From the Division of Microbiology, School of
Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2
9JT, United Kingdom and the ¶ Institute of Medical Technology,
Tampere University Hospital, Tampere, Finland FIN-33014
Received for publication, October 24, 2002, and in revised form, February 26, 2003
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ABSTRACT |
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The hepatitis C virus nonstructural 5A (NS5A)
protein is a pleiotropic phosphoprotein that has been shown to
associate with a wide variety of cellular signaling proteins. Of
particular interest is the observation that a highly conserved
C-terminal Class II polyproline motif within NS5A mediated association
with the Src homology 3 domains of members of the Src family of
tyrosine kinases and the mitogenic adaptor protein Grb2 (A. Macdonald,
K. Crowder, A. Street, C. McCormick, and M. Harris, submitted
for publication). In this study, we analyzed the consequences of NS5A
expression on mitogenic signaling pathways within a variety of cell
lines. Utilizing a transient luciferase reporter system, we observed that NS5A inhibited the activity of the mitogenic and stress-activated transcription factor activating protein-1 (AP1). This inhibition was
dependent upon a Class II polyproline motif within NS5A. Using a
combination of dominant active and negative mutants of components of
the MAPK signaling pathways, selective inhibitors, together with
immunoblotting with phospho-specific and phosphorylation-independent antibodies, we determined the signaling pathways targeted by NS5A to
inhibit AP1. These studies demonstrated that in both stable NS5A-expressing cells and Huh-7-derived cells harboring subgenomic hepatitis C virus (HCV) replicons, this inhibition was mediated through
the ERK signaling pathway. Importantly, a comparable inhibition of AP1
reporter activity was observed in hepatocyte-derived cell lines
transduced with a baculovirus vector driving expression of full-length
HCV polyprotein. In conclusion, these data strongly suggest a role for
the NS5A protein in the perturbation of mitogenic signaling pathways in
HCV-infected hepatocytes.
Hepatitis C virus (HCV)1
can establish a persistent infection, often leading to chronic
liver disease and cirrhosis. There is considerable interest in HCV,
because chronic infection is strongly associated with development of
hepatocellular carcinoma. Indeed, HCV infection is the leading
indicator for liver transplantation in the United States (1).
HCV is a small, enveloped Hepacivirus classified within the family
Flaviviridae, most closely related to the Pestiviruses (e.g. bovine viral diarrhea virus). The genome is a
single-stranded positive sense RNA molecule of ~9.5 kilobases in
length with a single large open reading frame encoding for a
polyprotein of ~3000 amino acids (2). The open reading frame is
flanked by 5'- and 3'-untranslated regions, which have been shown to be
essential in both initiation of translation and viral replication (3). Processing of the precursor polyprotein requires both host and viral
proteases to produce the structural (core, E1, E2, and p7) and
nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (4).
The mature NS5A protein is generated by the action of the NS3/4A serine
protease. NS5A is localized to cytoplasmic and perinuclear regions of
the cell and exists in a basal or hyperphosphorylated state (p56 or
p58), with the degree of serine/threonine phosphorylation accounting
for the difference in size (5). The exact function of NS5A is unknown,
although it is speculated to form part of a multiprotein replication
complex located on the cytosolic face of the endoplasmic reticulum
membrane. An increasing body of evidence has demonstrated that NS5A
interacts with a number of cellular proteins and may also interfere
with host cell signaling pathways; for example, NS5A was shown to
interact directly with the interferon-induced double-stranded
RNA-activated protein kinase PKR, and this interaction seems to
correlate with inhibition of PKR function (6). NS5A and NS5B form a
complex with a human membrane-associated protein, hVAP (7), indicating
the membrane association of the replication complex. Recent reports
suggest that NS5A may also regulate cell cycle progression, resulting
in a reduced S phase and an increase in the G2/M phase
(8).
One indicator that NS5A may have a role in cell signaling is the
presence of polyproline (PXXP) motifs, which are
highly conserved throughout a range of HCV genotypes. These motifs form
extended helical structures and are found in a number of viral and
cellular proteins involved in signal transduction. They bind to Src
homology 3 (SH3) domains found in a diverse group of signal-transducing molecules (9), and indeed it was previously shown that one of these
PXXP motifs in NS5A interacts directly with the SH3 domains of the growth factor receptor-bound protein 2 (Grb2) (10), a component
of the mitogen-activated protein kinase (MAPK) cascade (see below).
The MAPK cascades are among the best characterized of intracellular
signaling pathways. These cascades consist of a three-kinase module
that includes a MAPK/ERK kinase (MEK) kinase (MEKK), which activates a MEK that in turn activates a MAPK (11). Three distinct groups of MAPK have been discovered in mammalian cells, including the
mitogen-responsive ERK (extracellular signal-regulated kinases), the
stress-activated c-Jun N-terminal kinase (JNK)/stress-activated protein
kinases, and the less understood p38 MAPKs (12). Phosphorylation on
both threonine and tyrosine residues is required to activate MAPKs.
Once activated, MAPKs translocate to the nucleus, where they
phosphorylate and activate transcription factors and other target
proteins (13, 14).
Activating protein-1 (AP1) is a collective term referring to homo- and
heterodimeric transcription factors composed of Jun, Fos, or ATF2
subunits that bind to a common DNA site, the AP1 site (15). MAPKs
contribute to the activation of AP1 activity in response to a diverse
array of extracellular stimuli including cytokines, growth factors,
T-cell activators, neurotransmitters, and UV irradiation. Each of the
three types of MAPK kinases affects AP1 activity through
phosphorylation of a different substrate. Whereas the ERKs
phosphorylate TCF/Elk1 and thereby induce c-Fos synthesis, they do not
phosphorylate c-Jun. In addition, the ERKs do not appear to be involved
in ATF2 phosphorylation. The JNKs, on the other hand, phosphorylate the
stimulatory sites of both c-Jun and ATF2 but generally do not
phosphorylate c-Fos (15). Activated AP1 triggers transcription of many
genes involved in the cell response to external stimuli.
