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INTRODUCTION |
Hepatitis C virus
(HCV),1 a member of the
Flaviviridae family, is one of the major causes of chronic
hepatitis, which can result in cirrhosis and finally hepatocellular
carcinoma (1). Its genome consists of a linear, positive-strand RNA
molecule of ~9,500 nucleotides (nt) encoding a single polyprotein
precursor of ~3,000 amino acids (aa) that is processed into three to
four structural proteins at the amino terminus (Core, E1, and E2/p7)
and six nonstructural proteins at the carboxyl terminus (nonstructural
proteins 2, 3, 4a, 4b, 5a, and 5b) (2, 3). The genomic region of the
putative core protein encodes 191 aa and has an apparent molecular mass of 21 kDa (2). The core protein, relatively conserved among all
identified HCV isolates (4), may be the fundamental unit for the
encapsidation of genomic RNA to help in virus morphogenesis. In
addition, previous studies suggested that HCV core protein has various
biological properties, one of which is its effect on the nuclear factor
B (NF-
B) pathway (5-9).
NF-
B belongs to a highly conserved Rel-related protein family, which
includes RelA (p65), RelB, c-Rel, NF-
B1 (p105/p50), and NF-
B2
(p100/p52). Of these, the p50/p65 heterodimer, commonly referred to as
NF-
B, is the most abundant and ubiquitous. One of the most
intensively studied signals to the NF-
B is induced by tumor necrosis
factor (TNF), a proinflammatory cytokine associated with inflammation,
immune response, and apoptosis. Currently, this signal transduction
pathway is understood as follows (10-15); when TNF binds and activates
the TNF receptor 1 (TNFR1), TNFR-associated death domain (TRADD),
TNFR-associated factor 2 (TRAF2), and receptor interacting protein
(RIP) form a complex with TNFR1. Subsequently NF-
B inducing kinase
(NIK) and/or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEKK1) are activated. Activated NIK
and/or MEKK1 phosphorylate and activate both I
B kinase (IKK)
and
IKK
. Activated IKK
/
phosphorylate I
B
, which associates with and sequesters NF-
B in the cytoplasm. Phosphorylated I
B
is ubiquitinated and degraded, and then NF-
B translocates into the
nucleus and binds to DNA to initiate the transcription of various genes
associated with inflammation, the immune response, cell growth, and survival.
There have, however, been conflicting reports up until now about the
effect of HCV core protein on this NF-
B pathway. Recently, it was
shown that HCV core protein activated the NF-
B pathway (7-9). On
the contrary, it was previously shown that core protein suppressed
TNF-induced NF-
B activation (5, 6). At present, the mechanism for
core protein's effect on the NF-
B pathway remains unclear;
therefore, we focused our attention on the effect HCV core protein has
on the NF-
B pathway and tried to determine how core protein affects
NF-
B signaling.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
Human cervical carcinoma cells (HeLa), human
hepatoma cells (HepG2), and monkey kidney cells (COS-7) were obtained
from the RIKEN cell bank (Tsukuba Science City, Japan). HeLa Tet-Off
cells, which constitutively express the tetracycline-controlled
transactivator, were purchased from CLONTECH (Palo
Alto, CA). Cells were grown in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal
bovine serum.
HCV Core Protein-expressing Plasmids--
Type 1b HCV core
region (nt 1-575 and aa 1-191 of the prototype HCV type 1b, HCV-J;
Ref. 16) was amplified by reverse transcription-polymerase chain
reaction (PCR) using the HCV RNA extracted from the sera of a patient
with chronic hepatitis C as a template. Reverse transcription-PCR was
performed as previously described (17) using the following primers
having an XhoI restriction site (underlined) (the nucleotide positions in HCV-J are shown in parentheses): F1 (a sense primer, nt
1-20), 5'-CCGCTCGAGACCATGAGCACGAATCCTAAACC-3', and R573
(an antisense primer, nt 555-573),
5'-CCGCTCGAGTCAAGCGGAAGCTGGGATGGTC-3'. The amplified
product was digested with XhoI and then cloned into the
XhoI site of pCXN2 (kindly provided by J. Miyazaki, Osaka University, Osaka, Japan), a mammalian expression vector having a
-actin promoter and cytomegalovirus enhancer (18), to generate pCXN2-core.
