Article

Regulation of Hepatitis C virus replication and gene expression by the MAPK-ERK pathway

Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
Virologica Sinica 10/2012; 27(5). DOI: 10.1007/s12250-012-3257-6

ABSTRACT

The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle. In the present study using a Huh7 cell line Con1 with an HCV replicon, we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-α signalling. Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells. It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site. Consistently, a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays. Thus, the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication. In addition, cyclin-dependent kinases (CDKs) downstream of ERK may also be involved in the modulation of HCV replication since roscovitine, an inhibitor of CDKs had a similar effect to that of U0126. Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels. Further, the replication of HCV replicon in Con1 cells was inhibited by IFN-α. The inhibitory effect of IFN-α could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs. It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.

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Available from: Zhongji Meng, Dec 28, 2013
VIROLOGICA SINICA, October 2012, 27 (5):278-285
DOI 10.1007/s12250-012-3257-6
© Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2012
Research Article
Regulation of Hepatitis C Virus Replication and Gene Expression by the
MAPK-ERK Pathway
*
Rongjuan Pei
1,3
, Xiaoyong Zhang
2
, Song Xu
1,3
, Zhongji Meng
2
, Michael Roggendorf
2
, Mengji Lu
2
and Xinwen
Chen
1
1. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China;
2. Institute of Virology, University Hospital of Essen, University of Duisburg-Essen, Essen 45122, Germany;
3. Graduate School of the Chinese Academy of Sciences, Beijing 100049, China
The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation
of multiple cellular processes including the cell cycle. In the present study using a Huh7 cell line Con1 with an HCV replicon,
we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein
expression and might influence IFN- signalling. Epithelial growth factor (EGF) was able to stimulate ERK activation and
decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A
protein amounts in Con1 cells. It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated
the translation directed by the HCV internal ribosome entry site. Consistently, a U0126 treatment enhanced activity of the
HCV reporter replicon in transient transfection assays. Thus, the MAPK-ERK pathway plays an important role in the
regulation of HCV gene expression and replication. In addition, cyclin-dependent kinases (CDKs) downstream of ERK may
also be involved in the modulation of HCV replication since roscovitine, an inhibitor of CDKs had a similar effect to that of
U0126. Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA
levels. Further, the replication of HCV replicon in Con1 cells was inhibited by IFN-. The inhibitory effect of IFN- could be
partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs. It could be shown
that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.
Hepatitis C Virus (HCV); Mitogen activated protein kinases-extracellular signal regulated kinase (MAPK-ERK); Cell cycle
progression
epatitis C virus (HCV) is an enveloped positive RNA
virus and belongs to the family Flaviviridae
[8]
. HCV
causes persistent infection in 50 %-80 % of infected persons
and may lead to the development of fibrosis, cirrhosis, and
hepatocellular carcinoma
[2,33,37]
. The establishment of a HCV
subgenomic replicon system
[21]
was a major breakthrough in
research on HCV. Furthermore, an HCV infection system was
established based on the HCV JFH-1 molecular clone and
Huh-7 derived cell lines
[3,20,39,41]
. These systems facilitate
studies on the HCV life cycle and HCV-host cell interaction.
Since the viral replication is dependent on the host cell
machinery, activation or suppression of cell signalling pathways
may modulate virus replication. In the case of HCV, a number
of intracellular signalling pathways, such as PI3K/Akt, IRF3,
and the JAK-STAT pathway, have been shown to influence
HCV replication
[4,7,10,11,13,15,26,34]
. HCV has developed different
strategies to interfere with host cell signalling pathways. It has
been demonstrated that HCV NS3/4A blocks retinoic
acid-inducible I gene (RIG-I) mediated signalling by cleaving
the signalling adaptor Cardif and TLR3 mediated IRF3
activation through cleavage of the adaptor Toll/interleukin
receptor domain-containing adapter-inducing interferon
(TRIF), thereby interfering with the induction of innate
responses
[12,19,28,35]
. The blockage of the RIG-I mediated
activation of innate responses and subsequent interferon (IFN)
production is essential for the maintenance of HCV replication,
as HCV is highly sensitive to the antiviral action of IFN.
H
Received: 2012-04-26, Accepted: 2012-08-17
* Foundation items: Partly supported by a joint grant of Chinese
Academy of Science and Deutsche Akademische Austausch
Dienst, and the the National Basic Research Priorities Program o
f
China (2009CB522501, 2005CB522901, 2007CB512901).