Grb2 acts as an adaptor in the ERK pathway linking growth factor
receptors to the MEKK cascade via Sos and Ras. Although it has been
shown that in cells expressing NS5A phosphorylation of ERK resulting
from EGF stimulation was reduced (10), the exact mechanism for this is
unclear, and that study failed to observe a reduction in Grb2
interactions with Sos. Given the importance of mitogenic signaling
pathways for cell survival and proliferation, we set out to determine
the mechanism and downstream consequences of the previously observed
inhibition of ERK phosphorylation by NS5A. Using a luciferase reporter
containing an AP1-responsive promoter sequence, in transient NS5A
expression systems, we demonstrate that a C-terminal PXXP
motif within NS5A is required for the inhibition of AP1. We show that
this inhibition acts directly through the ERK MAPK pathway and not the
JNK or p38 pathways. Phorbol 12-myristate 13-acetate stimulation and
expression of dominant active Ras, Raf, and PKC isoforms abrogated
inhibition, implying that NS5A interferes with the ERK MAPK cascade at
a very early stage. In both Huh-7 cells harboring a subgenomic HCV
replicon and stable osteosarcoma cells expressing NS5A alone, we also
observed an inhibition of phosphorylation of members of the ERK MAPK
pathway and a concomitant reduction in levels of the c-Fos
transcription factor. Most significantly, we find a reduction in AP1
reporter activity in cells expressing the full-length HCV genome under the control of an authentic HCV IRES. Thus, our data provide new insight into the signaling pathways modulated by the HCV NS5A protein
and may account for the oncogenic and immunomodulatory effects observed
during a HCV infection.
DNA Manipulations and Plasmids--
NS5A sequences from HCV
genotype 1a (H77) (16) (kindly supplied by Dr. Jens Bukh, National
Institutes of Health, Bethesda, MD) were amplified by PCR with
appropriate sequence-specific primers (sequences available on request)
and Pfu polymerase (Promega). PCR-amplified fragments were
subcloned into the mammalian expression vector, pSG5 (17). A mutant of
NS5A in which proline residues 350, 353, and 354 were substituted for
alanine (PA2) was generated by the PCR overlap method (18) using an
appropriate template and overlapping internal oligonucleotide primers.
Briefly, internal forward and reverse primers containing the modified
sequence were used in conjunction with flanking primers to create two
primary PCR products. The primary amplification products were agarose gel-purified and used together as template in a secondary round of
amplification using flanking primers to produce full-length product
containing the required mutation. All constructs were verified by
dideoxy sequencing. The pSG5.NS3/4A construct has been previously
described (19). The IL-6 luciferase reporter constructs (20) were
kindly provided by Dr. Derek Mann (University of Southampton).
cAMP-response element (CRE)-luciferase and serum-response element
(SRE)-luciferase constructs were obtained from Stratagene; Sp1-luciferase was previously described (19). A luciferase reporter construct regulated by a promoter sequence responsive to AP1 (pAP1-luc) and dominant active Ras, Raf, PKC, and p21-activated kinase (PAK) expression constructs were previously described (21-23).
Cell Culture--
COS-7 (African Green Monkey kidney cells) and
HEK 293T (human embryonic kidney cells) were propagated in Dulbecco's
modified Eagle's medium supplemented with 10% FCS, 2 mM
L-glutamine, 100 IU/ml penicillin, and 100 µg/ml
streptomycin. Huh-7 (human hepatoma cells) were cultured in minimal
essential medium supplemented with 10% FCS, 1% nonessential amino
acids, 2 mM L-glutamine, 100 IU/ml penicillin,
and 100 µg/ml streptomycin. To generate a replicon, cell line
transcripts were produced from the ScaI-linearized plasmid pFK-I389neo/NS3-3'/5.1 (kindly provided by Dr. Ralf
Bartenschlager, University of Heidelberg, Germany) (24) using T7
RNA polymerase (New England Biolabs) according to the manufacturer's
recommendations, treated with DNase (Promega), extracted with acid
phenol/chloroform, and spun through a mini-QuickSpin column (Roche
Applied Sciences). 5 µg of transcript was transfected into 400 µl
of Huh-7 cells (1 × 107 cells/ml in
phosphate-buffered saline) using a Bio-Rad Gene Pulser II (270 V, 950 microfarads). Transfected cells were allowed to recover for 24 h
before the addition of 1 mg/ml G418 to the culture medium. Selection
with G418 was maintained for 16 days, during which time the medium was
changed every 3 or 4 days. G418-resistant colonies formed were
maintained as a polyclonal cell line in medium supplemented with 250 µg/ml G418. Western analysis confirmed that the cell line expressed
NS3, NS5A, and NS5B, and Northern analysis demonstrated the presence of
a replicon transcript. All cell lines were incubated at 37 °C in a
humidified 5% CO2 incubator.
Transfection of Plasmid DNA and Luciferase Assays--
One day
prior to transfection, cells were seeded into six-well dishes (2 × 105 cells/dish). Cells were transfected using the
commercially available Lipofectin reagent (Invitrogen) according to the
manufacturer's instructions. Briefly, 1 µg of expression vector was
transfected together with 1 mg of luciferase reporter construct. As an
internal control for transfection efficiency, an expression plasmid
encoding the Renilla luciferase gene driven by the herpes
simplex virus thymidine kinase promoter (pRLTK) was also
transfected (0.05 µg). For co-transfections, the final DNA
concentration in all groups was kept constant by the addition of empty
expression vector as appropriate. Cells were incubated with
transfection reagent for 6 h, after which the transfection reagent
was removed, and the cells were overlaid with growth medium. For
most experiments, transfected cells were maintained under low serum
growth conditions (0.5% serum) prior to stimulation with 10% serum
containing growth medium. Where appropriate, cells were treated with
100 ng/ml phorbol 12-myristate 13-acetate, 10 µM
2'-amino-3'-methoxyflavone (PD98059), or 10 µM
2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one (p38 MAPK inhibitor) (all obtained from Calbiochem) 1 h prior to
growth factor stimulation. EGF and hepatocyte growth factor (HGF) were
obtained from Sigma and were used at a final concentration of 20 ng/ml.