In addition to the plasmid expressing full-length core protein (aa
1-191), we constructed three more plasmids expressing deletion mutant
forms of HCV core protein, aa 1-173 (pCXN2-core173), aa 1-151
(pCXN2-HAcore151), and aa 92-191 (pCXN2-HAcore92-191). The core
fragments were amplified by PCR using pCXN2-core as a template and then
cloned into pCXN2 or pCXN2-HA for the expression of hemagglutinin (HA;
YPYDVPDYA)-tagged core protein. For construction of pCXN2-core173, the
region of HCV core aa 1-173 was amplified with primers F1 and R519 (an
antisense primer, nt 501-519),
5'-CCGCTCGAGTCAAGCGGAAGCTGGGATGGTC-3', and then cloned into
pCXN2. For construction of pCXN2-HAcore151, the region of HCV core aa
1-151 was amplified with F1 and R453 (an antisense primer, nt
438-453), 5'-CCGCTCGAGTCACAGGGCCCTGGCAGCG-3', and then
cloned into pCXN2-HA. Similarly, pCXN2-HAcore92-191, which encodes
N-terminal 91-aa deleted core protein, was constructed by using F276 (a
sense primer, nt 276-292),
5'-CCGCTCGAGACCATGGGGTGGGCAGGATGGCT-3', and R573.
To generate a tetracycline-regulated HCV core expression plasmid
(pTRE2-core), full-length HCV core cDNA was prepared by digesting pCXN2-core with XhoI and then subcloned into the
SalI site of pTRE2 (CLONTECH). This
construct allowed for the expression of HCV core protein under the
transcriptional control of a tetracycline-controlled transactivator-dependent promoter.
A series of core expression plasmids were sequenced using a cycle DNA
sequencing system (PE Applied Biosystems, Foster City, CA), as
described previously (19), to confirm the integration of core genes.
Expression of core proteins was examined by the ECL-plus Western
blotting detection system (Amersham Pharmacia Biotech, Buckinghamshire,
United Kingdom), using extracts of COS-7 cells transfected with core
expression plasmids and mouse anti-HCV core antigen IgG fraction
(Austral Biologicals, San Ramon, CA) or anti-HA polyclonal antibody
(Santa Cruz Biotechnology, Santa Cruz, CA).
NF-
B Pathway Reporter Plasmids--
To examine the effect of
HCV core protein on the NF-
B pathway, NF-
B-inducible reporter
plasmid (pNF-
B-Luc) containing the Photinus pyralis
(firefly) luciferase reporter gene driven by a basic promoter element
(TATA box) plus five repeats of
B cis-enhancer element
(TGGGGACTTTCCGC) (Stratagene, La Jolla, CA) was utilized. pFC-MEKK
(Stratagene), which expresses constitutively active MEKK1 (amino acids
360-672) driven by a cytomegalovirus promoter, was used as a positive
control plasmid to activate the NF-
B pathway. pRL-TK (Promega,
Madison, WI), a control plasmid that expresses Renilla
reniformis (sea pansy) luciferase driven by the herpes simplex
virus thymidine kinase promoter, was used to monitor the efficiency of transfection.
To elucidate the mechanism of how core protein affects the NF-
B
pathway, the expression vectors for catalytically inactive IKK
(pIKK
(K44A)), containing an alanine substitution of a
conserved lysine residue in its kinase domain (13), and IKK
(pIKK
(K44A)) (20) (kindly provided by Goeddel DV,
Tularik, CA) were utilized. In addition, the expression vectors for the
dominant negative form of TRAF2 (pTRAF2-(87-501)), lacking the RING
finger motif (21), and TRAF6 (pTRAF6-(289-522)), lacking either the
entire zinc-binding region or zinc fingers 3-5 (22), were kindly
provided by Goeddel DV and utilized. Moreover, pCMV-TAK1 (K63W) (kindly provided by K. Matsumoto, University of Tokyo, Tokya, Japan), which
expresses catalytically inactive TAK1, was utilized with pCMV-TAB1
(kindly provided by K. Matsumoto), which expresses TAK1 activator
(23).
Reporter Plasmids Having Interleukin (IL)-1
and TNF
Promoters--
In addition to the reporter plasmid having synthetic
B cis enhancer element, luciferase reporter plasmids
having IL-1
or TNF
promoter containing NF-
B binding sites,
were constructed. A fragment of 691 base pairs (positions
585 to
+106) of the TNF
gene was amplified by PCR using a primer set of
5'-ggggtaccgcttgtcctgctacccc-3' and 5'-cccaagcttgtcaggggatgtggcgt-3'.
The amplified fragment was digested with KpnI and
HindIII, and then cloned into pGL3 basic vector (Promega)
(pTNF
-Luc). Similarly, a fragment of 1125 base pairs (
1110 to +15)
of IL-1
gene was amplified by PCR using primer set of
5'-ggggtacccctgtagtcccagctg-3' and 5'-ctagctagctcgaagaggtttggtatct-3'. Those fragments were digested with KpnI and NheI,
and then cloned into pGL3 basic vector (pIL-1
-Luc).
All cloned plasmids were purified using the Endfree plasmid kit
(Qiagen, Hilden, Germany). Nucleotide sequencing of constructed plasmids was performed using an autosequencer (PE Applied Biosystems) and the dye termination method as described previously (19) to confirm
gene expression.