Corresponding author.
Phone: +86-27-87199106, Fax: +86-27-87199106,
Email: chenxw@wh.iov.cn
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The mitogen activated protein kinases (MAPKs) are widely
expressed serine/threonine kinases and mediate signals for the
regulation of important cellular functions such as gene
transcription, post transcriptional regulation, and cell cycle
progression
[32]
. There are three main groups of MAPKs:
extracellular signal regulated kinases (ERK), the p38 family
kinases, and the JUN amino terminal kinases. The activation
of the MAPK-ERK pathway is pivotal for the cell cycle
progression
[22,27]
. Mitogenic stimulation of cells causes
phosphorylation of ERK and translocation of active ERK to
the nucleus
[9]
. This translocation is necessary for
ERK-dependent activation of DNA synthesis and progression
from G1 into S phase
[6]
. It has been shown that the HCV NS5A
protein may interfere with the activation of the MAPK-ERK
pathway by altering the trafficking of epithelial growth factor
(EGF) receptor
[23,25]
, thus attenuating the cellular response to
EGF.
However, the significance of the MAPK pathway activation
in HCV replication is not clear. In a previous study, HCV
translation mediated by the internal ribosome binding site
(IRES) was shown to be enhanced by inhibitors of the MAPK
pathway
[29]
. However, this study did not clarify the influence of
the MAPK pathway on HCV replication. Huang et al.
examined the role of the MAPK pathway in the antiviral action
of interferon- against HCV
[16]
. In a luciferase-based reporter
replicon system, a blockage of the MAPK pathway activation
could partially interfere with the inhibition of the reporter
replicon by IFN-. It was suggested that the phosphorylation of
HCV NS5A plays an important role in the HCV replication and
is influenced by the MAPK activity.
In the current study, we examined the influence of the
MAPK-ERK pathway on HCV replication. Using the HCV
replicon system, we examined whether HCV replication and
HCV IRES-dependent translation could be modulated by the
inhibition or activation of the MAPK-ERK pathway. Furthermore,
we investigated how the modulation of the MAPK-ERK
pathway influences the IFN signalling and the inhibition of
HCV replication by IFN. As the MAPK-ERK pathway is able
to directly influence cell progression by activation of
cyclin-dependent kinase 2 (CDK2), an important key protein of
cell cycle control, we tested whether a blockage of cdk2 by an
inhibitor roscovitine would also modulate HCV replication. A
cell cycle inhibitor aphidicolin and an siRNA of a cellular
negative regulator CDKN2B were used to modulate the cell
cycles and to test the influence on HCV replication.
MATERIALS AND METHODS
Cell culture
Huh7-lunet cells (kindly provided by Dr. Ralf Bartenschlager)
were cultured in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10 % fetal bovine serum (FBS), 2 mmol/L
of glutamine, 100 U/mL of penicillin and 100 U/mL of streptomycin
at 37 °C in a 5 % CO
2
atmosphere. The subgenomic replicon system
based on Huh7-lunet cells and the subgenomic HCV replicon
pFKI389neo/NS3-3´ was kindly provided by Dr. Ralf
Bartenschlager. Con1 cells with subgenomic HCV replicon were
maintained in the same medium supplemented with 0.5 mg/mL
G418 (Cytogen).
Reagents
The inhibitor of MAPK-ERK pathway U0126 and the CDK
inhibitor roscovitine were purchased from Sigma and InvivoGene,
respectively. Epidermal growth factor (EGF) was purchased
from BD Biosciences. Aphidicolin was purchased from Sigma.
Polyclonal antibodies to HCV NS5A were kindly provided by
Dr. Ralf Bartenschlager. Antibodies to ERK1 (K-23) and pERK
(E-4) were purchased from Santa Cruz Biotechnology, Inc. A
monoclonal mouse anti-actin (α-Sarcomeric) antibody was
provided by Sigma-Aldrich and the secondary antibodies
peroxidase affinipure goat anti-mouse IgG (H+L) and
peroxidase affinipure goat anti-rabbit IgG (H+L) by Jackson
ImmunoResearch Inc.
Electroporation and transient HCV replication assay
1×10
7
lunet cells were transfected by electroporation using
5 µg of a luciferase
replicon pFKI389luc/NS3-3’ as described
[18]
.
