From the Department of Biochemistry and Molecular
Biology, University of Kansas Medical Center, Kansas City, Kansas
66160,
Laboratory of Molecular Biology, NCI, National Institutes
of Health, Bethesda, Maryland 20892,
Laboratory of Molecular Biology, NINDS,
National Institutes of Health, Bethesda, Maryland 20892, and
§ Department of Pathology and Cell Biology, University of
Occupational and Environmental Health, Kitakyushu 807, Japan
Received for publication, September 12, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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Hepatitis C virus nonstructural protein, NS5A, is
a phosphoprotein produced from the processing of the viral polyprotein
precursor. NS5A associates with several cellular proteins in mammalian
cells, and the biological consequences of this interaction are
currently unknown. To this end, five stable NS5A-expressing murine and
human cell lines were established. Tetracycline-regulated NIH3T3
cells and rat liver epithelial cells as well as the
constitutive, NS5A-expressing, human Chang liver, HeLa, and NIH3T3
cells all exhibited cell growth retardation compared with the control
cells. Cell cycle analysis by flow cytometry indicated that the
NS5A-expressing human epitheloid tumor cells had a reduced S phase and
an increase in the G2/M phase, which could be
explained by a p53-dependent induction of p21Waf1/Cip1 protein and mRNA levels. NS5A interacts
with Cdk1 in vivo and in vitro, and a
significant portion of the p21Waf1/Cip1 was found to be in
a complex with Cdk2 in the NS5A-expressing human hepatic cell line.
Cdk1 and cyclin B1 proteins were also reduced in human Chang liver
cells consistent with the increase in G2/M phase. Our
results suggest that the NS5A protein causes growth inhibition and cell
cycle perturbations by targeting the Cdk1/2-cyclin complexes.
Hepatitis C virus
(HCV),1 a member of the
positive strand RNA viruses of the family Flaviviridae, is a
major etiologic, causative agent of non-A, non-B hepatitis worldwide
(1, 2). Approximately 85% of persons infected with HCV are estimated
to develop chronic hepatitis as determined by persistently elevated
serum alanine aminotransferase and/or viremia (HCV RNA) (3). Persistent
HCV infection often leads to liver cirrhosis with its attendant risks of liver cancer (4, 5). However, research on the pathogenesis and viral
replication as well as development of therapeutic strategies for
control of HCV infections has been limited. This is attributed to a
lack of an appropriate cell culture system or an adequate animal model
for HCV infection and propagation (6-10). The HCV virion contains an
~9.5-kilobase single-stranded RNA genome that encodes a single
precursor polyprotein of about 3010 amino acids. The precursor
is processed by a combination of host and viral proteases to yield
mature structural (C, E1, and E2/p7) and nonstructural (NS2, NS3, NS4A,
NS4B, NS5A, and NS5B) proteins (11-13).
NS5B has been shown to be the viral RNA-dependent RNA
polymerase (14). NS5A is a phosphoprotein that exists in differentially phosphorylated forms of 56 and 58 kDa with modifications of serine residues (15). The 58-kDa form is produced by additional
phosphorylation of the 56-kDa form (16-18). NS5A interacts with a
number of cellular proteins in mammalian cells, some of which have been
identified and partially characterized. NS5A was shown to interact
directly with the interferon-induced double-stranded RNA-activated
protein kinase PKR, and this interaction seems to correlate with PKR
function (19). NS5A also associates with cellular serine/threonine
kinase and is phosphorylated by this kinase (21, 22, 43). Other interactions of NS5A were identified by using a yeast two-hybrid assay.
NS5A binding with growth factor receptor-bound protein 2 (Grb2) adaptor
protein has been implicated in interference with cell signaling (24).
NS5A and NS5B form a complex with the human vesicle-associated membrane
protein-associated protein, hVAP (25). In addition, NS5A associates
with a cellular transcription factor, SRCAP (26), as well as with the
human karyopherin Eukaryotic cell cycle progression and proliferation are regulated by
activation of a number of cyclin-dependent kinases (Cdks) that consist of a catalytic subunit of about 34 kDa in complex with a
cyclin-regulatory subunit (for a review, see Ref. 28). The activities
of the Cdks are tightly regulated by a family of Cdk-inhibitory
proteins of which there are two classes. The first class consists of
INK4 proteins (p16, p15, p18, p19). The INK4 proteins interact with
Cdk4 and Cdk6 and prevent these Cdks from interacting with cyclin D and
subsequent phosphorylation of retinoblastoma protein (pRB) (29-31).
The other class of Cdk inhibitors includes p21Waf1/Cip1,
p27Kip1, and p57 (32). p21Waf1/Cip1 interacts
with Cdk-cyclin complexes (cyclin D complexed with Cdk4 or Cdk6,
Cdk2-cyclin E, and Cdk1 complexed with cyclin A or cyclin B (33, 34).
In a number of studies in both yeast and mammalian cells, it has been
shown that ectopic expression of p21Waf1/Cip1 causes
G1 and G2 arrest and, in some studies,
predominantly G2 arrest (35-37). Dulic et al.
(38) proposed that cyclin A and Cdk2 are the primary targets of
p21Waf1/Cip1 in premitotic G2/M phase and may
inhibit its kinase activity or Cdk-activating kinase (38).
In this study, we sought to examine the effect of NS5A
expression on cell cycle progression and proliferation in a variety of
cell lines. To this end, we established five stable, NS5A-expressing cell lines: two cell lines using the tetracycline-regulated (Tet-off) system, the murine NIH3T3 and a rat liver epithelial (RLE) cell line,
and three constitutively expressing NS5A cell lines (two of which are
of human origin, Chang liver and HeLa, and the third, NIH3T3). The
results of this study clearly indicated that NS5A expression caused
growth retardation in all cell lines examined. The cell cycle
distribution, analyzed by flow cytometry, showed that the two human
cell lines constitutively expressing NS5A exhibited a shortened S phase
and a prolonged G2/M phase progression, resulting in a
significant increase in population doubling time. Interestingly, this
property of the NS5A protein was most prominent in human Chang liver
cells. Moreover, the induced expression of p21Waf1/Cip1
protein and mRNA in both human cell lines was more pronounced in
Chang liver than in HeLa cells constitutively expressing NS5A. A
significant por-tion of the p21Waf1/Cip1 was found to be in
a complex with Cdk2 in the human Chang cells expressing NS5A
concomitant with reduced levels of Cdk1 and cyclin B1 proteins, which
is consistent with the increase in G2/M phase. NS5A was
also found to interact specifically with Cdk1 in both human and mouse
cell lines. These results suggest that NS5A-mediated growth retardation
with a pause of G2/M transition is through targeting
Cdk1/2-cyclin complexes by two mechanisms. Thus, the human Chang liver
cell line, which expresses NS5A constitutively, appears to be a good
model system to study the function of NS5A in liver-specific gene expression.