For luciferase reporter assays, cells were harvested in 200 µl of
passive lysis buffer (Promega). Quantitation of relative light units
was determined using the dual luciferase Stop & Glo reagent (Promega)
and a Berthold luminometer (EG & G Berthold) with a dual injector
system. All assays were performed in triplicate, and each experiment
was repeated a minimum of three times.
Western Blotting--
To analyze the activity status of MAPK
pathway proteins, cells were lysed in radioimmune precipitation assay
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing
protease and phosphatase inhibitors at 4 °C for 30 min. Lysates (50 µg of total protein) were resolved by SDS-PAGE and probed with
antibodies specific to the phosphorylated forms of ERK1/2 (Upstate
Biotechnology, Inc., Lake Placid, NY) or with a phosphorylation
status-independent ERK1/2 antibody (New England Biolabs). Cell
lysates were also probed by Western blot using an in-house polyclonal
antiserum to NS5A or antibodies to c-Fos (Upstate Biotechnology), c-Jun
(Upstate Biotechnology), phospho-c-Jun (Upstate Biotechnology), PKC
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Ras (Upstate
Biotechnology), Raf1 (New England Biolabs), and hemagglutinin tag for
the PAK constructs (Santa Cruz Biotechnology) as per the
manufacturer's recommendations. Immunoblots were visualized using an
ECL system (Amersham Biosciences).
Baculovirus-mediated Delivery and Expression of the Full-length
HCV Genome--
Huh-7 cells were seeded at 2.5 × 104
cm Inhibition of AP1 by NS5A Is Cell Line-independent and Requires a
C-terminal Polyproline Motif within the NS5A Protein--
A number of
studies have shown that NS5A is able to transcriptionally regulate
cellular genes and to thereby modulate cell growth (8, 26). Because the
predicted amino acid sequence of NS5A contains no DNA binding motifs,
it is likely that NS5A regulates the transcription of cellular genes
either by direct interaction with cellular transcription factors or
through modulation of cellular signaling pathways (27).
NS5A has been shown previously to interact with the SH3 domains of the
mitogenic adaptor protein Grb2 via a highly conserved PXXP motif within the C terminus of the NS5A protein (10).
Furthermore, our recent data have shown that NS5A also interacts with
the SH3 domains of members of the Src family of tyrosine
kinases.2 Both Src kinases
and Grb2 are involved in regulating MAPK cascades that ultimately lead
to activation of members of the AP1 family of transcription factors.
To further understand the mechanisms by which NS5A modulates mitogenic
signaling cascades, we utilized a transient reporter assay in COS-7
cells. NS5A-expressing plasmids were co-transfected with a reporter
construct in which expression of the luc gene was regulated
by three tandem sequences corresponding to the binding site for the AP1
transcription factor (pAP1-luc) (28). Incubation of cells in medium
containing 10% serum resulted in a 5-fold stimulation of the AP1
luciferase reporter construct (Fig.
1a), compared with cells
maintained in low serum (0.5%) growth conditions. In cells expressing
the NS5A protein, however, both basal and serum-stimulated levels of
luciferase were reduced by ~60% (Fig. 1a). To identify the region(s) of NS5A responsible for mediating the inhibition of AP1
activity in COS-7 cells, we analyzed levels of luciferase in cells
expressing a NS5A derivative (NS5A(PA2)), in which the highly conserved
C-terminal PXXP motif was mutated to abrogate binding to SH3
domains (10).2 Fig. 1a clearly illustrates that
NS5A(PA2) failed to inhibit AP1 reporter activity, implicating a role
for this motif in NS5A-mediated inhibition of AP1.
To ensure that the results obtained were not an artifact of the COS-7
cell line, we repeated these experiments in two other cell lines, 293T
and the human hepatoma cell line Huh-7 (Fig. 1, b and
c, respectively). Expression of NS5A(wt), but not NS5A(PA2), resulted in a similar inhibition of AP1 reporter activity in both cases.
NS5A Expression Does Not Result in a Global Inhibition of
Transcription--
Expression of NS5A in transformed cell lines
resulted in an inhibition of an AP1 luciferase reporter construct;
however, the mode of inhibition was unknown. It was therefore crucial
to rule out the possibility of a global inhibition of cellular
transcription. To investigate this possibility, we analyzed the
activity of luciferase reporter constructs responsive to the
cAMP-responsive element-binding protein (CREB) or the transcription
factor Sp1. COS-7 cells were transfected with NS5A expression plasmids
and either CREB-luc or Sp1-luc. In this case, all cells were incubated
in the presence of 10% FCS prior to analysis of luciferase activity.
Levels of both CREB and Sp1 reporter activity were not significantly
affected by expression of either NS5A(wt) or NS5A(PA2) (Fig.
2). Cotransfection of a plasmid
expressing the HCV NS3/4A protein complex, however, impaired CREB
reporter activity by up to 50%, in agreement with our previously
published data (19). As expected, CREB was also inhibited by incubation
of cells with a well characterized inhibitor of
cAMP-dependent protein kinase, a myristoylated
peptide corresponding to residues 14-22 of the heat stable PKA
inhibitor protein. Appropriate expression of NS5A or NS3/4A was
confirmed by immunoblot analysis (data not shown). These data confirmed
that NS5A did not mediate a global inhibition of transcription and
therefore acted to specifically inhibit AP1 activity. This encouraged
us to further investigate the mechanism of AP1 inhibition.
Repression of an IL-6 Reporter Construct by NS5A Is Mediated by AP1
Promoter Sequences--
The above experiments were performed using a
reporter construct containing three tandem repeats of the AP1 binding
sequence. It was important to demonstrate that AP1 inhibition occurred
when the binding site was present in a more physiologically relevant setting. To this end, we utilized a reporter expressing luciferase under the control of the IL-6 promoter. The IL-6 promoter has a single
AP1 binding sequence and is known to be responsive to a variety of
transcription factors including AP1 (29). Therefore, IL-6 was a
suitable candidate to study the downstream effects of AP1 inhibition.