12 mL of complete DMEM were then added to cells. 1 mL
aliquots of the cell suspension were seeded in each well of a
24-well-plate and harvested at indicated time points for
luciferase assay. Luciferase activity was expressed as relative
light units (RLU). These values represent the percentage of
luciferase activity determined at a given time point relative to
the one measured 4 h after transfection.
RNA extraction and quantification of HCV RNA by real
time RT-PCR
Total RNAs from cultured cells were extracted using Trizol
(Invitrogen) according to the manufacturer’s instructions. The
quantification of HCV RNA was performed by real-time
RT-PCR using a QuantiFast SYBR Green RT-PCR kit (Qiagen).
The copy numbers of human beta-actin mRNAs were
determined and used for normalization of real-time RT-PCR
detection of other RNAs. The following primers were used:
5’-atcactcccctgtgagga act-3’ (nt 36-56) and 5’- gcgggttgatccaagaaagg-
3’ (nt 192-172) for HCV (Numbering of the sequence is according
to GenBank entry AJ242652), A1 5´-tccctggagaagagctacga-3´
(nt 879-905) and A2 5´- agcactgtgttggcgtacag-3´ (nt 1224-1200)
for -actin (Numbering of the sequence is according to
GenBank entry NM_001101.2). The primers for real time
RT-PCR of CDKN2B were validated and provided by Qiagen
(Order number QT00203147).
The HCV RNA levels in untreated Con1 cells ranged
between 0.5 to 2 copies/beta-actin RNAs under normal culture
conditions, as determined by real time RT-PCR. For real time
RT-PCR, HCV RNA copy numbers per reaction were around
10
6
in the majority of the samples.
Treatment of cells with EGF, inhibitors, and IFNs
Cells were seeded at a density of 7×10
4
per well in 24-well
plates in DMEM omitting G418. After 18 h incubation, cells
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280
reached a confluence of 80 %. Different compounds at
appropriate concentrations were then added to the wells at
indicated time points.
Detection of HCV NS5A and cellular proteins by Western
blotting
The detection of HCV NS5A protein was performed as
described previously
[5]
. Cells were lysed in 50 mmol/L Tris-
HCl pH 7.4, 150 mmol/L NaCl, 0.5 % (v/v) Triton-X100, 1
mmol/L EDTA, and 1 mmol/L PMSF or directly in Laemmli
sample buffer. Proteins were separated by SDS-PAGE and
transferred to a polyvinylidene difluoride membrane. After
blocking with 5 % non-fat milk in Phosphate Buffered Saline
with Tween 20 (PBST), membranes were incubated with
specific primary antibodies. Peroxidase-conjugated secondary
antibodies matched to the primary antibodies were used for
detection. Detected proteins were visualized using ECL
Western blot detection reagents (GE Healthcare).
Reporter assays based on the CoA-acetyl transferase (CAT)
and luciferase systems
Huh7-lunet or Con-1 cells were transfected with reporter
plasmids (pISRE-Luc (Clontech) or a dicistronic vector pHCV-
IRES_D128) using lipofectamine 2000 (Invitrogen). Plasmid
pHCV-IRES_D128 was constructed based on pD128 (kindly
provided by Dr. M Niepmann) and consisted of a chloramphenicol
acetyltransferase (CAT) reporter gene directed by the cap-
dependent CMV promoter and a luciferase gene directed by
HCV IRES. The HCV 5’ noncoding region (nt 1-362) was
amplified with suitable primers and cut with restriction
enzymes, and cloned into pre-digested pD128 vector. Luciferase
reporter assays were performed using the luminescence reporter
gene assay system (PerkinElmer) according to the manufacturers
instructions. The levels of CAT in transfected cells were
determined by using the Roche CAT ELISA Kit.
Treatment of cells with siRNA
Cells were grown for 24 h to a confluence of 80 % and then
transfected with siRNAs. Lipofectamine 2000 (Invitrogen) was
used according to the manufacturer’s instructions. Twenty pmol
siRNA and 1 μL of Lipofectamine 2000 per well were applied
in a final volume of 0.5 mL Opti-MEM. After 5 h, the medium
was replaced by fresh culture medium. The siRNAs were
purchased from Qiagen. The siRNA siHCV 5-ggucucgua
gaccgugcacTT-3 targeted the HCV sequence nt 331 to 351
(numbered according to the sequence with GenBank accession
number AJ242654)
[40]
. Another siRNA to CDKN2B is a
validated siRNA from Qiagen (Ordering number SI00288281).