Cell Culture--
NIH3T3 cells (mouse fibroblasts), Chang cells
(Chang liver cells), and HeLa cells (human cervical cancer cells) were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. The diploid rat liver epithelial (RLE) cell line, a
single cell clone derived from parental cells from a 10-day-old Fischer
344 rat (39) (a generous gift of Dr. S. S. Thorgeirsson, NCI, National Institutes of Health) was cultured in a 1:1 mixture of
Dulbecco's modified Eagle's medium and F-12 medium supplemented with
10% fetal bovine serum. All media contained 50 µg/ml each of
penicillin G and streptomycin. Cells were cultured at 37 °C in a
humidified atmosphere of 5% CO2.
Plasmid Construction--
The full-length wild-type HCV NS5A
coding sequence derived from the HCV-H strain was cloned into the
SalI and XhoI sites of pTet-Splice (Tet-off
system, Life Technologies, Inc.) and the EcoRI and
XhoI sites of pCI-neo (Promega) to yield pTet-Splice/NS5A and pCI-neo/NS5A, respectively. Cotransfection of pTet-Splice/NS5A and
pTet-tTAK (Life Technologies) plasmids into cells allowed the induction
of NS5A protein under conditions of tetracycline withdrawal from the
growth medium. The pCI-neo/NS5A plasmid was used for the generation of
stable cell lines constitutively expressing NS5A protein.
Establishment of Stable NS5A-expressing Cell Lines--
For
tetracycline-regulated NS5A expression, cells grown in 60-mm tissue
culture dishes (30-50% confluence) were cotransfected with
pTet-Splice/NS5A (5 µg), pTet-tTAK (5 µg), and pCI-neo (0.1 µg)
for 6 h in serum-free medium in the presence of cationic lipids as
previously described (40). For constitutive expression of NS5A, cells
were transfected with the pCI-neo/NS5A plasmid in a similar way. Clones
of G418 (Geneticin; Life Technologies)-resistant cells were isolated
using cloning rings and screened for expression of NS5A by Western or
Northern blot analysis. For tetracycline-regulated expression of NS5A,
cells were continuously maintained in the medium containing 500 ng/ml
of tetracycline to prevent the expression of a tetracycline-controlled
transactivator protein (tTA) and then harvested at the indicated time
after withdrawal of tetracycline from the medium. G418-resistant stable
cell clones carrying both pTet-splice and pTet-tTAK vectors or pCI-neo
vector alone were also generated as controls for analysis of NS5A
effects in each expression system.
Cell extract was prepared by using Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% Nonidet
P-40) containing 1 mM each of dithiothreitol and
phenylmethylsulfonyl fluoride. Cells were also lysed with H buffer (20 mM Hepes-KOH, pH 7.5, 5 mM KCl, 0.5 mM MgCl2) and N buffer (50 mM
Hepes-KOH, pH 7.5, 10% sucrose, 5 mM KCl, 0.5 mM MgCl2, 0.2 M NaCl). The two
fractions were combined and were used as total cell lysates. Proteins
were separated by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
Western Blot Analysis--
Immunoblotting was carried out either
subsequent to immunoprecipitation of cell extracts with a specific
antibody, as indicated, or with cell extracts directly fractionated by
SDS-PAGE. Proteins from the SDS-PAGE were electrotransferred onto
nitrocellulose membrane and probed with the monoclonal antibodies
against HCV NS5A (a gift from Dr. Jade Chin, Ortho Clinical
Diagnostics), human p53 (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), mouse p53 (PAB 122, which can recognize both mutant and wild type
p53; Pharmingen), cyclin A (Calbiochem), cyclin B1 and Cdk2 (Santa Cruz
Biotechnology), pRB (Upstate Biotechnology, Inc., Lake Placid, NY), and
polyclonal antibodies against p21Waf1/Cip1 and Cdk1 (Santa
Cruz). In some experiments, immunoprecipitation of extracts from NIH3T3
cells was carried out first with the monoclonal anti-p53 antibody that
can recognize either p53 in the native (PAB 246) or mutant form (PAB
240) followed by immunoblotting with PAB 122, which can recognize both
forms. Horseradish peroxidase-labeled anti-mouse or anti-rabbit Ig was
used for a detection of bound primary antibody by enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Northern Blot Analysis--
Total RNA was prepared from cells at
80% confluency using Trizol reagent (Life Technologies). RNA (25 µg
each) was fractionated on a 1% agarose, 6% formaldehyde gel and
transferred onto nitrocellulose membrane. After baking the membrane at
80 °C for 2 h, the membrane was prehybridized and then
hybridized with a 32P-labeled probe using the Quick-Hyb
hybridization solution (Stratagene). Full-length HCV NS5A cDNA (1.3 kilobase pairs), human p53 cDNA, mouse p21Waf1/Cip1
cDNA, and a 0.8-kilobase pair DNA fragment obtained by digestion of
pTet-tTAK plasmid at SphI and NcoI sites were
used as probes.
Cell Growth and Colony Formation Efficiency Assays--
For cell
growth analysis, 1.0 × 104 cells were seeded onto
each 35-mm tissue culture dish at day 0. Cells were lysed with 0.1 N NaOH for the measurement of absorbance
(A260) (41) or trypsinized for the cell
count. The same number of cells (100 cells) from different cell clones
were seeded onto each 60-mm tissue culture dish for determination of
colony formation efficiency and then cultured for 10-14 days until
colonies could be visualized. Colonies were stained with 0.25%
Coomassie Brilliant Blue (R-250) in 50% methanol and 10% glacial
acetic acid as described previously (42).