Construction of HeLa Cells Induced to Express Core
Protein--
HeLa cells induced to express HCV core protein were
generated with use of a tetracycline-regulated gene expression system (Tet-Off gene expression system, CLONTECH). HeLa
Tet-Off cells were cotransfected with pTRE2-core and pTK-Hyg
(CLONTECH), a selection vector that confers
hygromycin resistance, followed by selection in culture medium
containing 200 µg/ml hygromycin (CLONTECH) and 1 µg/ml doxycycline (Sigma). Hygromycin-resistant clones, termed HeTOC,
were examined for expression of the core protein upon withdrawal of
doxycycline by Western blotting using mouse anti-HCV core antigen IgG
fraction, as described previously. Positive clones were
expanded and rescreened by Western blotting of cells grown in the
presence and absence of doxycycline.
Transfection--
We used the Effectene transfection reagent
(Qiagen) for all transfection experiments. Approximately 4 × 105 HeLa cells were plated into the well of a six-well
tissue culture plate (Iwaki Glass, Chiba, Japan) 24 h before
transfection. To examine the effect of HCV core protein on the NF-
B
pathway, HeLa cells were transfected with a total of 0.4 µg of
plasmids consisting of 0.19 µg of pNF-
B-Luc, 0.01 µg of pRL-TK,
and 0.2 µg of pCXN2 or pCXN2-core. As a positive control, pFC-MEKK
was added to the transfection complexes containing pCXN2, or human
TNF
(Strathmann Biotech GmbH, Hamburg, Germany) was added to the
medium of transfected HeLa cells at a concentration of 20 ng/ml 6 h before harvest. The effect of HCV core protein on the NF-
B pathway
in HeLa cells was examined using HepG2 cells with the same protocol as
that used for HeLa cells. To examine the dose-dependent
effect of core protein on the NF-
B pathway, HeLa cells were
transfected with a total of 1.2 µg of plasmids consisting of 0.38 µg of pNF-
B-Luc, 0.02 µg of pRL-TK, 0-0.8 µg of pCXN2-core,
and 0-0.8 µg of pCXN2 adjusted to total 1.2 µg.
In addition to the HeLa cells, which express core protein transiently,
HeTOC cells, which can be induced to express core protein under the
control of doxycycline, were used to examine the effect of HCV core
protein on the NF-
B pathway. Approximately 4 × 105
HeTOC cells were plated into the well of a six-well tissue culture plate containing 1 µg/ml doxycycline 24 h before transfection. Cells were transfected with 0.39 µg of pNF-
B-Luc and 0.01 µg of
pRL-TK and cultured in medium with or without doxycycline.
To elucidate how HCV core protein affects the NF-
B pathway, dominant
negative forms of IKK
/
, TRAF2/6, and TAK1, and an IKK
-specific
inhibitor were used. To initially examine the effect of the dominant
negative form of components of the NF-
B pathway, HeLa cells were
transfected with a total of 1.2 µg of plasmids consisting of 0.38 µg of pNF-
B-Luc, 0.02 µg of pRL-TK, 0.4 µg of pCXN2-core, and
0.4 µg of one of the following pIKK
(K44A), pIKK
(K44A), pTRAF2-(87-501), or pTRAF6-(289-522).
Similarly, HeLa cells were transfected with a total of 1.6 µg of
plasmids consisting of 0.38 µg of pNF-
B-Luc, 0.02 µg of pRL-TK,
0.4 µg of pCXN2-core, 0.4 µg of pCMV-TAK1(K63W), and 0.4 µg of
pCMV-TAB1. Second, we added 5 mM acetyl salicylic acid
(Sigma), which inhibits cyclooxygenase (24) and IKK
(25), to the
medium of HeLa cells transfected with 0.19 µg of pNF-
B-Luc, 0.01 µg of pRL-TK, and 0.2 µg of pCXN2 or pCXN2-core. Acetyl salicylic
acid was added 1 h after the transfection of plasmids. Instead of
acetyl salicylic acid, 25 µM indomethacin (Sigma), a
non-steroidal anti-inflammatory drug, which inhibits cyclooxygenase but
not IKK
(24, 25), was added to the medium as a control.
Luciferase Assay--
The entire cell lysate was collected
36 h after transfection. A luciferase assay was performed with the
PikkaGene dual sea pansy system (Toyo Ink, Tokyo, Japan) using a
luminometer (Lumat LB9507, EG&G Berthold, Bad Wildbad, Germany). Assays
were conducted at least in triplicate. Firefly luciferase activity and
sea pansy luciferase activity were measured as a relative light unit.
Firefly luciferase activity was then normalized for transfection
efficiency based on sea pansy luciferase activity.