Two IL-6 reporter constructs were used: a parental plasmid in which a
651-bp fragment of the IL-6 gene including the full-length promoter
sequence was cloned into the luciferase reporter construct (pIL6-luc)
and a deletion mutant lacking the consensus binding sequence for AP1
(pIL-6(
COS-7 cells were transiently transfected with combinations of NS5A
expression plasmids and IL-6-luc reporters and incubated in low serum
growth conditions. Following stimulation with 10% FCS, the parental
IL-6 reporter construct exhibited 40% inhibition in the presence of
NS5A(wt) but was unaffected by NS5A(PA2) (Fig. 3a, gray
bars). Although this level of inhibition is less than that
of the AP1 luciferase reporter, it is consistent with previous results
(Fig. 1) indicating that NS5A(wt) inhibits AP1 activity. In order to
confirm that the AP1-responsive cis sequence was responsible for the NS5A-dependent down-regulation of IL-6-promoter
driven luciferase, the assay was also performed with the
IL6( NS5A Down-regulates AP1 Activity in EGF- and HGF-stimulated
Cells--
Fetal calf serum is a complex mixture of proteins including
a variety of growth factors and immunomodulatory compounds. This undefined mixture of mitogens makes it difficult to determine which
pathways are stimulating the levels of AP1 activity in the absence of
NS5A. In order to define these pathways more precisely, cells were
transiently transfected with NS5A(wt)- or NS5A(PA2)-expressing plasmids
together with pAP1-luc, incubated in low serum growth medium, and
stimulated with EGF or HGF. These are potent mitogens for a variety of
cell lines including epithelial and mesenchymal cells. They have been
shown to be effective activators of AP1 stimulation via the ERK MAPK
pathway (30), exerting their influence through the EGF receptor and
c-Met receptor, respectively. Transfected cells were stimulated with a
20 ng/ml concentration of either EGF or HGF for 2 min, and cells were
harvested 2, 4, and 6 h poststimulation. In COS-7 cells, treated
over the 6-h time period, NS5A(wt) reduced levels of AP1 reporter
activity by up to 40% in comparison with control cells transfected
with empty vector (Fig. 4a).
Conversely, the NS5A(PA2) mutant failed to inhibit AP1 activity in
EGF-stimulated cells. Interestingly, HGF-induced AP1 reporter activity
was also inhibited in the presence of NS5A(wt) (Fig. 4b),
although the extent of the inhibition was not as pronounced. In cells
stimulated with HGF, inhibition by NS5A was ~25%. The experiments
were also performed in Huh-7 cells transfected with NS5A-expressing
vectors. Similar results were obtained with both EGF and HGF treatment (Fig. 4, c and d). In all cases, the increase in
luciferase activity over time was a result of growth factor
stimulation, since control untreated cells did not exhibit a
time-dependent increase.
These data confirm that NS5A-induced inhibition of AP1 reporter
activity in both COS-7 and Huh-7 cells could be observed following growth factor stimulation implicating the ERK pathway. Indeed, the data
observed followed a similar trend to that obtained from serum stimulation.
Involvement of the Ras-ERK Pathway in NS5A-mediated AP1
Inhibition--
Stimulated EGF receptor activates multiple downstream
targets including Grb2 and Src kinases. Formation of multiprotein
complexes at the membrane leads to activation of Ras, Raf1, and MEKK1
(31, 32). Raf1, in turn, activates the ERK1 and ERK2 MAPK through MEK1
and MEK2 (33, 34). To evaluate the role of the ERK pathway in the
NS5A-mediated inhibition of AP1 reporter activity, we transfected COS-7
cells with pAP1-luc, pSG5-NS5A(wt), and plasmids expressing dominant
active (DA) forms of Ras, Raf1, and PKC. Both DA-Ras and DA-Raf1
significantly increased the EGF-stimulated AP1 reporter activity (Fig.
5, b and c,
gray bars), indicating that these DA proteins
acted synergistically with growth factor stimulation to up-regulate AP1
activity. Expression of DA-PKC isoforms
In addition to the Raf1 cascade, ligand stimulation can also activate
the MEKK1 signaling cascade (36). In this pathway, PAKs are
activated by the small membrane-bound GTPases, Rac1 and Cdc42 (21), and
in turn stimulate MEKK1. MEKK1 activates MEK4, which activates JNK and
p38 MAPKs (37). To evaluate whether the NS5A mediated inhibition was
transduced through alternative MAPK pathways, we co-transfected cells
with pAP1-luc, pSG5-NS5A(wt), and plasmids expressing either DA-PAK2 or
a dominant negative form of PAK2 (DN-PAK2) (38). Co-transfection of
DA-PAK2 with AP1 did not significantly increase basal AP1 reporter
activity and had no effect on the inhibition mediated by NS5A(wt) (Fig. 5h), suggesting that NS5A-mediated inhibition does not
function via PAK2-mediated pathways. Expression of DN-PAK2 did not
decrease serum stimulated AP1 reporter activity and did not alter the
NS5A mediated AP1 inhibition (Fig. 5i). To provide further
evidence for our hypothesis that only the ERK MAPK pathway was being
inhibited, we transfected cells with pAP1-luc with or without
pSG5-NS5A(wt) and incubated these cells in the presence of a specific
p38 MAPK inhibitor
(2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol3-one). Incubation of cells with this compound did not significantly
reduce serum-stimulated AP1 levels, and importantly, the NS5A-mediated AP1 inhibition was maintained in the presence of the inhibitor (Fig.
5j). Appropriate expression of the DA or DN proteins and NS5A was confirmed by immunoblot analysis (Fig. 5, a-j,
lower panels). In all of these experiments, cells
were growth factor-stimulated to maintain consistency throughout the
study, and similar data were obtained in nonstimulated cells (data not shown).
Phosphorylation of Ras-ERK Pathway Members Is Inhibited in HCV
Replicon Cells--
The data obtained from the use of specific
mitogens and mutant signaling proteins suggested that the NS5A-mediated
inhibition of AP1 involved only the ERK pathway. To test this
hypothesis more directly, we investigated the expression and
phosphorylation of individual components of the ERK and JNK pathways.