Coimmunoprecipitation--
For the immunoprecipitation assay, we
also employed a transient NS5A expression in HeLa or Chang liver cells
by infection with recombinant vaccinia virus encoding T7 RNA
polymerase, vTF7-3, followed by transfection with the pTM1-NS5A-FLAG
expression plasmid (FLAG epitope tag located at the C-terminal end of
NS5A). HeLa and Chang liver cells infected with vTF7-3 alone were used
as negative controls for NS5A expression. The total cell lysates were
prepared using H and N buffers 24 h postinfection. After precleaning lysates with normal mouse or rabbit serum, 200 µg of
extract was incubated with monoclonal anti- FLAG antibody (IBI), polyclonal anti-PSTAIRE antibody (raised against the highly conserved peptide motif present in all Cdks identified to date; obtained from
Oncogene Science), polyclonal anti-Cdk1 antibody (Santa Cruz Biotechnology), or anti-p21Waf1/Cip1 antibody (Santa Cruz
Biotechnology) for 2 h at 4 °C. Immunocomplexes bound to
protein A-agarose were washed four times with the IP washing buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.5% Nonidet P-40, 200 mM NaCl, 1 mM phenylmethylsulfonyl
fluoride) and fractionated by SDS-PAGE for immunoblotting.
In Vitro GST Pull-down Assay--
Escherichia coli
XL1-Blue strain transformed with either pGEX-3X (Amersham Pharmacia
Biotech) or pGST-NS5A plasmid (43) was cultured at 37 °C until the
cell density reached a mid-log phase (A600 = 0.6), followed by 1 mM
isopropyl-1-thio- Histone H1 Kinase Assay--
Total cell lysates (50 µg of
protein from each sample) from Chang liver neor
NS5A-expressing cell clones or CL2-4 cells grown in the presence or the
absence of tetracycline were immunoprecipitated by either rabbit
polyclonal anti-Cdk1 or mouse monoclonal anti-Cdk2 antibody or by
rabbit polyclonal anti-PSTAIR antibody. The anti-PSTAIR antibody can
recognize both Cdk1 and Cdk2. Immunocomplexes bound to protein
A-agarose were suspended in a 50 µl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2,
10 µCi of [ Cell Cycle Analysis--
Cells in 50-80% confluency were
trypsinized 48 h after seeding and then fixed with 67% cold
methanol in phosphate-buffered saline. Before flow cytometric analysis,
cells were washed and resuspended in phosphate-buffered saline
containing 20 µg/ml of RNase A and 50 µg/ml propidium iodide. Cell
cycle analysis was performed on a Becton-Dickinson FACSort cytometer
using CellQuest software. DNA distributions were analyzed with ModFit
LT version 2.0 software.
Establishment of Tetracycline-regulated NS5A-expressing Stable Cell
Lines and the Effects of NS5A Expression on Cell Growth--
To
elucidate the effect of HCV NS5A protein on cell growth, we first used
the tetracycline-regulated system (44) to express NS5A protein in
NIH3T3 cells. Individual G418-resistant stable cell clones were
screened by Western blot analysis. The expression levels of NS5A
protein varied in these clones. One clone (CL2-4) that showed
detectable levels of NS5A expression in a tetracycline-regulated manner
was chosen for further analysis. The appearance of NS5A protein was
first detectable 8 h after withdrawal of tetracycline from the
medium, and the levels of NS5A increased in a
time-dependent fashion (Fig.
1A) as shown by Western blot
analysis. The addition of tetracycline at a concentration of 63 ng/ml
in the medium dramatically repressed the expression of NS5A to a barely
detectable level (Fig. 1B). The induction of tTA gene
expression after withdrawal of tetracycline from the medium of the
NS5A-expressing CL2-4 cells and the control cells expressing tTA from
the empty vector was at the same level as confirmed by Northern blot
analysis (Fig. 1C).
To study the effect of NS5A protein on cell growth, we used the
G418-resistant, clonally selected (CL2-4) population of early passage
cells (5-10 passages). The induction of NS5A protein in CL2-4 cells
markedly reduced cell growth, as compared with that of the same cells
grown in the medium containing tetracycline in which NS5A expression
was shut off (Fig. 2, left).
On the other hand, the control cells carrying both empty pTet-Splice
and pTet-tTAK plasmids showed no significant difference in cell growth
in the presence or absence of tetracycline in the culture medium (Fig. 2, right). The expression of NS5A in the absence of
tetracycline declined gradually from 7 days up to 1 month and was still
detectable by Western blots, but the growth retardation of
NS5A-expressing cells continued during this period compared with the
cells in the presence of tetracycline (data not shown). We also
examined the colony formation efficiency of control NIH3T3 cells
carrying the empty vector as well as the NIH3T3-CL2-4 cells expressing NS5A in the presence and absence of tetracycline. The results indicate
that colony formation efficiency was greatly reduced in NIH3T3-CL2-4
cells when the expression of NS5A protein was induced by withdrawal of
tetracycline, compared with the control cells in the presence or
absence of tetracycline (data not shown). Although the number of
colonies in the control cells was not significantly affected under the
Tet-off conditions, the colony size was reduced, probably due to
the effect of tTA-VP16 activator protein. This is consistent with an
earlier report that the tTA-VP16 protein confers morphological changes
to hamster glioblastoma cells (45). These results indicated that the
expression of HCV NS5A protein was growth-inhibitory to NIH3T3 cells
under the tetracycline-regulated expression system.
We sought to extend this observation from the NIH3T3 fibroblasts to the
rat liver epithelial cell line, RLE. Expression of NS5A in the
tetracycline-regulated stable RLE cell line also conferred growth-inhibitory properties (RLE13 in Fig.
3A, left), whereas in the control cells carrying empty vector (RLEC2 cells in Fig. 3A, right) there was no significant difference in
cell growth in the presence or absence of tetracycline. The expression
of NS5A in RLE13 cells under the Tet-off conditions was confirmed by
Western blot analysis (Fig. 3B, left).
In this regard, it is of interest to note that a previous study that
used the tetracycline-regulated expression system reported that the
expression of all of the HCV proteins from the processing of the
polyprotein precursor in osteosarcoma cells was also inhibitory to cell
growth and colony formation efficiency (46, 47). However, no data were
presented to evaluate which of the HCV proteins was responsible for
growth inhibition in that study.