RESULTS
Inhibition of MAPK-ERK pathway led to an increased
HCV replication and protein expression
To investigate the role of MAPK-ERK pathway in HCV
replication, we first examined whether HCV replication and
protein expression could be modulated by inhibiting the
MAPK-ERK pathway. Con1 cells were treated with U0126, an
inhibitor of MAPK-ERK pathways, at different concentrations
for 48 h. The U0126 treatment prevented the phosphorylation
of ERK upon stimulation with EGF (data not shown). HCV
RNA and protein levels in cells were determined by real time
RT-PCR and Western blot (Fig 1. A and B). U0126, at a high
concentration of 10 mol/L, led to a more than twofold
increase of HCV RNA load. The abundance of NS5A protein
also increased at the lower concentration of 2.5 mol/L. The
HCV RNA levels in U0126-treated Con1 cells increased
gradually up to 72 h (Fig. 1C). These results indicated that
inhibition of the MAPK-ERK pathway leads to an enhancement of
HCV replication and protein expression.
The influence of the MAPK-ERK pathway on HCV
replication was also tested in a transient transfection system
using a HCV replicon based on the luciferase reporter gene
[18]
.
U0126 was added to cells 4 h after electroporation and HCV
replication was assessed by measuring the luciferase activity in
transfected cells. Consistently, U0126 was able to enhance the
luciferase expression in transfected cells in a dose-dependent
manner, indicating an enhancement of HCV replication activity
(Fig. 1D). However, a high concentration of 10 mol/L of U0126
abolished HCV replication completely, probably due to a strong
Fig. 1. The ERK inhibitor U0126 increases HCV RNA and protein
level in Con1 cells. Con1 cells were treated with U0126 at
concentrations of 2.5, 5, and 10 mol/L for 48 h. HCV RNA (A) and
NS5A protein (B) levels in cells were detected by real time RT-PCR
and Western blot, respectively. HCV RNA levels in cells were
determined as the ratio of HCV RNA/beta-actin RNA (see Material and
methods section for details). Relative HCV RNA levels were calculated
as the untreated control was set as 100%. Beta-actin was detected by
using specific antibodies for normalization. The NS5A protein levels
were quantified by densitometry, normalized against beta-actin and
expressed in arbitory units. C: Con1 cells were treated with U0126 and
harvested for RNA extraction at indicated time points. HCV RNA
levels in cells were detected by real time RT-PCR. D: Transfection of
HCV reporter replicon in the presence of U0126 at concentrations of 0,
2.5, and 5 mol/L. The transfected cells were harvested for determination of
luciferase expression at indicated time points. The significant
differences of the different groups are shown as *(p<0.05).
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inhibition of recovery of lunet after electroporation (data not
shown).
Activation of MAPK-ERK pathway by EGF modulates the
HCV replication
Next, we tested whether activation of the MAPK-ERK pathway
influences HCV replication. First, we confirmed that EGF was
able to activate the MAPK-ERK pathway in our system. The
steady-state level of phosphorylated ERK and the activation of
ERK by EGF were determined in naïve Lunet cells and Con1
cells under normal culture conditions. As shown in Fig. 2A, the
amounts of ERK1/2 and the phosphorylated form of ERK1/2 in
both cell lines were comparable. Furthermore, similar kinetics
of EGF-directed ERK1/2 phosphorylation were observed in
both Lunet and Con1 cells. The phosphorylation of ERK1/2
occurred rapidly and reached a peak level 5 min after EGF
stimulation. The dephosphorylation of ERK1/2 followed and
led to a gradual decrease of phosphorylated ERK1/2. Thus, the
presence of HCV replicon did not change the steady-state level
of phosphorylated ERK1/2 and the response to EGF in our
system. Previously, activation by short term stimulation by
EGF of the MAPK pathway in Huh7 cells harboring HCV
replicon was shown to be suppressed
[23]
. Interestingly, the HCV
RNA level in cells declined transiently with a maximum
reduction of 35% at 24 h and returned to a normal level after 48 h
(Fig. 2B). A slight treatment and returned to the normal level after
48 h if EGF change in HCV protein abundance occurred shortly
after the with was not added after 24 h (Fig. 2C). An additional
treatment EGF after 24 h did not reduce further the HCV RNA
level but prevented its return to the pre-treatment level (data not
shown), consistent with published data
[16]
. It could be concluded
Fig. 2. EGF modulates HCV replication and gene expression. Con1
cells were treated with 50 units of EGF for indicated time and
harvested for RNA extraction and Western blotting analysis.