Indirect Immunofluorescence Staining of HCV Core
Proteins--
Indirect immunofluorescence staining was performed as
previously described (26); COS-7 cells were transfected with
pCXN2-core, pCXN2-core173, or pCXN2-HAcore151. Forty-eight hours after
transfection, COS-7 cells were washed with phosphate-buffered saline
(PBS) and subsequently fixed with 3.7% formaldehyde in PBS for 1 h at room temperature. After washing with PBS, cells were then
permeabilized with 0.1% Tween 20 in PBS for 1 h. The cells were
then blocked with PBS containing 2% normal rabbit serum for 1 h
and incubated with mouse anti-core antigen IgG fraction (1:500) for
1 h at room temperature. After washing three times with PBS, cells
were incubated with fluorescein isothiocyanate-conjugated rabbit
anti-mouse IgG antibody (1:40) (Dako, Carpinteria, CA) for 1 h at
room temperature. Cells were then washed with ice-cold PBS, coated with
fluorescent mounting medium (Dako), covered with glass, and observed by
microscope using an epifluorescent attachment (AX80, Olympus, Tokyo, Japan).
Electrophoretic Mobility Shift Assay--
Approximately 2 × 106 HeTOC-22 cells were plated into a 10-cm dish (Iwaki
Glass) and cultured in medium with or without 1 µg/ml doxycycline.
Forty-eight hours later, the cells were harvested and their nuclear
extracts were prepared according to mini-nuclear extraction methods
(27). The concentration of the nuclear extracts was determined by a
Micro BCA protein assay reagent kit (Pierce) and was adjusted to give
equal concentrations. Electrophoretic mobility shift assay (EMSA) was
performed using a Gel shift assay system (Promega) according to
the manufacturer's protocol. Briefly, a synthetic double-stranded
oligonucleotide having a
B site (5'-AGTTGAGGGGACTTTCCCAGGC-3') was
end-labeled with [32P]ATP using T4 polynucleotide kinase
and incubated with 10 µg of nuclear extracts for 20 min at room
temperature. For competition, an unlabeled competitor oligonucleotide
or an unlabeled noncompetitor oligonucleotide
(5'-GATCGAACTGACCGCCCGCGGCCCGT-3') was added to the reaction mixture in
100-fold osmolar excess over the labeled probe to examine the binding
specificity. Reaction mixtures were loaded onto a 4% polyacrylamide
gel and then separated by electrophoresis in 0.5× Tris borate/EDTA
electrophoresis buffer (0.045 M Tris borate and 0.001 M EDTA).
Statistics--
Data were expressed as means ± S.D.
Statistical analyses were performed using the t test. A
p value of less than 0.05 was considered statistically significant.
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RESULTS |
Detection of Transiently Expressed HCV Core Proteins by Western
Blotting--
Expression of full-length and three deleted core
proteins was examined in the soluble protein cell extracts of
transiently transfected COS-7 cells by Western blotting (Fig.
1, A and B). Full-length (pCXN2-core) and aa 1-173 core (pCXN2-core173) were detected by mouse anti-HCV core antigen IgG fraction (Fig.
1A). HA-tagged core aa 1-151 (pCXN2-HAcore151) and aa
92-191 (pCXN2-HAcore92-191) were detected by anti-HA polyclonal
antibody (Fig. 1B). The size of each core protein was
consistent with the expected size.

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Fig. 1.
Detection of HCV core protein expression by
Western blotting. A, COS-7 cells were transfected with
pCXN2, pCXN2-core, and pCXN2-core173. Whole cell lysate was collected
36 h after transfection and analyzed by Western blotting using
mouse anti-HCV core antigen IgG fraction. Arrows indicate
expressed HCV core proteins. B, COS-7 cells were transfected
with pCXN2, pCXN2-HAcore151, and pCXN2-HAcore92-191. Whole cell lysate
was collected 36 h after transfection and analyzed by Western
blotting using anti-HA polyclonal antibody. Arrows indicate
expressed HCV core proteins.
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Doxycycline-regulated HCV Core Protein Expression in HeTOC
Cells--
Expression of HCV core protein was detected by Western
blotting in HeTOC cells, a clone termed HeTOC-22, which can be induced to express core protein under the control of doxycycline, 48 h after the removal of doxycycline. The induction ratio was greater than
1,000 when the relative amount of expressed HCV core protein with or
without doxycycline was analyzed using a LAS-1000 image analyzer (photo
film from Fuji, Tokyo, Japan) (data not shown).
Activation of the NF-
B Pathway by the HCV Core Protein--
In
HeLa cells, HCV core protein (0.2 µg of pCXN2-core) significantly
activated the NF-
B pathway at a value 6.2 ± 3.4 (mean ± S.D.) times higher than that of the control, whereas TNF-
(20 ng/ml)
activated the pathway at a value 8.6 ± 2.0 times higher (Fig.
2A). Activation of the NF-
B
pathway increased in relation to the amount of plasmid utilized for
pCNX2-core (Fig. 3). In HepG2 cells, HCV
core protein activated the pathway at a value 4.3 ± 0.9 times
higher than the control, whereas TNF-
activated the pathway at a
value 4.5 ± 2.5 (Fig. 2A).