Establishment of Stable Cell Lines Constitutively Expressing NS5A
and Their Growth Properties--
To ensure that the growth-inhibitory
effect in the early passage cells was specific to NS5A expression but
was not due to tTA-VP16 expression, we constructed three stable cell
lines constitutively expressing NS5A protein. The
NS5A gene was under the control of the
cytomegalovirus immediate early promoter in the pCI-neo vector. The
NIH3T3, human Chang liver, and HeLa cells were transfected with this
NS5A expression plasmid or the pCI-neo vector plasmid. Stable
G418-resistant cell lines expressing NS5A or the
neor from the vector plasmid were isolated. More
than 10 clonal cell lines were isolated from each of the different cell
types. The expression of NS5A protein and mRNA levels were screened
by Western and Northern blot analyses, respectively, and more than five
NS5A-expressing stable cell clones from each of the three different
cell types were isolated. The expression level of NS5A protein was low
in these cell clones (Fig. 4A)
compared with the relatively high levels achieved with the
tetracycline-regulated system (Fig. 1A). Finally, two clones
of Chang cells and one clone each of HeLa and NIH3T3 cell lines were
selected for further analyses.
As shown in Fig. 4B, Northern blot analysis clearly
confirmed NS5A expression in cells stably transfected with the pCI-neo NS5A plasmid but not with the pCI-neo vector plasmid. The steady state
levels of NS5A protein are comparable with the corresponding mRNA
levels in each of the NS5A-expressing cell lines.
We determined the cell growth rates of these stable cell lines. For
these experiments, triplicate pairs of control versus NS5A-expressing cells (1 × 104 cells/plate) from each
cell line were analyzed. The results shown in Fig.
5A indicated that the growth
of these constitutive NS5A-expressing Chang, HeLa, and NIH3T3 cell
lines was also slower when compared with their pCI-neo vector control
cell counterparts. The population doubling time was increased from
33.9 h to an average of 55 h in two different clones of Chang
cell lines, and from 18.5 to 26 h in HeLa or NIH3T3 cells (Fig.
5A). Interestingly, growth inhibition due to NS5A expression
was most prominent in Chang liver cell clones. The results shown in
Fig. 4, A and B, indicate that NS5A was still
being produced after 7 days, the longest time point taken in cell
growth experiments (Fig. 5A). The effect of NS5A protein on
cell growth was not due to apoptosis when examined both visually and by
flow cytometric analysis.
We also determined the colony formation efficiency of NS5A-expressing
Chang, NIH3T3, and HeLa cells. The same number of cells was seeded onto
culture dishes, and the colonies were visualized as described under
"Experimental Procedures" (Fig. 5B). The results shown
in Fig. 5B indicated that the colony formation efficiency was greatly reduced in NS5A-expressing Chang cells but to a lesser extent in NIH3T3 cells, whereas it was essentially unchanged in HeLa
cell clones compared with their respective G418-resistant control cells
not expressing NS5A.
Since the evidence thus far indicated that NS5A expression increased
the population doubling time by inhibiting cell proliferation, we
sought to determine the effect of NS5A expression on cell cycle distribution by flow cytometric analysis. For these experiments, cells
were grown to 50-80% confluence and prepared for cell cycle distribution analysis as described under "Experimental Procedures." The results indicate that the effect of NS5A expression on cell cycle
distribution depends on the expression system chosen (Table I). For example, in the Tet-off
cells expressing both NS5A and tTA-VP16 (CL2-4), a predominant
accumulation of cells in G0/G1 phase with a
decreased S phase was observed compared with the control cells. This
pattern of cell cycle distribution is probably due to the combined
effects of NS5A and tTA-VP16 on cell growth and cell cycle control.
However, in the human Chang liver and HeLa cells constitutively
expressing NS5A, a consistently increased proportion of cells were
found in the G2/M phase with a shortened S phase (Table I,
bottom). These cell cycle distribution data are in agreement
with their corresponding attenuated growth rates. However, in NIH3T3
cells constitutively expressing NS5A, there was no apparent difference
in the cell cycle distribution of the NS5A-expressing versus
control cells (Table I, bottom), although they show a clear difference
in their respective growth rates. In this regard, the effect of NS5A on
growth retardation resembles that of the human immunodeficiency virus
Vpr protein (48, 49). Vpr protein also interfered with the
proliferation of NIH3T3 cells by increasing the doubling time but
failed to cause the G2 arrest. In HeLa cells, however, the
Vpr protein caused growth retardation and G2 arrest.
Expression of Cell Cycle Regulatory Genes in NS5A-expressing
Cells--
Overexpression of p21Waf1/Cip1 and/or p53 is
often associated with the repression of cell proliferation. Therefore,
we sought to examine whether the inhibitory effect of NS5A on cell
growth found in this study is linked to an elevated expression of these
genes. The results shown in Fig.
6A indicated that
p21Waf1/Cip1 protein was significantly increased in the
constitutive NS5A-expressing cells from all three different cell types
to different extents (Chang liver > HeLa > NIH3T3).
Comparing the two different NS5A-expressing clones of Chang liver
cells, we found that the p21Waf1/Cip1 protein level of
clone 1 was much higher than that of clone 3. The mRNA levels of
the p21Waf1/Cip1 gene were also elevated in these cells
(Fig. 7A). Thus, the increased p21Waf1/Cip1 expression level seems to closely correlate
with their cell cycle distribution in this stable human cell line.
Since the p21Waf1/Cip1 gene expression is regulated by p53,
we examined the p53 protein and mRNA levels in these
NS5A-expressing cells. The results shown in Figs. 6B and
7B indicate that the p53 protein and mRNA levels are
significantly increased in human Chang liver cells expressing NS5A.
However, in HeLa cells, although the p53 mRNA level was slightly
increased (Fig. 6B), the p53 protein level was not (Fig.