Phosphorylated and unphosphorylated ERK were detected using
specific antibodies (A). HCV RNA (B) and NS5A protein (C) were
detected by real time RT-PCR and Western blot, respectively. HCV
RNA levels in cells were determined as the ratio of HCV
RNA/beta-actin RNA. Relative HCV RNA levels were calculated as
the untreated control was set as 100%. Beta-actin was detected by using
specific antibody for normalization. The significant differences of the
different groups are shown as * (p<0.05).
that activation of the MAPK-ERK pathway by EGF leads to a
slight and transient decrease in HCV replication in Con1 cells.
Modulation of the HCV IRES-dependent gene expression
The inhibition of the MAPK-ERK pathway led to changes in
the HCV RNA and protein abundance. Thus, it is possible that
HCV translation is influenced by the MAPK-ERK pathway.
HCV translation is specifically mediated by an internal
ribosome entry site (IRES) at the 5’ non-coding region of HCV.
To study the IRES-dependent translation, a dicistronic vector
was constructed. The dicistronic vector consisted of a CAT
reporter gene directed by the CMV promoter and a firefly
luciferase gene directed by HCV IRES (Fig. 3A). Lunet cells
were transfected with the dicistronic vector and then treated
with U0126 at indicated concentrations for 24 h. Luciferase and
CAT activities were detected separately. The results showed
that the rate of the Cap-dependent translation decreased in cells
treated with U0126 (Fig. 3B). However, the ratio of IRES-
dependent translation to Cap-dependent translation was raised
in the presence of U0126 (Fig. 3C). Therefore, the IRES-
dependent translation was preferred in comparison with the
cellular cap-dependent translation by the inhibition of the
MAPK-ERK pathway. This result indicated that the inhibition
of the MAPK-ERK pathway facilitates the IRES-directed HCV
translation and contributes to enhanced HCV replication.
Inhibition of MAPK-ERK pathway modulates the interferon-
stimulated response element (ISRE)-dependent gene expression
and influences the inhibitory effect of IFN on HCV
Previously, Huang et al.
[16]
showed that a modulation of
MAPK-ERK pathway influenced the anti-HCV action of IFN-,
Fig. 3. Modulation of the HCV IRES-dependent gene expression by
U0126. A: The schema of dicistronic vector with HCV IRES. B: The
dicistronic vector were transfected into Con1 cells. The cells were
treated with U0126 at concentrations of 1 and 5 mol/L. The activity of
luciferase and CAT enzyme were determined by luciferase assay and
CAT ELISA, respectively. The untreated controls were set as 100%. C:
The ratio of the HCV IRES-dependent to Cap-dependent gene
expression. The significant differences of the different groups are
shown as * (p<0.05).
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indicating that a cross-talk between the MAPK-ERK pathway
andthe IFN signalling pathway may exist. Therefore, we investigated
whether the modulation of MAPK-ERK pathway influences
IFN- signalling and the antiviral action of IFN- in Con1
cells. First, the effect of inhibitors of the MAPK-ERK pathway
on the ISRE-dependent gene expression was examined. Con1
cells were transfected with a reporter plasmid pISRE-Luc
vector. The transfected cells were treated with U0126 for 2 h
and then stimulated with IFN- for 6 h. Clearly, U0126 was
able to inhibit the IFN--stimulated reporter gene expression in
a dose-dependent manner (Fig. 4A). These results indicate that
1000 units per mL) for 48 h. Both HCV RNA and protein levels
decreased in Con1 cells after the treatment with IFN- (Fig. 4B
and C). In cells treated with U0126, the levels of HCV RNA
and HCV NS5A protein were significantly higher compared
with the control. However, IFN- was still effective in U0126
an inhibition of MAPK-ERK pathway influences IFN signalling.
We addressed the question whether an inhibition of MAPK-
ERK pathway reduces the ability of IFN- to suppress HCV
replication in Con1 cells. Con1 cells were treated with U0126
and then incubated with IFN- at different concentrations(0 to
treated cells and was able to reduce the HCV RNA and protein
levels in a dose-dependent manner (Fig. 4B and C). These results
Fig. 4. U0126 inhi5A protein (C) were detected by real time RT-PCR
and Western blobited the activation of ISRE reporter gene by IFN- (A)
and attenuated the anti-HCV effect of IFN- (B and C). Con1 cells
were pre-incubated with different inhibitors before IFN- treatment.