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Fig. 2.
Activation of the
NF- B pathway by HCV core protein.
A, HCV core protein activated the NF- B pathway in HepG2
and HeLa cells. Luciferase activities were normalized by assuming the
activity of pCXN2-transfected cell lysate to be 100% (relative
luciferase activity). Results are expressed as the mean
(bar) + S.D. (line) of at least three
experiments. B, HeTOC cells were cultured in the medium with
or without doxycycline. Reporter plasmids were transfected 48 h
before assay. TNF (20 ng/ml) was added to the medium of HeTOC cells
6 h before assay. Results are expressed as the mean
(bar) + S.D. (line) of at least three
experiments.
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Fig. 3.
Dose-dependent activation of the
NF- B pathway by HCV core protein. HeLa
cells were transfected with various amounts of pCXN2 or pCXN2-core.
Luciferase activities were measured and expressed as described in the
legend for Fig. 2. pFC-MEKK, expressing constitutively active MEKK1,
served as a positive control for the NF- B pathway.
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HeTOC cells, which can be induced to express core protein under the
control of doxycycline, were used to examine activation of the NF-
B
pathway. Core-expressing HeTOC-22 cells cultured in a medium without
doxycycline showed a pathway activation value 5.2 ± 1.5 times
higher than that found with HeTOC-22 cells cultured in a medium with
doxycycline. Addition of TNF
to core-expressing HeTOC-22 cells did
not affect significantly the activation of NF-
B pathway by core
protein (Fig. 2B).
Core Protein Enhances NF-
B-DNA Binding Activity--
To examine
whether core protein enhances NF-
B-DNA binding, EMSA was performed
using the nuclear extracts of HeTOC-22 cells, which were induced to
express core protein. As shown in Fig. 4, NF-
B-DNA binding activity was enhanced in HeTOC-22 cells expressing core protein (about 2.9 times) as compared with that in HeTOC-22 cells
without expressing core protein under the existence of doxycycline. This NF-
B-DNA binding activity observed in these assays was ablated by an excess of unlabeled competitor but not by an excess of unlabeled noncompetitor (Fig. 4). Addition of an antibody directed against p65 or
p50 (Santa Cruz Biotechnology) generated a supershifted band,
suggesting that this NF-
B-DNA complex was containing p65 and p50
(data not shown).

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Fig. 4.
Enhancement of the
NF- B DNA-binding activity by HCV core
protein. Approximately 2 × 106 HeTOC-22 cells
were plated into a 10-cm dish and cultured in medium with or without 1 µg/ml doxycycline. Forty-eight hours later, nuclear extracts were
prepared and assayed for the NF- B DNA-binding activity by
electrophoretic mobility shift assay. The following were added to the
reaction: lane 1, nuclear extracts from HeTOC-22
cells with doxycycline (no expression of HCV core protein);
lane 2, nuclear extracts from HeTOC-22 cells
without doxycycline (expressing HCV core protein); lanes
3 and 4 contained the same nuclear extracts as
lane 2 except either excess unlabeled competitor
oligonucleotide probe or excess unlabeled noncompetitor oligonucleotide
probe was added for competition, respectively.
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Mapping the Region of HCV Core Protein Responsible for Activation
of the NF-
B Pathway--
To determine the region of HCV core
protein responsible for activation of the NF-
B pathway, we
constructed three plasmids, which expressed deletion mutant forms of
core protein: pCXN2-core173, pCXN2-HAcore151, and pCXN2-HAcore92-191.
HeLa cells were transfected with full-length or each deletion mutant
form of HCV core expressing plasmids in combination with pNF-
B-Luc
(Fig. 5). None of the deletion mutant
forms of core protein activated the NF-
B pathway, whereas
full-length HCV core protein did activate the pathway. These results
suggest that both the N terminus (aa 1-91) and C terminus (aa 174 to
191) of the core protein may play an important role in activation of
the NF-
B pathway.

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Fig. 5.
Mapping the region of HCV core protein
responsible for activation of the NF- B
pathway. HeLa cells were transfected with pCXN2, pCXN2-core,
pCXN2-core173, pCXN2-HAcore151, pCXN2-HAcore92-191, and reporter
plasmids. Luciferase activities were measured and expressed as
described in the legend for Fig. 2. Any deletion of mutant forms of
core protein did not activate the NF- B pathway, whereas full-length
HCV core protein activated the pathway.
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We then examined subcellular localization of full-length and two
deletion mutants of HCV core protein by indirect immunofluorescence assay. As shown in Fig. 6, full-length
core protein (aa 1-191) was located diffusely in the cytoplasm, in
contrast to the perinuclear localization of the C-terminal 18 aa-deleted core protein (aa 1-173) and the nuclear localization of the
C-terminal 40 aa-deleted core protein (aa 1-151).