6B). This phenomenon has been observed earlier by others
(see for example, Ref. 37). Although HeLa, a cervical cancer cell line
with human papilloma virus etiology, is considered to be pRB-positive
and p53-positive, pRB is inactivated by association with E7
oncoprotein, and p53 does not accumulate in these cells because of the
expression of the E6 oncoprotein, which is known to target p53 protein
to ubiquitin-mediated degradation (50, 51). The increase in p53
expression was the least in the NIH3T3 cell line constitutively
expressing NS5A, which is consistent with the low level of
p21Waf1/Cip1 found in these cells as well as with the lack
of any effect on cell cycle distribution (Table I, bottom). Western
blot analysis of the NIH3T3 cell extracts using monoclonal antibodies
that can recognize p53 in native conformation (PAB 246) and in mutant
conformation (PAB 240) indicated that the NIH3T3 cell line used in this
study has dysfunctional p53 (data not shown). These results are also consistent with the low level of p21Waf1/Cip1 found in
these cells as well as with the lack of any effect on cell cycle
distribution in response to NS5A expression.
The Cdk1 is expressed in G1
Next, we examined whether NS5A interacts with any cell cycle-regulatory
proteins such as Cdk1, Cdk2, or the cyclin components, and titrates out
their availability for the normal G2
It has been suggested that the increased expression of
p21Waf1/Cip1 may target the cyclin A-Cdk2 complex to
attenuate mitosis (60). We sought to determine whether the
significantly increased expression of p21Waf1/Cip1
accompanied by the prolonged G2/M phase in the
NS5A-expressing Chang liver cells might be targeting the cyclin A-Cdk2
complexes, which are required for the activation of Cdk1-cyclin A or
Cdk1-cyclin B1 complexes and entry into mitosis (61). Extracts from
Chang liver cells expressing NS5A or from control cells expressing the G418 resistance marker from the vector plasmid were immunoprecipitated with the anti-p21Waf1/Cip1 antibody. The immunoprecipitates
were analyzed by SDS-PAGE and immunoblotting with the anti-Cdk2
antibody. The results, shown in Fig.
9A, indicated that there was
an increased association of Cdk2 with p21Waf1/Cip1 in the
NS5A-expressing Chang liver cells compared with the control cells
(lanes 2 and 3 versus
lane 1). There was no significant difference in
the total Cdk2 levels in the NS5A-expressing cells versus
the control cells (data not shown), which suggests that NS5A targets
the Cdk2 indirectly through the up-regulation of p21Waf1/Cip1.
We next sought to determine whether the interaction between
p21Waf1/Cip1 and Cdk2 would affect the histone H1 kinase
activity of cyclin-Cdk2 complexes in NS5A-expressing cells. To verify
this, extracts from Chang liver cells expressing NS5A and control cells
were immunoprecipitated with anti-PSTAIRE antibody or anti-Cdk2
antibody. The immunoprecipitates were assayed for histone H1 kinase
activity. The results shown in Fig. 9, B and C,
indicated that the histone H1 kinase activity was indeed inhibited in
the extracts from NS5A-expressing Chang liver cells compared with the
control cell extracts. This inhibition of histone H1 kinase activity
was not significant in HeLa cells expressing NS5A (data not shown). One
possible explanation is that the levels of p21Waf1/Cip1 are
much lower in HeLa cells expressing NS5A, and the Cdk1 and cyclin B1
levels are also higher (Fig. 6).
The results presented in this study indicate that expression of NS5A
causes growth retardation with a delay in the G2 In this study, we demonstrate that human liver cell lines
constitutively expressing NS5A exhibited a pronounced retardation of
cell proliferation and a reduced colony formation efficiency concomitantly with a shortened S phase and a prolonged G2/M
phase of the cell cycle. NS5A inhibits cell proliferation, as shown by
an increase in the population doubling time in different cell types
examined, when expressed under inducible as well as constitutive expression systems. However, the specific effects of NS5A on the cell
cycle are dependent on the p53 status and the conditions chosen for
expression of NS5A. Thus, unlike the original tetracycline- inducible
system (44) in which tTA-VP16 expression is constitutive and expressed
at low levels, in the modified tetracycline-regulated system (62),
tTA-VP16 expression is autoregulatory and depends on the expression of
small amounts of tTA-VP16 protein made from the leakiness of the
minimal promoter subsequent to withdrawal of tetracycline. In NIH3T3
cells, higher levels of tTA-VP16 protein and NS5A are produced in this
modified expression system, which cause accumulation of cells
predominantly with G0/G1 DNA content.
In contrast, there was no apparent change in the cell cycle
distribution of the NIH3T3 cells expressing NS5A constitutively, although there was growth retardation of the NIH3T3 cells expressing NS5A compared with the control cells expressing the G418 resistance marker. The NIH3T3 cell line used in this study expresses p53, which is
predominantly of the mutant form. In this respect, NS5A protein
functions in a similar manner to the human immunodeficiency virus Vpr
protein, which also interfered with proliferation of NIH3T3 cells by
increasing the doubling time but failed to cause G2 arrest
(48). In HeLa cells, Vpr caused growth retardation and G2
arrest (49). Our results indicate that NS5A caused growth retardation
and prolongation of G2/M phase in both HeLa and Chang liver
human cell lines but only growth retardation in NIH3T3 cells. The
effect of NS5A on cell cycle progression was most pronounced in Chang
liver cells that were originally derived from human hepatocytes, a cell
type thought to be the primary target for HCV replication.
Moreover, in Chang liver cells constitutively expressing NS5A, both
p21Waf1/Cip1 mRNA and protein levels are up-regulated
concomitant with elevation of the p53 mRNA and protein levels. Due
to the expression of the E6 protein of human papilloma virus,
which targets p53 by the ubiquitin-mediated degradation pathway (51),
expression of p21Waf1/Cip1 was only modestly increased in
HeLa cells, and a corresponding increase of p53 protein was not
observed. It has been shown previously that human papilloma virus 16 E6-expressing human fibroblast cells also failed to arrest in
G1 upon exposure to ionizing radiation due to inefficient
induction of p21Waf1/Cip1 (63). A higher number of
mitotic cells in G2 phase was also observed, and cells
entered mitosis more rapidly in E6-expressing cells (63).