This analysis was undertaken in three types of cells: stably
transfected human osteosarcoma cells (UTA6) in which expression of NS5A
was driven by the tetracycline-responsive promoter, stably transfected
Huh-7 cells expressing NS5A alone, and also Huh-7 cells harboring the culture-adapted FK5.1 subgenomic replicon (i.e. expressing
the HCV nonstructural proteins NS3 to NS5B). We show here data obtained from a comparison of naive Huh-7 cells and replicon FK5.1 cells (Fig.
6); however, identical data were obtained
from cells expressing only NS5A (data not shown).
Naive Huh7 and Huh7-FK5.1 cells were stimulated with EGF and lysed at
various points over a 4-h time course. Samples were analyzed by
immunoblotting with a range of commercially available antibodies to
detect both overall expression of ERK1/2, c-Fos, and c-Jun and the
phosphorylated forms of MEK1/2, ERK1/2, and c-Jun. Fig. 6a
shows that, following EGF stimulation, phosphorylation of MEK1/2 by Raf
was significantly reduced in FK5.1 cells in comparison with naive Huh7.
Similarly, immunoblotting with an antibody directed against the dually
phosphorylated, activated form of ERK1/2 revealed that phosphorylation
of these proteins was also reduced in FK5.1 cells (Fig. 6b),
although the time course of phosphorylation was similar in both cell
lines (maximal ERK1/2 phosphorylation occurred within 30 min of EGF
stimulation). Blotting with a phosphorylation state-independent
antibody showed similar levels of ERK1/2 in both cell lines (Fig.
6c), confirming that the reduction in ERK1/2 phosphorylation
was not due to a reduction in the abundance of ERK1/2. Once
phosphorylated, ERK1/2 can enter the nucleus and phosphorylate the Elk1
transcription factor; Elk1 then binds to the SRE of the
c-fos promoter and activates expression of c-Fos. Fig.
6d shows that levels of c-Fos induction were dramatically reduced in FK5.1 cells in comparison with naive Huh7. In the latter, maximal c-Fos expression occurred at 4 h post-EGF treatment,
whereas, interestingly, in FK5.1 cells c-Fos expression peaked at
2 h post-EGF and declined thereafter.
In parallel, we also examined the expression and phosphorylation of
c-Jun in naive Huh7 and FK5.1 cells. Overall levels of c-Jun remained
stable throughout the 4-h time course, and there were no differences
between the two cell lines in the kinetics or amounts of c-Jun
phosphorylation following EGF stimulation. This result is consistent
with our previous observation that transfection of DA- or DN-PAKs had
no effect on NS5A-mediated inhibition of AP1 activity (Fig. 5,
h and i). Appropriate expression of NS5A in FK5.1
cells was confirmed by immunoblot analysis (Fig. 6g).
NS5A Inhibits Activation of the Serum-response Element of the c-Fos
Promoter--
The c-fos gene promoter contains an SRE that
is bound by the phosphorylated ETS family transcription factor Elk1
(39). To provide further evidence that the reduction in c-Fos
expression was the result of a reduction in SRE-dependent
transcription, COS-7 cells were transiently transfected with NS5A
expression plasmids together with a reporter construct containing the
SRE upstream of luciferase (pSRE-luc) and maintained under low serum growth conditions. As expected, EGF stimulation caused an increase in
SRE-driven luciferase expression; however, EGF-stimulated levels of
luciferase were reduced by ~60% in the presence of NS5A(wt) (Fig.
7). This reduction was not observed in
the presence of NS5A(PA2). We conclude, therefore, that the
down-regulation of ERK1/2 phosphorylation mediated by NS5A results in
decreased transcription from the SRE of the c-fos promoter,
with a concomitant reduction in the expression of c-Fos.
Inhibition of AP1 by Full-length HCV in Hepatocyte-derived Cell
Lines--
A major hindrance to HCV research is the lack of an
efficient and convenient culture system. Although this has, in part,
been overcome by the advent of subgenomic HCV replicons (40, 41), use
of this system has the disadvantage of only expressing the HCV
nonstructural proteins NS3-NS5B. Given that the HCV core protein has
recently been reported to up-regulate all three MAPK pathways (42), we
considered it of importance to determine whether AP1 activity was modulated in the context of the entire HCV polyprotein. To
address this possibility, we took advantage of a baculovirus delivery
system capable of expressing the full-length HCV genome in
hepatocyte-derived cell lines, under the control of a
tetracycline-responsive promoter, that has been developed in our
laboratory (25).
Huh-7 cells were transduced with baculoviruses expressing the tTA
tetracycline transactivator protein (BACtTA) together with a second
virus encoding either The lack of a robust in vitro replication system for
HCV has meant that the exact role of the nonstructural NS5A protein in HCV infection is as yet unknown. To date, information about potential NS5A functions has derived, almost exclusively, from the use of in vitro transient or constitutive expression systems. In
one such previous study, HeLa cells stably expressing NS5A exhibited a
reduction in the levels of EGF-activated ERK activity compared with
control cells (10). However, the mechanism of this reduction and the
concomitant effect of NS5A on the activity of mitogenically modulated
transcription factors were not analyzed. We therefore set out to
dissect the effects of NS5A on both mitogenic and stress-stimulated transcription factors using a variety of cell lines and expression systems, including in the context of a full-length HCV genome delivery
vector in hepatocyte-derived cell lines.
The ability of viruses to modulate transcription factors involved in
cell proliferation, differentiation, and stress response has been
studied in great depth. AP1 transcription factor activity is regulated
in a complex manner involving different patterns of Fos and Jun protein
expression. The type of AP1 complex formed partly accounts for the
conflicting cell functions associated with induction of the
fos and jun genes, which range from proliferation and transformation to differentiation and growth arrest (43). We
therefore characterized the signaling pathways and the mechanism by
which NS5A inhibits AP1 and the specific family members involved.