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Fig. 6.
Subcellular localization of full-length and
two deletion mutants of the core proteins by indirect
immunofluorescence assay. COS-7 cells were transfected with
three types of core protein expression plasmid, pCXN2-core,
pCXN2-core173, and pCXN2-HAcore151. After 48 h, cells were fixed
and incubated with mouse anti-core antigen IgG fraction and then
stained with fluorescein isothiocyanate-conjugated rabbit anti-mouse
IgG antibody. Full-length core protein (aa 1-191) was located
diffusely in the cytoplasm (A), whereas the C-terminal 18 aa-deleted core protein (aa 1-173) was located in the perinuclear
region (B) and the C-terminal 40 aa-deleted core protein (aa
1-151) was found mainly in the nucleus (C).
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HCV Core Protein Activates the NF-
B Pathway through
IKK
--
To examine whether activation of the NF-
B pathway by
HCV core protein is transduced through IKK
or IKK
, HeLa cells
were cotransfected with pCXN2/pCXN2-core, pNF-
B-Luc, and
pIKK
(K44A)/pIKK
(K44A). Expression of
IKK
(K44A), catalytically inactive IKK
, significantly reduced HCV core induced NF-
B activation to about one-tenth, whereas
expression of IKK
(K44A), catalytically inactive IKK
, reduced the activation to about two-fifths (Fig.
7). To confirm the participation of
IKK
in activation of the NF-
B pathway by HCV core protein, we
added 5 mM acetyl salicylic acid, an IKK
-specific inhibitor (28), to the HeLa cells transfected with pCXN2/pCXN2-core and
pNF-
B-Luc. Activation of the pathway by core protein was significantly inhibited by acetyl salicylic acid but not by
indomethacin, a cyclooxygenase inhibitor (Fig.
8). These results suggest that HCV core
protein activates the NF-
B pathway through IKK, especially IKK
.

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Fig. 7.
Catalytically inactive IKK reduced the
NF- B activation by HCV core protein. HeLa
cells were transfected with pCXN2-core/pCXN2,
pIKK (K44A)/pIKK (K44A), and pNF- B-Luc.
Luciferase activities were measured and expressed as described in the
legend for Fig. 2. Catalytically inactive IKK blocked activation of
the NF- B pathway by HCV core protein more significantly than
catalytically inactive IKK . TNF (20 ng/ml) was added 6 h
before harvest to function as an inducer of the pathway (positive
control).
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Fig. 8.
Acetyl salicylic acid reduced
NF- B activation by HCV core protein.
Acetyl salicylic acid, an IKK -specific inhibitor, was added to the
medium of HeLa cells transfected with pCXN2/pCXN2-core and pNF-kB-Luc
at a concentration of 5 mM. Luciferase activities were
measured and expressed as described in the legend for Fig. 2.
Activation of the pathway by HCV core protein was significantly
inhibited by acetyl salicylic acid but not indomethacin, a
cyclooxygenase inhibitor.
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Dominant Negative Forms of TRAF2/6 Reduced the Activation of
the NF-
B Pathway by HCV Core Protein--
To examine whether
activation of the NF-
B pathway by HCV core protein was transduced
through TRAF2/6, HeLa cells were cotransfected with pCXN2/pCXN2-core,
pNF-
B-Luc, and pTRAF2-(87-501)/pTRAF6-(289-522). Expression of the
dominant negative form of TRAF2 (aa 87-501) significantly reduced
core-induced NF-
B activation to about two-fifths in HeLa cells (Fig.
9). Expression of the dominant negative
form of TRAF6 (aa 289-522) also reduced core-induced NF-
B
activation (Fig. 9). These results suggest that HCV core protein
activates the NF-
B pathway through TRAF2/6.

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Fig. 9.
Dominant negative TRAF2/6 reduced
NF- B activation by HCV core protein. HeLa
cells were transfected with pCXN2-core/pCXN2,
pTRAF2-(87-501)/pTRAF6-(289-522), and pNF- B-Luc. Luciferase
activities were measured and expressed as described in the legend for
Fig. 2. Dominant negative TRAF2 blocked activation of the NF- B
pathway by HCV core protein. TNF (20 ng/ml) was added 6 h
before harvest to function as an inducer of the pathway (positive
control).
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Catalytically Inactive TAK1 Has No Effect on Activation of the
NF-
B Pathway by HCV Core Protein--
The kinase TAK1 was shown to
act upstream of NIK in the IL-1-activated signaling pathway and
associate with TRAF6 during IL-1 signaling (23). To examine whether
activation of the NF-
B by HCV core protein was transduced through
TAK1, HeLa cells were cotransfected with pCXN2/pCXN2-core,
pNF-
B-Luc, pCMV-TAK1, and pCMV-TAB1. Although expression of
catalytically inactive TAK-1 and its activator, TAB1, efficiently
suppressed IL-1-induced activation of the NF-
B pathway, they had no
effect against core-induced activation of the pathway (Fig.