p21Waf1/Cip1 is induced in a
p53-dependent pathway in response to a variety of
DNA-damaging agents. The expression of p21Waf1/Cip1 is also
enhanced when cells undergo senescence or differentiation (64, 65). In
some mammalian cells, p21Waf1/Cip1 predominantly causes
G2 arrest (35-37), suggesting that
p21Waf1/Cip1 is a negative regulator of the
G2/M transition. In Xenopus egg extracts, it has
been shown that Cdk2 functions as a positive activator of the cyclin
B1-Cdk1 complex, whereas p21Waf1/Cip1 inactivates Cdk2 and
blocks progression into mitosis (61). The results of the study by Dulic
et al. (38) suggest that cyclin A-Cdk2 is the primary target
of p21Waf1/Cip1 in the premitotic G2/M phase
either by inhibition of its kinase activity or activation of the
Cdk-activating kinase. Induction of p21Waf1/Cip1
expression in NS5A-expressing Chang liver cells may also target the
cyclin A-Cdk2 complexes as shown by increased association of Cdk2 with
p21Waf1/Cip1 in these cells compared with the control
cells. The levels of Cdk1 and cyclin B1 are also very much reduced in
NS5A-expressing Chang liver cells compared with control cells. However,
in NS5A-expressing HeLa cells, the Cdk1 and cyclin B1 levels were only
slightly reduced compared with the control cells. This is consistent
with a previous report that due to the presence of human papilloma
virus 18 oncoprotein and low p21Waf1/Cip1 levels in HeLa
cells, the levels of cyclin B1-Cdk1 kinase activity are
sufficiently high due to incomplete inhibition of Cdk2 by p21Waf1/Cip1 (63). This inherent property of HeLa cells
would explain our observation that immobilized GST-NS5A binds more Cdk1
from HeLa cell extracts than from Chang liver cell extracts when the
same amount of total protein was used for binding experiments. (Fig. 8A). Consistent with these findings, the histone H1 kinase
activity is also lower in Chang liver cells expressing NS5A compared
with the control cells, whereas this inhibitory effect on the kinase activity could not be detected in HeLa cells (data not shown). Moreover, a novel finding in this study is that NS5A physically interacts with Cdk1, and this interaction is specific. Our results, taken together, suggest that NS5A appears to attenuate mitosis in a
cell type-specific manner by two independent mechanisms: by targeting
Cdk1 via direct interaction and by inhibiting the activity of Cdk2 by
inducing the expression of p21Waf1/Cip1.
In our previous study, we demonstrated that NS5A associated with
a serine/threonine kinase and was phosphorylated by the associated kinase, which was independently confirmed by another group (21, 22,
43). Therefore, direct interaction of NS5A with Cdk1 as well as the
up-regulation of p53 and concomitant activation of p21Waf1/Cip1 in response to NS5A expression in hepatic
cells may probably be linked to the perturbation of normal function
of the cellular kinase in the host as a consequence of HCV infection,
which could play an important role in the pathogenesis of hepatitis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 protein (27). However, the physiological role of
NS5A protein is still largely unknown.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside induction for
4 h. Bacterial pellets were resuspended in cold phosphate-buffered
saline containing 1 mM phenylmethylsulfonyl fluoride and
lysed by mild sonication. The soluble fraction was incubated with 60 µl of a 50% slurry of glutathione-Sepharose beads (Amersham
Pharmacia Biotech) at 4 °C for 1 h. After washing the beads
four times with cold phosphate-buffered saline, 200 µg of total HeLa
or Chang liver cell extract was incubated with the immobilized GST-NS5A
at 4 °C overnight. Finally, Sepharose beads were washed four times
with cold phosphate-buffered saline to remove proteins bound
nonspecifically before loading onto SDS-PAGE.
-32P]ATP, and 50 µg/ml of histone H1
and incubated at 30 °C for 30 min. The reaction was terminated by
the addition of 6× SDS-PAGE sample loading buffer. The phosphorylated
products were separated by SDS-PAGE and electrotransferred onto
nitrocellulose membrane, followed by autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Tetracycline-regulated expression of NS5A in
cloned NIH3T3 (CL2-4) cells. A, time course of NS5A
expression. Cells were cultured in the presence of tetracycline (500 ng/ml) until time point 0, when tetracycline was withdrawn from the
medium. Cells were subsequently harvested at indicated time periods up
to 72 h. Total cell lysates (20 µg) were fractionated by 8%
SDS-PAGE, and the proteins were analyzed by immunoblotting using
monoclonal HCV NS5A antibody (A and B).
B, NS5A expression regulated by tetracycline concentration
in the medium. Cells were grown in the presence of 500 ng/ml
tetracycline and then subcultured for 24 h after removal of the
original medium and replacement with a medium containing various
concentrations of tetracycline. Total cell lysates (20 µg) were
analyzed by immunoblotting using anti-NS5A monoclonal antibody as
described for A. C, the induction of tTA
expression in tetracycline-regulated cell lines. CL2-4 and control
NIH3T3 cells were cultured in the medium containing (+) or lacking ( )
tetracycline for 24 h. Induction of tTA mRNA was analyzed by
Northern blot using 32P-labeled tTA cDNA probe. 28 and
18 S rRNA stained by methylene blue is shown.
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Fig. 2.
NS5A retards cell growth rate in
tetracycline-regulated CL2-4 cells. For cell growth
analysis, CL2-4 (left) or control cells (right)
(1.0 × 104 of each) were grown in 35-mm dishes in the
presence ( ,
) or in the absence (
,
) of tetracycline in
culture medium containing 10% fetal bovine serum. Cells were lysed
with 0.1 N NaOH at the day indicated, followed by the
measurement of absorbance (A260) in each sample.
Each point represents the average value from duplicate dishes.
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Fig. 3.
Inhibition of cell growth by NS5A expression
using a tetracycline-regulated system in RLE cells. A,
cell growth rate in tetracycline-regulated RLE cells expressing NS5A.
NS5A-expressing (RLE13) cells or control (RLEC2) cells (1 × 104 of each) were grown in the presence ( ,
) or
absence (
,
) of tetracycline in the medium. Cells were
trypsinized and counted at the time periods indicated. Each time point
represents the average of duplicate dishes. The population doubling
time obtained from day 1 to day 3 is indicated. B,
tetracycline-regulated expression of NS5A in RLE13 cells. RLE13 or
RLEC2 cells were cultured for 24 h in the presence or absence of
tetracycline in the medium. Total cell extracts (20 µg of protein)
were fractionated by SDS-PAGE (8%), and the NS5A was detected by
immunoblotting using the anti-NS5A monoclonal antibody.