The main conclusion from the data presented here is that NS5A
inhibition of AP1 transcription factor activity is mediated via
specific perturbation of the Ras-ERK pathway and does not involve the
other MAPK pathways (JNK and p38). The effect of NS5A was abrogated by
expression of dominant active forms of proteins in the Ras-ERK pathway
(Ras, Raf-1, and PKC) but not the JNK pathway (PAK2). Our data both
confirm and significantly extend previous observations pertaining to
mitogenic signaling via ERK (10, 44) but appear to contradict a further
study by the same group showing inhibition of p38 MAPK signaling by
NS5A (44). Since the latter study exclusively used HeLa cells, it is
possible that cell type differences in the MAPK cascades may account
for this discrepancy. However, our data do not formally exclude an
effect of NS5A on p38 signaling but do demonstrate that any inhibition of p38 does not impact on AP1 activity. The observation that the p38
inhibitor has no effect on NS5A-mediated AP1 inhibition suggests that
if NS5A does indeed perturb p38 signaling it must target the pathway
below p38 itself (i.e. in a very different manner from the
effect on the ERK pathway). This is consistent with the observations
that NS5A inhibits PKR (6) and PKR phosphorylates p38 MAPK (45).
Our data show that although the effect of NS5A on AP1 activity was
significant, it was clear that activity of the transcription factor was
never completely inhibited. This may be due to the inability of NS5A to
completely overcome the strong stimulation of AP1 activity achieved
with growth factor stimulation, a hypothesis supported by the
observation that NS5A inhibition of basal AP1 activity (Fig. 1) was
more marked than inhibition of serum-stimulated activity. One other
possible explanation may lie in the specific inhibition of c-Fos by
NS5A. The active AP1 transcription factor is composed of either a
Jun-Jun homodimer or a Fos-Jun heterodimer. Interestingly, the
specificity of biological effects induced by upstream stimuli is
determined by the composition of AP1 DNA binding complexes in a
particular signal transduction cascade. The reasons for specific
targeting of c-Fos are unclear. It is known that Fos-Jun heterodimers
bind DNA elements containing AP1 consensus sequences up to 50 times
more efficiently than Jun-Jun homodimers (43). It has also
been observed that JunB isoforms can act as negative regulators of AP1
function and inhibit transcription by a negative interaction with AP1
consensus sequences (46). Thus, by inhibiting c-Fos expression, NS5A
may shift the AP1-mediated response to external stimuli by modulating
the composition of specific AP1 complexes. The exact composition of AP1
dimers in hepatoma cells is currently unknown but requires
clarification in order to fully understand the effect of NS5A on AP1
transcription factor activity. The precise mechanism of AP1 inhibition
by NS5A remains to be elucidated; at this point, it is perhaps
pertinent to note that we do not observe an increase in NS5A
phosphorylation following serum or growth factor stimulation (data not
shown). Thus, it is unlikely that NS5A functions as a substrate for one of the kinases in the Ras-ERK pathway, thereby sequestering specific members of the pathway.
Why should HCV possess a mechanism for inhibition of AP1 signaling via
the Ras/ERK pathway? Several potential explanations can be proposed. As
well as contributing to AP1 activity, ERK has a number of independent
functions including regulation of the STAT3 transcription factor (47),
which itself plays a key role in interferon signaling and cell
proliferation. Phosphorylation by ERK negatively regulates STAT3 by
inhibiting STAT3 tyrosine phosphorylation (48); thus, it would be
expected that inhibition of ERK activation by NS5A might stimulate
STAT3. In this regard, our data are consistent with two recent reports:
activation of STAT3 by both the HCV core protein and another (as yet
undefined) HCV gene product (49) and a study demonstrating activation
of STAT3 by NS5A (50). However, an earlier report (51) showed inhibition of STAT3 DNA binding activity in human osteosarcoma cells
expressing the entire HCV genome, so clearly this discrepancy must be
addressed in the future.
Growth factor-mediated activation of AP1 is critical in the control of
hepatocyte proliferation; indeed, HGF is the most potent hepatocyte
mitogen, mediating the transition from G1 phase through to
S phase and mitosis. One plausible scenario, therefore, is that
NS5A-mediated inhibition of AP1 might slow the exit of infected hepatocytes from the G1 phase. Induction of entry into cell
cycle, followed by a block in cell cycle progression, is a
common mechanism utilized both by viruses that establish chronic
infections (52) and more acute viruses, such as Reovirus (53, 54).
Presumably, this is because the G1 phase provides the
optimal environment for viral replication. In the case of Reovirus,
cell cycle arrest in G1 has been reported to occur as a
result of inhibition of the Ras-MAPK pathway (55).
Two other hepatotrophic viruses, hepatitis B and hepatitis E, both
encode proteins that deregulate MAPK signaling pathways (56, 57).
Although both the hepatitis B HBx protein and hepatitis E virus ORF3
target the Ras-ERK cascade, unlike NS5A, they lead to an increase in
ERK kinase activity. The effects of ORF3 on AP1 signaling have not been
described; however, there is a wealth of literature describing an
HBx-mediated increase in AP1 activity (58-60). It is thought that, in
comparison with other chronic viruses, HBx up-regulates AP1 in order to
stimulate cell proliferation and rapid progression through the cell
cycle in order to aid viral replication (61). Other viral gene products
that target the Ras-ERK pathway include the human immunodeficiency
virus Nef protein (62, 63), the polyoma virus middle T antigen (64),
and the Epstein-Barr virus latent membrane protein 1 (65). In all three cases and that of HBx, evidence for a role of Src kinases in mediating the activation of the Ras-ERK pathway has been presented. In this regard, it is pertinent to note that our recent data have demonstrated functional interactions between NS5A and members of the Src family of
tyrosine kinases.2 This raises the possibility that the
effects of NS5A on the Ras-ERK pathway might be mediated by
interactions with multiple proteins within the pathway.
Infection with HCV is increasingly linked to the development of
hepatocellular carcinoma, although, unlike hepatitis B, no direct link
between viral replication and tumorigenesis has been unambiguously
identified. Intriguingly, a naturally occurring transforming mutant of
the EGF receptor, EGFRvIII, down-regulates the ERK MAPK pathways while
simultaneously activating PI3-kinase (66). PI3-kinase plays a key role
in regulating both cell proliferation and apoptosis, and several
studies have indicated that ERK acts as a negative regulator of
PI3-kinase (67). While this manuscript was in preparation, He et
al. (68) published data suggesting that NS5A was capable of
up-regulating PI3-kinase phosphorylation in stable cell lines.