10), suggesting that HCV core protein activates the NF-
B pathway independent of TAK1.

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Fig. 10.
Catalytically inactive TAK1 had no effect on
activation of the NF- B pathway by HCV core
protein. HeLa cells were transfected with pCXN2/pCXN2-core,
pNF- B-Luc, pCMV-TAK1, and pCMV-TAB1. Luciferase activities were
measured and expressed as described in the legend for Fig. 2.
Catalytically inactive TAK-1 and its activator, TAB1, had no effect on
core-induced activation of the pathway. IL-1 was added 6 h before
harvest to function as an inducer of the pathway (positive
control).
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The Effect of HCV Core Protein on IL-1
or TNF
Promoter--
HCV core protein activated the IL-1
promoter 2.5 ± 0.6 times higher than the control. However, core protein did not
have significant influence on the TNF
promoter. This activation of IL-1
promoter was cancelled by catalytically inactive IKK
, or dominant negative TRAF2/6 (Fig. 11),
suggesting HCV core protein activates IL-1
promoter mainly through
NF-
B signaling.

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Fig. 11.
HCV core protein activated the
IL-1 promoter. HeLa cells were
transfected with pCXN2-core/pCXN2,
pIKK (K44A)/pIKK (K44A),
pTRAF2-(87-501)/pTRAF6-(289-522), and pIL-1 -promoter-Luc.
Luciferase activities were measured and expressed as described in the
legend for Fig. 2. Dominant negative TRAF2/6 completely blocked
activation of the NF- B pathway by HCV core protein. MEKK1 (0.01 µg) was expressed as an inducer of the pathway (positive
control).
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DISCUSSION |
In this study, HCV core protein clearly activated the NF-
B
pathway not only in a dose-dependent manner in mammalian
cells transiently transfected with core protein expression plasmid, but
also in HeLa cells inducibly expressing core protein. In fact, activation of NF-
B signaling by transfection of 0.4 µg of
pCXN2-core corresponded to activation by 20 ng/ml TNF
(Fig. 2; Ref.
7-9). This concentration of TNF
, which is widely used to study
responses to TNF, is enough to induce cytolysis in murine fibrosarcoma
L929 cells (29). However, there are contradicting data regarding the
effect core protein has on the NF-
B pathway; two studies demonstrated that core protein enhanced NF-
B signaling in cells stably expressing the core protein when using an EMSA (8) or in cells
transiently expressing the core protein when using a reporter assay (7,
9), while other studies showed that HCV core protein suppressed
TNF-induced NF-
B activation in cells stably expressing HCV core
protein with use of an EMSA (5, 6). These contradicting results may be
due to differences in the type of cells stably or transiently
expressing core protein and the method for detecting NF-
B
activation. To solve this problem, we adopted a tightly regulated, high
level core protein expression system responsive to doxycycline (30).
This system allowed us to overcome the problem of clonal selection of
permanent transfectants that differ in characteristics from parental
cell lines, as well as the problem of low transfection efficiency in
the transient transfection assay. Thus, we could analyze the effect of
HCV core protein on NF-
B signaling in the same cells with or without
doxycycline by use of both a reporter assay and an EMSA.
This study also demonstrated that HCV core protein activates the
NF-
B pathway through IKK
/
(Fig. 7). Core protein may
predominantly modulate the activity of one of the two I
B kinases,
IKK
. The dominant negative mutant of IKK
completely abrogated
activation of the NF-
B pathway by HCV core protein, in contrast to
the minor role played by the comparable mutant IKK
. Moreover, acetyl
salicylic acid, an IKK
-specific inhibitor (25), significantly
blocked the action of HCV core protein on NF-
B. Recently, it was
shown that only IKK
phosphorylation contributes to IKK activation by proinflammatory cytokines or by cotransfected NIK and MEKK1 (31). This
result is consistent with the genetic analysis of IKK function; whereas
disruption of the IKK
locus has no effect on IKK activation and
I
B degradation in response to proinflammatory stimuli, disruption of
the IKK
locus results in a major defect in both events (32, 33). In
fact, the dominant negative mutant of IKK
blocked activation of the
pathway by HCV core protein and TNF more efficiently than IKK
(Fig.
7). These results suggest that HCV core protein mimics proinflammatory
cytokine activation of the NF-
B pathway.