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Fig. 4.
Expression of NS5A protein and mRNA in
different cell lines constitutively expressing NS5A. A,
Western blot analysis. Cell extracts (30 µg of protein) from both
neor control and NS5A-expressing cells were
separated by SDS-PAGE (8%) and analyzed by immunoblotting using
monoclonal anti-NS5A antibody. B, Northern blot analysis.
Total cellular RNA (25 µg) from different cell types were loaded onto
denaturing agarose (1%) gel, and after electrophoresis the RNAs were
transferred onto nitrocellulose membrane. Hybridization was performed
using 32P-labeled full-length HCV NS5A cDNA probe, and
the bands were detected by autoradiography. 28 and 18 S ribosomal RNA
stained by methylene blue are shown.
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Fig. 5.
NS5A inhibits cell growth and colony
formation efficiency in different cell types constitutively expressing
NS5A. A, cell growth analysis.
neor control ( ) or NS5A-expressing cells (
or
). 1 × 104 cells from different cell types were
seeded onto 35-mm dishes and were grown in medium containing 10% fetal
bovine serum at 37 °C in a CO2 (5%) incubator as
described under "Experimental Procedures." The number of cells was
counted at the day indicated. The population doubling time was obtained
from the number of cells grown at days 3-5 for each cell line. Each
point represents the mean ± SD of triplicate dishes.
B, colony formation efficiency assay. Cells of
neor control or NS5A-expressing cells (5 × 102) were seeded onto 60-mm dishes and cultured until
colonies were visually seen. Colonies were stained as described under
"Experimental Procedures. " The assay was carried out in triplicate
dishes for each cell line, and the results were reproducible.
Effect of NS5A expression on cell cycle
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Fig. 6.
Expression of p21Waf1/Cip1, p53,
Cdk1, and cyclin B1 proteins in different cell clones constitutively
expressing NS5A. Extracts containing 30 µg of total protein from
each cell clone were separated by SDS-PAGE and analyzed by
immunoblotting using antibodies against p21Waf1/Cip1
(A), p53 (B), Cdk1 (C), or cyclin B1
(D).
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Fig. 7.
Determination of p21Waf1/Cip1 and
p53 mRNA levels in NS5A-expressing cells by Northern blot
analysis. Total cellular RNAs (25 µg) from each cell clone were
loaded onto 1% denaturing agarose gel, and after electrophoresis, the
RNAs were transferred onto nitrocellulose membrane. Hybridization was
performed using 32P-labeled mouse p21Waf1/Cip1
and human p53 cDNA probes, and the bands were detected by
autoradiography. 28 and 18 S ribosomal RNAs stained by methylene blue
are shown.
S phase and is primarily
required for G2
M phase transition (see Refs. 52-54;
for reviews, see Refs. 55-57). Cdk1 also plays a role in
phosphorylation of key substrates involved in G1
S
phase transition (for reviews, see Refs. 28 and 58). Since the cell
cycle distribution of human cells expressing NS5A showed an increase in
G2/M phase, we examined whether there were any changes in
the levels of Cdk1 and cyclin B1. Extracts from NS5A-expressing and
control cells were analyzed by Western blot using anti-Cdk1 and
anti-cyclin B1 antibodies. The results shown in Fig. 6, C
and D, indicated that the levels of both Cdk1 and cyclin B1
were greatly reduced in human Chang liver cells expressing NS5A but
much less so in HeLa cells. In NIH3T3 cells, however, there was no
change in the Cdk1 level caused by NS5A expression. This result is
again consistent with the lack of any effect of NS5A expression on the
cell cycle distribution of NIH3T3 cells (Table I, bottom).
M transition. For
this experiment, HeLa cells were infected with vTF7-3, the recombinant
vaccinia virus encoding T7 RNA polymerase, either alone (Fig.
8A, lane
3) or together with transfection of the pTM1-NS5A-FLAG expression plasmid (Fig. 8A, lane 4).
In the pTM1-NS5A expression plasmid, the NS5A gene was cloned
under the control of the T7 promoter and encephalomyocarditis virus
5'-leader sequence for cap-independent translation (59). The cell
extracts were immunoprecipitated with anti-FLAG antibody and analyzed
by SDS-PAGE and immunoblotting with the anti-Cdk1 antibody. The results
shown in Fig. 8A indicated that the anti-FLAG
immunoprecipitates also included Cdk1. To confirm this interaction
between NS5A and Cdk1, the infected HeLa or Chang liver cell lysates
were also immunoprecipitated with either anti-PSTAIRE antibody or
normal rabbit serum, and the immunoprecipitates were analyzed by
SDS-PAGE and immunoblotting with the monoclonal anti-NS5A antibody. The
results shown in Fig. 8B indicate that the anti-PSTAIRE antibody, which recognizes both Cdk1 and Cdk2, immunoprecipitated NS5A
from the vTF7-3-infected, pTM1-NS5A-FLAG plasmid-transfected cell
extracts. To further confirm this interaction, we expressed GST-NS5A in
E. coli as described in our previous report (43) and
immobilized it to glutathione-Sepharose beads. As a negative control,
GST was also expressed and immobilized in a similar manner. Normal HeLa
cell extracts (Fig. 8A, lanes 5 and
6) or Chang liver cell extracts (Fig. 8A,
lanes 7 and 8) containing equal
amounts of total protein were batch-bound to either GST- or
GST-NS5A-immobilized beads. After washing the beads, the proteins bound
to the beads were analyzed by SDS-PAGE and immunoblotting with the
anti-Cdk1 antibody. Additionally, NS5A was also coimmunoprecipitated
together with Cdk1 when NS5A was induced in Tet-off NIH3T3 cells (Fig. 8C). The results confirmed that NS5A specifically interacts
with Cdk1 in vivo and in vitro. Furthermore,
using the GST and GST-NS5A-immobilized beads, no interaction between
NS5A and Cdk2, cyclin B1, pRB, p21Waf1/Cip1, or cyclin A
was observed (data not shown), suggesting that the interaction between
NS5A and Cdk1 is very specific. Next, we examined whether this binding
of NS5A to Cdk1 affects the kinase activity of cyclin-Cdk1 complexes.