HCV RNA (B) and NSt, respectively, 48 h later. HCV RNA levels in
cells were determined as the ratio of HCV RNA/beta-actin RNA.
Relative HCV RNA levels were calculated as the untreated control was
set as 100%. Beta-actin was detected by using specific antibody for
normalization. The NS5A protein levels were quantified by densitometry,
normalized against beta-actin and expressed in arbitory units. The
significant differences of the different groups are shown as * (p<0.05).
imply that IFN and MAPK-ERK pathways may synergistically
regulate HCV replication.
Inhibition of CDK2 enhanced HCV replication in the
replicon system
One important function of activated ERK 1/2 is to activate
the cell cycle regulator CDK2 by inducing degradation of
p27
[1,17,36]
. Thus, a blockage of MAPK-ERK activation by
U0126 may prevent cell cycle progression. The question has
been raised as to whether the modulation of the cell cycle
progression by prevention of CDK2 activation causes changes
of the HCV replication levels. Therefore, HCV replication and
protein expression was tested during the inhibition of CDK2
activity by roscovitine. An incubation of Con-1 cells with
roscovitine led to a significant increase of the HCV RNA levels
and HCV NS5A expression (Fig. 5A and B). Similarly to
U0126, a roscovitine treatment modulated the effect of IFN-
on HCV replication in lunet cells (Fig. 5C, D and E). These
data suggested that MAKP-ERK pathway may modulate HCV
replication and protein expression by influencing the cell cycle
progression.
To verify the influence of the cell cycle progression on HCV
replication, two different approaches were applied. First, the
cell cycle progression was arrested by aphidicolin, an inhibitor
of cellular DNA polymerase (Fig. 6A). A treatment of Con1
cell with this compound for 24 h led to an increase of HCV
RNA levels compared to in those untreated cells. Second,
CDKN2B, a negative regulator of cell cycle progression, was
targeted by RNAi. A transfection of Con-1 cells with siRNA to
CDKN2B resulted in a decrease of the level of the specific
mRNAs to less than 50% of the controls within 72 h (Fig. 6B).
At the same time, the HCV RNA level was reduced to about
40% of that measured in control cells (Fig. 6C). This result is
consistent with the previous findings that a treatment with EGF
transiently reduced HCV RNA and protein levels (Fig. 1B).
Either a treatment with EGF or knock down of CDKN2B
facilitated the cell cycle progression.
DISCUSSION
In this study, we demonstrate that HCV replication could be
modulated by activation or inhibition of MAPK-ERK pathway.
While an inhibition of MAPK-ERK pathway enhanced HCV
translation and replication, the activation of this pathway by
EGF led to a reduction of HCV replication. Trujillo-Murillo et
al.
[38]
showed that treatment of cells with acetylsalicylic acid
led to an inhibition of HCV replication and protein expression
through cyclooxygenase 2 signalling pathways with involvement
of ERK1/2. Thus, different mediators leading to the activation
or inhibition of ERK1/2 may possess the ability to modulate
HCV replication. The blockage of MAPK-ERK pathway
facilitates the HCV IRES-dependent over the cap-dependent
translation. Thus, HCV protein levels were elevated in Con1
cells treated with the MAPK-ERK pathway inhibitors. It is also
possible that the increased viral translation contributed to HCV
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283
Fig. 5. Roscovitine modulated HCV replication and attenuated the anti-HCV effect of IFN-. Con1 cells were treated with roscovitine for 48 h at the
concentrations indicated in the absence (A, B) or presence (C, D) of IFN-. HCV RNA (A, C) and NS5A protein (B, D) were detected by real time
RT-PCR and western blot, respectively. HCV RNA levels in cells were determined as the ratio of HCV RNA/beta-actin RNA. Relative HCV RNA
levels were calculated as the untreated control was set as 100%. E: Con1 cells were transfected with pISRE-Luc and maintained in culture medium in
the presence of roscovitine for 48 h with the concentrations indicated. The relative luciferase activities were determined by luciferase assay. The
untreated controls were set as 100%. The significant differences of the different groups are shown as * (p<0.05).