Furthermore, the dominant negative forms of TRAF2/6 significantly
blocked activation of the NF-
B pathway by HCV core protein. TRAF
proteins are known to function as signal transducers for distinct
receptor families. TRAF2 is thought to be a common mediator of TNFR and
CD40 signaling (21), whereas TRAF6 is thought to be a signal transducer
for IL-1 (22). Because CD40 is not expressed and CD40 signaling is not
activated by signal-activating anti-CD40 antibody (34) (monoclonal
antibody 89, Immunotech, Marseille, France) in HeLa and HepG2 cells
(data not shown), TNFR or IL-1 signaling is the temporary candidate for
the target of HCV core protein. Although TRAF6 is thought be involved
in IL-1 signaling and not in TNFR signaling, previous (35) and present
studies have shown that the dominant negative form of TRAF6 suppresses the activation of TNF
activation of NF-
B (Fig. 9). On the other hand, the dominant negative form of TRAF2 suppresses only TNFR signaling and not IL-1 signaling. Moreover, catalytically inactive TAK1, which links TRAF6 to the NIK-IKK cascade in the IL-1 signaling pathway, has no effect against the core-induced activation of NF-
B.
These data may imply that HCV core protein mimics proinflammatory cytokine activation of the NF-
B pathway, especially TNFR signaling through TRAF2/6.
Recently, HCV core protein was shown to interact with the cytoplasmic
tail of lymphotoxin-
receptor (36, 37), a member of the TNFR family,
and also with the cytoplasmic domain of TNFR1 (6), where TRADD and RIP
interact to produce TNF-induced NF-
B activation (11, 12). There may
be the possibility that HCV core protein activates the NF-
B pathway
through interaction with TNFR1. Our finding that the N-terminal 91-aa
deleted core protein did not activate the NF-
B pathway may support
this idea because the N-terminal 115 aa of HCV core protein is
important for the interaction with TNFR1 (6). However, we could not
detect an in vivo interaction between core protein and
TNFR1, TRADD, and TRAF2 by coimmunoprecipitation assay (data not
shown). Deletion analysis of the core protein showed that the
C-terminal 18 aa, a highly hydrophobic region (38), is also important
for NF-
B activation. It was suggested that this region is
responsible for the association of HCV core protein with intracellular
membranes, and the C-terminal deleted core protein translocates into
the nucleus (39, 40). As shown in Fig. 6, the C-terminal deleted core
proteins are located in the perinuclear region or the nucleus, whereas
full-length core protein was diffusely located in the cytoplasm. These
data may support our finding that core protein activates NF-
B in the
cytoplasm through TRAF2/6.
The NF-
B pathway is known to be activated by oncogenic viral
proteins such as X protein of the hepatitis B virus, Tax of human
T-cell leukemia virus type 1, and latent membrane protein 1 (LMP-1) of
the Epstein-Barr virus (41). Hepatitis B virus X protein interacts
directly with I
B
to probably prevent the reassociation of
I
B
with NF-
B (42). Tax was shown to interact with components
of the IKK complex, such as MEKK1 (28), IKK (43), and the NF-
B
essential modulator (44), thereby activating the NF-
B pathway. Tax
has been shown to also associate with I
B
, p105, p100, RelA, and
c-Rel (41). Thus, more than one molecule may be the target of HCV core
protein to activate the NF-
B pathway as well as Tax.
TNF
activates not only NF-
B signaling, but also activator protein
(AP)-1 signaling through TRAF2 (45). TRAF2 activates germinal center
kinase (46)/germinal center kinase-related (47) or MEKK1 (48), which
subsequently activate c-Jun N-terminal kinase-AP-1 cascade (49). Since
HCV core protein activates both NF-
B- and AP-1-associated pathways
(9), as well as LMP-1 (51), the shared components between these
pathways may be the target molecules for HCV core protein. This also
supports our finding that HCV core protein activates NF-
B in the
cytoplasm through TRAF2/6.
The NF-
B pathway plays an important role in cellular response to
proinflammatory cytokines such as TNF-
and IL-1 and induces an
inflammatory response by the up-regulation of many cytokines, including
IL-1, -2, -6, -8, and -12, and TNF-
(10). In this study, HCV core
protein is shown to mimic proinflammatory cytokine activation of the
NF-
B pathway, especially TNFR signaling., and actually activates
IL-1
promoter mainly through NF-
B signaling pathway. Recently we
have shown that HCV core protein activates also IL-8 promoter through
NF-
B pathway (9).Therefore, it is quite conceivable that HCV core
protein could induce an inflammatory response and cause
"hepatitis." In fact, the eradication of HCV by interferon leads to
the rapid resolution of acute and chronic hepatitis (52, 53). Moreover,
serum or intrahepatic expression of IL-1
, 2, 6, 8 and TNF-
are
elevated from 2 to 10 times higher in patients with active chronic
hepatitis C than those of a control group, and reduced after
eradication of the virus by interferon treatment (54-58). Although the
host immune response caused by cytotoxic T lymphocytes is believed to
play a pivotal role in the pathogenesis of C-viral hepatitis (50), our
findings suggest that HCV core protein directly induces hepatitis
through inflammatory cytokine production. Therefore, blockage of the
activation of the NF-
B pathway may become an attractive option for
the treatment of chronic hepatitis C in the future.