The same amount of cell extract from NS5A-expressing or control cells
was immunoprecipitated with the anti-Cdk1 antibody, followed by
in vitro kinase assay using histone H1 as a substrate.
The results, as shown in Fig. 8, D and
E, clearly demonstrated that the histone H1 kinase activity
by cyclin-Cdk1 complexes was inhibited in Chang liver cells
constitutively expressing NS5A as well as NIH3T3 cells expressing NS5A
under the Tet-off condition.
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Fig. 8.
NS5A physically interacts with Cdk1 and
inhibits histone H1 kinase activity. GST-NS5A was expressed in
E. coli and immobilized to glutathione-agarose
beads as described (43). The recombinant vaccinia virus expression
plasmid encoding the NS5A gene (pTM1-NS5A) described
previously (20) was modified to include in-frame fusion of FLAG epitope
by standard molecular biology techniques. A, HeLa cells were
infected with vTF7-3, the recombinant vaccinia virus encoding the T7
RNA polymerase, and then transfected with pTM1-NS5A-FLAG. Samples of
HeLa cell extracts (6 or 12 µg of total protein) were loaded in
lanes 1 and 2. Extracts from HeLa
cells infected with the vTF7-3 either alone (lane
3) or together transfected with pTM1-NS5A-FLAG expression
plasmid (lane 4) were immunoprecipitated with
anti-FLAG monoclonal antibody, and the immunoprecipitates were loaded.
Uninfected HeLa cell lysates bound to either GST beads (lane
5) or GST-NS5A immobilized beads (lane
6) and uninfected Chang liver cell lysates bound to either
GST beads or GST-NS5A immobilized beads (lanes 7 and 8, respectively) were applied to SDS-PAGE and analyzed
by immunoblotting with the anti-Cdk1 antibody. B, HeLa or
Chang liver cells were infected with vTF7-3 and transfected with
pTM1-NS5A-FLAG expression plasmid. Cell lysates were immunoprecipitated
with either normal rabbit serum or polyclonal anti-PSTAIRE antibody
(which recognizes both Cdk1 and Cdk2) as indicated. Subsequent to
SDS-PAGE, immunoblotting was carried out using the anti-NS5A monoclonal
antibody. C, NIH3T3 cells expressing NS5A (CL2-4) were grown
in the presence or absence of tetracycline. Cell extracts (200 µg of
total protein) were immunoprecipitated with anti-Cdk1 antibody, and the
immunoprecipitates were fractionated by SDS-PAGE. Immunoblotting of the
membranes was carried out using the monoclonal anti-NS5A antibody.
D and E, extracts (50 µg) from control
neor Chang liver and NS5A-expressing cells, 5A-1
and 5A-3 (D), or CL2-4 cells grown in the presence or
absence of tetracycline in the culture medium (E) were
immunoprecipitated with polyclonal anti-Cdk1 antibody. Immunocomplexes
bound to protein A-agarose were subjected to in vitro kinase
assay using histone H1 (50 µg/ml) as substrate. The phosphorylated
products were separated by SDS-PAGE (10%) and electrotransferred onto
nitrocellulose membrane, followed by autoradiography.
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Fig. 9.
p21Waf1/Cip1 is bound to Cdk2 in
NS5A-expressing Chang liver cells and inhibits histone H1 kinase
activity. A, total cell lysates (200 µg of protein)
from control neor Chang liver cells or
NS5A-expressing 5A-1 and 5A-3 cloned cells were immunoprecipitated with
anti-p21Waf1/Cip1 antibody. Immunocomplexes bound to
protein A-agarose were separated by SDS-PAGE and transferred onto a
nitrocellulose membrane, followed by immunoblotting with the anti-Cdk2
antibody. B and C, cell lysates (50 µg of
protein) from stable clones of control G418-resistant Chang liver cells
(lane marked neo) or the
NS5A-expressing clones (5A-1 and 5A-3) were immunoprecipitated with
polyclonal anti-PSTAIRE antibody (B) or monoclonal anti-Cdk2
antibody (C). The immunoprecipitates were analyzed for
histone H1 kinase activity as described below. Immune complexes bound
to protein A-agarose were resuspended in a 50-µl reaction mixture
containing 50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 10 mCi of [ -32P]ATP, and 50 µg/ml
histone H1 and incubated at 30 °C for 30 min. The reaction was
terminated by the addition of 6× SDS-PAGE sample-loading buffer.
Phosphorylated products were separated by SDS-PAGE (10%),
electrotransferred to nitrocellulose membrane, and subjected to
autoradiography.
M transition through a two-pronged mechanism: a p53-dependent
p21Waf1/Cip1 pathway by targeting Cdk2 as well as through
direct interaction of NS5A with Cdk1. This effect of NS5A on
attenuation of cell growth and cell cycle control is more pronounced in
human Chang liver cells, which are the probable biological target of
HCV, than in HeLa cells.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Jade Chin (Ortho Diagnostic Systems, Johnson & Johnson) for the gift of the monoclonal antibody against NS5A, Dr. Snorri Thorgeirsson (NCI, National Institutes of Health) for the gift of RLE cells, and Dr. Gigi Lozano (University of Texas M. D. Anderson Cancer Center) for the mouse p21Waf1/Cip1 and human p53 cDNAs. We thank Dr. Mary Wetzel for critical reading and careful editing of the manuscript.
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FOOTNOTES |
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* This work was supported in part by NIAID, National Institutes of Health, Grant R03-AI-44036 and by a Focus Giving grant from the Johnson & Johnson Foundation (to R. P.).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.
¶ Present address: Sigma-Aldrich Life Science, 3300 S. Second St., St. Louis, MO 63178.
** Present address: Dept. of Internal Medicine III, Klinikum Rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, D-81675, Munich, Germany.
§§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421. Tel.: 913-588-7018; Fax: 913-588-7440; E-mail: rpadmana@kumc.edu.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M008329200
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ABBREVIATIONS |
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The abbreviations used are: HCV, hepatitis C virus; RLE, rat liver epithelial cells; tTA, tetracycline-controlled transactivator protein; Cdk, cyclin-dependent kinase; pRB, retinoblastoma protein; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
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