Fig. 6. Modulation of the cell cycle progression led to changes of HCV
RNA levels. A: Treatment with aphidicolin. Con1 cells were incubated
with aphidicolin at a concentration of 10 μg/mL for 24 h. Aphidicolin
was washed away to avoid the possible toxicity. Cells were then
harvested for RNA extraction at the indicated time points. B and C:
Silencing of CDKN2B. ConI cells were transfected with siRNAs
targeting CDKN2B and HCV or unrelated siRNA and harvested after
72 h later for RNA extraction. CDKN2 (B) and HCV RNA levels (C)
were determined by real time RT-PCR and expressed as the ratio to
beta-actin RNA respectively. Relative HCV RNA levels were
calculated as the untreated control was set as 100%. The significant
differences of the different groups are shown as * (p<0.05).
replication, resulting in a higher level of HCV RNA. However,
this hypothesis needs to be experimentally proven.
The activation of the MAPK-ERK pathway has a multiple
effect on cellular processes
[32]
. A critical function of ERK is to
promote the cell cycle progression by degradation of p27 and
release of CDK2/cyclin E
[17]
. In the current study, roscovitine,
the inhibitor of CDKs, exerted a similar or even a stronger
effect on HCV replication and protein expression than U0126.
Two different approaches with aphidicolin and siRNA to
CDKN2B demonstrated consistently the correlation between
the cell cycle progression and HCV replication. This
phenomenon could be explained by the fact that HCV usually
replicates in non-proliferating hepatocytes. It could be
hypothesized that the growth arrest of hepatoma cells may lead
to a cellular status that is more favourable for HCV replication.
An inhibitory effect of cell confluence on HCV replication was
observed. However, this inhibitory effect was a result of the
reduced intracellular pools of nucleosides in confluent cells,
possibly through the shutoff of the de novo nucleoside
biosynthetic pathway when cells become confluent
[31]
. Adding
exogenous uridine and cytidine to the culture medium restored
HCV replication and expression in confluent cells.
An interesting finding was that the inhibition of the MAPK-
ERK pathway reduced the ISRE-dependent gene expression.
These results suggested that the MAPK-ERK and IFN
signalling pathways may crosstalk over some yet unknown
links. Thus, understanding of the mechanisms of interaction of
different cellular signalling events will be helpful for design of
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Virologica Sinica|www.virol.cn
284
new strategies directed at modulation of the cellular gene
expression related to antiviral activities. The modulation of
MAPK-ERK pathway led to a change of effectiveness of the
antiviral action of IFN but did not block the inhibition of HCV
replication by IFN signalling. MAPK-ERK and IFN pathways
may act synergistically to regulate HCV replication.
Controversial reports have been published on this topic.
While some reports claimed an enhanced replication of HCV
by inhibition of MEK-ERK pathway
[16,29,30]
, others indicated
that the inhibition of this pathway could reduce HCV
replication
[14]
. Here we demonstrated that MAPK-ERK pathway
and the downstream cell cycle regulators play a significant role
in the regulation of HCV replication and protein expression in
subgenomic containing cell lines. However, when we applied
the MEK inhibitor, U0126, in the HCVcc infectious system, a
reduction of infectious virus produced in the supernatant was
observed. This may indicate a complex role of the MEK-ERK
pathway in the HCV life cycle.
Acknowledgements
The authors would like to thank Dr. Ralf Bartenschlager and
Dr. M Niepmann for providing cell lines and other reagents.
The authors would also like to thank Dr. Simon Rayner for
kindly reading and criticizing the manuscript.
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    • "Activation of MAPK signalling cascades including p38 MAPK, ERK1/2 and JNK has been reported to increase HO-1 gene expression and anti-HCV activity (Huang et al., 2006; Yano et al., 2009; Pei et al., 2012). To investigate the involvement "
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    • "For instance, activation of the MAPKs MEK/ERK and JNK1/2 is required for productive infection of baculovirus (Katsuma et al. 2007). The MAPK-ERK pathway was also shown to play an important role in regulating gene expression and replication of hepatitis C virus (HCV) (Pei et al. 2012) and Kaposi's sarcoma-associated herpesvirus (Sharma-Walia et al. 2005). Many other studies have reported the activation doi: 10.1590/0074-0276108052013004 Financial support: FAPEMIG, CAPES, MCT-Brazil, CNPq APCS, JDA and LGA were recipients of a predoctoral fellowship from CNPq, LGA was recipient of premaster fellowship from CNPq, CAB, EGK and PCPF are recipients of research fellowships from the CNPq, JAPS-M was recipient of a predoctoral fellowship from CAPES. "
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