Down-regulation of the ERK MAPK pathway by NS5A may therefore be a
mechanism of up-regulating PI3-kinase activity in HCV-infected cells,
thus affording these cells protection against apoptotic stimuli. We are
currently addressing this question by investigating the physiological
consequences of NS5A-mediated Ras-ERK and concomitant AP1 inhibition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 in six-well plates and 24 h later were
transduced either with BACtTA and BACINDLacZtet or with
BACtTA and BACH77(HdV)tet for 4 h in the presence or
absence of 5 µg/ml tetracycline (each virus was at a final
concentration of 1 × 107 plaque-forming
units/ml) (25). The transduced cells were then transfected for
5 h with 1 µg of pAP1-luc and 0.05 µg of pRLTK using 5 µl of
Lipofectin (Invitrogen) according to the manufacturer's recommendations, in the presence or absence of 5 µg/ml tetracycline. To suppress constitutive activation of the AP1 pathway, cells were left
overnight in Dulbecco's modified Eagle's medium plus nonessential
amino acids plus 0.5% FCS and then stimulated with medium containing
10% FCS for 6 h before being harvested in passive lysis buffer
(Promega).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
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Fig. 1.
Inhibition of AP1 by NS5A is cell
line-independent and requires a C-terminal Class II polyproline motif
within NS5A. Cells were transfected with 1 µg of plasmid
pAP1-luc, which contains the luciferase reporter under the
transcriptional control of three tandem AP1 binding sites and a minimal
TATA box promoter, with or without co-transfection of
pSG5-NS5A(wt) or pSG5-NS5A(PA2), and placed in low serum (0.5%)
growth medium for 18 h. Cells were stimulated by the addition of
serum to 10% and harvested 6 h later. The level of expression of
the luciferase reporter was assayed using a luminometer and normalized
for transfection efficiency by using a co-transfected
Renilla luciferase control plasmid. Results are the average
of three independent experiments. The three graphs show results from
COS-7 (a), 293-T (b), and Huh-7 (c).
The expression of the different NS5A proteins from the 6-h time points
was verified by immunoblotting using a sheep polyclonal antiserum
(d). The faint bands co-migrating with NS5A in
lanes 4 and 7 are the result of
spill-over from adjacent lanes.
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Fig. 2.
NS5A does not modulate either CRE or Sp1
transcription factor activity in COS-7 cells. COS-7 cells were
transfected with 1 µg of either pCRE-luc or pSP1-luc with or without
co-transfection of pSG5-NS5A(wt), pSG5-NS5A(PA2), or pSG5-NS3/4A (19)
as indicated. Where appropriate, cells were treated with 10 µM myristoylated peptide corresponding to residues
14-22 of the heat stable PKA inhibitor protein (a kind gift from Dr.
Roger Clegg, Hannah Research Institute) immediately after transfection.
Cells were harvested at 18 h post-transfection, and the level of
expression of the luciferase reporter was assayed using a luminometer.
A co-transfected Renilla luciferase control plasmid was used
to normalize for transfection efficiency. Results are the average of
three independent experiments.
AP1)-luc) (20).
AP1)-luc construct. As expected, this mutant did not exhibit
NS5A mediated down-regulation and reached control levels of luciferase
activity upon serum stimulation (Fig. 3a, checked
bars). NS5A protein was expressed to similar levels in all
samples (Fig. 3b).
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Fig. 3.
Repression of an IL-6 reporter construct by
NS5A is mediated by AP1-responsive sequences. COS-7 cells were
transfected with 1 µg of either pIL6-luc or pIL6( AP1)-luc with or
without co-transfection of pSG5-NS5A(wt) or pSG5-NS5A(PA2) and placed
in low serum (0.5%) growth medium for 18 h. Cells were stimulated
by the addition of serum to 10% and harvested 6 h later. The
levels of expression of the luciferase reporters were assayed using a
luminometer and normalized for transfection efficiency by using a
co-transfected Renilla luciferase control plasmid. The
results shown in a are the average of three independent
experiments. The corresponding immunoblot in b demonstrates
appropriate expression of the NS5A protein in cells transfected with
pIL6-luc (lanes 1, 3, and
5) or pIL6(
AP1)-luc (lanes 2,
4, and 6).
View larger version (23K):
[in a new window]
Fig. 4.
NS5A down-regulates AP1 activity in EGF- and
HGF-stimulated cells. COS-7 (a and b) and
Huh-7 cells (c and d) were transfected with the
pAP1-luc reporter plasmid with or without pSG5-NS5A(wt) or
pSG5-NS5A(PA2) for 6 h and then placed in low serum (0.5%) growth
medium for 18 h prior to stimulation with 20 ng/ml EGF or HGF for
2 min. Luciferase assays were performed on samples treated with EGF
(a and c) or HGF (b and d)
or maintained in low serum growth medium (all
graphs). Cells were harvested prior to growth factor
stimulation (0-h time point) or at 2, 4, and 6 h poststimulation.
The level of expression of the luciferase reporter was assayed using a
luminometer and normalized for transfection efficiency by using a
co-transfected Renilla luciferase control plasmid. Results
are the average of three independent experiments. , control plus
growth factor;
, control minus growth factor;
, NS5A(wt) plus
growth factor;
, NS5A(wt) minus growth factor;
, NS5A(PA2) plus
growth factor;
, NS5A(PA2) minus growth factor.
and
also led to an
increase in AP1 reporter activity, although to a lesser extent than
DA-Ras and DA-Raf1 (Fig. 5, d and e). Consistent with PKC-mediated up-regulation of AP1, stimulation of cells with phorbol 12-myristate 13-acetate (a PKC activator) also led to a similar
increase in AP1 reporter activity (Fig. 5f). The observation that expression of each of these dominant active constructs abrogated the NS5A-mediated inhibition of AP1 reporter activity (Fig. 5, b-f, checkered bars) strongly
suggested that the NS5A-mediated inhibition of AP1 activity occurred
upstream of Ras and Raf1. To confirm that inhibition of ERK activity
can lead to reduced AP1 activity, we employed a specific MEK1/2
inhibitor, PD98059 (35). Incubation with 10 µM PD98059
led to a 70% decrease in AP1 reporter activity in the absence of NS5A,
demonstrating that specific perturbation of the ERK1/2 pathway was
capable of down-regulating AP1 activity to levels seen in
NS5A-expressing cells (Fig. 5g).
