcription factor (42) in that IRF-1?/?
mice display completely defective Th1 responses due to a lack
of IL-12 production by APC, accompanied by exclusive Th2
differentiation. In this respect, it is interesting that the study of
IRF-1 promoter polymorphisms in relation to the response to
IFN in chronic patients indicated that a lower viral load corre-
sponded to higher IRF-1 promoter activity and to a significantly
higher proportion of Th1 CD4?cells after IFN administration
(63). Interestingly, the establishment of a Th1 environment in
HCV-infected individuals is thought to be important in determin-
ing the outcome of the infection. As indicated by in vivo studies,
patients who demonstrate Th2 dominance tend to develop
chronic infection, whereas those with a Th1 phenotype can clear
FIG. 8. The HCV core protein inhibits transcriptional activity of the IL-12 p40 gene promoter. Naive Huh-7 (A) and Jurkat (B) cells were
cotransfected with a fragment of the proximal IL-12 p40 gene promoter and where indicated 0.5 ?g of an HCV core-expressing vector. After 24 h,
total cell extracts were processed for luciferase activity. Means ? standard deviations from three separate experiments were calculated after
normalization with the pAct Renilla activity.
VOL. 81, 2007HCV CORE PROTEIN INHIBITS IRF-1 EXPRESSION 211
by on November 1, 2008
the virus (2, 10, 32, 75, 76, 81). The immunosuppressive action
of core protein may thus be pivotal in viral escape by down-
modulating Th1 responses in favor of Th2 responses, a mech-
anism in which core-mediated inhibition of IRF-1 may be in-
Of the ISGs analyzed, IL-15, a unique target of IRF-1, was
also substantially reduced in both hepatic and T cells express-
ing either the entire HCV replicon or the structural proteins,
and to our knowledge this is the first study reporting a repres-
sion of IL-15 in cells bearing the entire HCV replicon. IL-15 is
involved in the activation and homeostatic maintenance of
cells of both the innate and adaptive immune systems, playing
a key role in CD8?T-cell homeostasis by promoting survival or
proliferation of naive and memory phenotype CD8 T cells and
in NK cell development, maturation, and survival (14, 40, 80).
Notably, it has been reported that impaired IL-15 production
is one of the mechanisms of the aberrant response of DC to
IFN in HCV-infected patients (28). In addition, a significant
reduction in serum IL-15 levels in HCV patients has also been
recently reported (46).
We have also shown that the HCV core, through inhibition
of IRF-1, specifically inhibits, at the transcriptional level, the
LMP2 gene promoter activity (Fig. 7B to D). LMP2 is a sub-
unit of immunoproteasomes, which are very efficient for the
generation of specific CTL epitopes. It has, in fact, been shown
that substitution of standard ?-subunits of the proteasome with
LMP2, LMP7, and MECL1 subunits improves the production
of peptide antigens with the correct C termini for binding to
MHC class I (53, 64, 66, 77). Since both basal and IFN-?-
induced LMP2 expression is absolutely dependent on IRF-1, it
is not surprising that inhibition of IRF-1 by the core protein
also results in suppression of LMP2 expression. Considering
that a reduction in the population and specific activity of CTL
(2, 32, 35) have been observed in chronically infected individ-
uals, we speculate that the HCV core protein down-modula-
tion of LMP2 may be partially responsible for these in vivo
observations. An analysis of the proteasome composition in
HCV patients would, therefore, be very useful.
Data reported in the present study suggest that HCV regu-
lation of IRF-1 may also impact other IRF-regulated pathways
influencing host gene expression on a more global scale. In this
respect, given that IRF-1 together with NF-?B is induced and
is also an effector of inflammatory cytokines, it is interesting
that the HCV core protein inhibits IRF-1 promoter activity
even after treatment with inflammatory cytokines (Fig. 4). This
raises the possibility that both locally, in hepatoma cells, and
systemically, by affecting PBMC, the core protein can depress
the inflammatory response induced by virus infection also
through repression of IRF-1. In agreement with this hypothe-
sis, a recent report (27) indicated that cyclooxygenase 2, an
enzyme that contributes to homeostasis and to inflammatory
pathways (56), is substantially repressed in core-expressing
cells. Notably, cyclooxygenase 2 is another gene tightly regu-
lated by IRF-1 (5).
Finally, it has been reported that other cellular and viral
genes altered by the core include p21waf1, p53, and human
immunodeficiency virus type 1 LTR (45, 73). As others and we
ourselves have reported (65, 70), these genes are all specific
targets of IRF-1.
Taking all these results into account and considering that
they well mirror clinical observations in chronically infected
patients, we conclude that IRF-1 repression by the HCV core
protein can be considered a unifying mechanism that recapit-
ulates most of the data so far reported on the dysregulation of
cellular processes induced by this protein and may at least
partially account for its role in evading the host response at
several levels: antiviral, inflammatory, and immune. Restora-
tion of the correct expression of IRF-1 in HCV-infected cells
could, therefore, represent a new avenue for therapeutic in-
We thank R. Bartenschlager for the Huh-7, Huh-21-5, Huh-22-6,
and Huh-20-1 cell lines, D. Moradpour for the UTHCNS3-43 cell line,
and J. Hiscott, K. L. Wright, T. Kitamura, and M. Li-Weber for
providing luciferase constructs. We thank Sabrina Tocchio for editorial
assistance and Roberto Gilardi for preparing graphs.
This work was supported by grants from the Italian AIDS Project,
the Italian Ministry of Health, and ISS-NIH Scientific Cooperation
agreement to A.B. and by grants from the “Integrated National Project
for the Study, the Prevention and the Treatment of the Chronic Hepa-
tology” (RF02.188), and the “Viral Hepatitis National Projects” of the
Istituto Superiore di Sanita ` (D. leg.vo no. 502) to M.R.
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