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Fig. 5.
NS5A-induced AP1 inhibition is mediated via
the Ras-ERK pathway. Subconfluent 90-mm diameter dishes of COS-7
cells were transfected with pAP1-luc in combination with the
appropriate DA- and DN-expressing plasmids in the presence or absence
of pSG5-NS5A(wt) for 6 h and then placed in low serum (0.5%)
growth medium. Specific MEK1 and p38 inhibitors were administered to
the appropriate cells 1 h prior to stimulation with EGF for 2 min.
Cells were harvested 2 h later. The level of expression of the
luciferase reporter was assayed using a luminometer and normalized for
transfection efficiency by using a co-transfected Renilla
luciferase control plasmid. Results are the average of three
independent experiments. The same lysates were adjusted for protein
concentration and immunoblotted with antibodies directed against the
various DA- and DN-expressed proteins as well as NS5A. WB,
Western blot; PMA, phorbol 12-myristate 13-acetate.
View larger version (47K):
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Fig. 6.
The Ras-ERK pathway is specifically inhibited
in replicon cells. Naive Huh-7 and FK5.1 cells were maintained in
low serum (0.5%) growth medium for 18 h. Samples were harvested
prior to treatment with 20 ng/ml EGF for 2 min (0-h time point) and at
0.5, 2, and 4 h post-EGF treatment for analysis. Cell lysates were
resolved by SDS-PAGE (12%) and subjected to immunoblot analysis with
antibodies specific to phosphorylated MEK (a), total ERK1/2
(b), phosphorylated ERK1/2 (c), c-Fos
(d), c-Jun (e), and phosphorylated c-Jun
(f). Appropriate expression of NS5A was determined by
immunoblot with a sheep polyclonal antiserum (g).
View larger version (39K):
[in a new window]
Fig. 7.
NS5A inhibits SRE-dependent
transcription. COS-7 cells were transfected with pSRE-luc with or
without pSG5-NS5A(wt) or pSG5-NS5A(PA2) for 6 h before being
placed in low serum (0.5%) growth medium. Cells were stimulated with
20 ng/ml EGF for 2 min and harvested 6 h later. The level of
expression of the luciferase reporter was assayed using a luminometer
and normalized for transfection efficiency by using a co-transfected
Renilla luciferase control plasmid. The results shown in
a are the average of three independent experiments. The
corresponding immunoblot in b demonstrates appropriate
expression of the NS5A protein prior to EGF stimulation
(lanes 1, 3, and 5) or
6 h poststimulation (lanes 2, 4,
and 6).
-galactosidase (BACINDLacZTET), or
the full-length HCV genome (BACH77(H
V)TET), under the
control of the tetracycline-responsive promoter. Cells were maintained
in the presence of absence of tetracycline to either repress or
activate expression from the tetracycline-responsive promoter,
respectively. The transduced cells were subsequently transfected with
pAP1-luc and serum-stimulated. At 6 h poststimulation, cells were
harvested, and the levels of luciferase were determined for each
sample. Fig. 8a illustrates
that transduction of cells with BACtTA and BACINDLacZTET
did not significantly affect levels of luciferase expression. Cells
infected with BACtTA and BACH77(H
V)TET, in the presence
of tetracycline, exhibited luciferase levels similar to control.
However, in cells where the tetracycline was removed, thus allowing HCV
genome transcription and polyprotein expression, levels of luciferase
were significantly reduced. The accompanying Western blot illustrates
that NS5A was expressed in the cells lacking tetracycline but not in
those treated with the antibiotic (Fig. 8b). This
observation clearly demonstrates that AP1 activity is reduced in
hepatoma cells expressing the entire HCV polyprotein. Taken together
with the transient expression data, we conclude that even in the
presence of the core protein, expression of the complete HCV
polyprotein is able to mediate inhibition of the AP1 pathway.
View larger version (32K):
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Fig. 8.
Inhibition of AP1 by full-length HCV in
hepatocyte-derived cell lines. Subconfluent Huh-7 cells were
transduced with BACtTA and BACINDLacZtet or BACtTA and
BACH77(H V)tet in the presence or absence of
tetracycline. Cells were then transfected with 1 µg of pAP1-luc and
50 ng of pRLTK and placed in low serum (0.5%) growth medium for
18 h. Cells were stimulated with serum and harvested 6 h
later for analysis. The level of expression of the luciferase reporter
was assayed using a luminometer and normalized for transfection
efficiency by using a co-transfected Renilla luciferase
control plasmid. Results shown in a are the average of three
independent experiments. The corresponding immunoblot in b
demonstrates appropriate tetracycline-regulated expression of the NS5A
protein. Lanes 1-5 correspond to the
bars in the graph in a.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Derek Mann (University of Southampton) and Dr. Herma Renkema (Institute of Medical Technology, University of Tampere, Finland) for plasmid reagents and helpful advice. We also thank Drs. Lucy Beales and Matthew Bentham for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by British Medical Research Council Grant G9801522 and a grant from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a Biotechnology and Biological Sciences Research Council Ph.D. studentship.
To whom correspondence should be addressed: Division of
Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Tel.: 44-113-343-5632; Fax:
44-113-343-5638; E-mail: mharris@bmb.leeds.ac.uk.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M210900200
2 A. Macdonald, K. Crowder, A. Street, C. McCormick, and M. Harris, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: HCV, hepatitis C virus; SH3, Src homology 3; Grb2, growth factor receptor-bound protein 2; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; AP1, activating protein 1; EGF, epidermal growth factor; IL-6, interleukin-6; CRE, cAMP-response element; SRE, serum-response element; PKC, protein kinase C; PAK, p21-activated kinase; FCS, fetal calf serum; HGF, hepatocyte growth factor; CREB, CRE-binding protein; ERK, extracellular signal-regulated kinase; DA, dominant active; DN, dominant negative; PI3-kinase, phosphatidylinositol 3-kinase.
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