Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A.

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 G(2)/M phase, which could be explained by a p53-dependent induction of p21(Waf1/Cip1) protein and mRNA levels. NS5A interacts with Cdk1 in vivo and in vitro, and a significant portion of the p21(Waf1/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 G(2)/M phase. Our results suggest that the NS5A protein causes growth inhibition and cell cycle perturbations by targeting the Cdk1/2-cyclin complexes.

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 ϳ9.5-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)(12)(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 receptorbound 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 proteinassociated protein, hVAP (25). In addition, NS5A associates with a cellular transcription factor, SRCAP (26), as well as with the human karyopherin ␤3 protein (27). However, the physiological role of NS5A protein is still largely unknown.
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 p21 Waf1/Cip1 , p27 Kip1 , and p57 (32). p21 Waf1/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 p21 Waf1/Cip1 causes G 1 and G 2 arrest and, in some studies, predominantly G 2 arrest (35)(36)(37). Dulic et al. (38) proposed that cyclin A and Cdk2 are the primary targets of p21 Waf1/Cip1 in premitotic G 2 /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, NS5Aexpressing 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 G 2 /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 p21 Waf1/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 p21 Waf1/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 G 2 /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 G 2 /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.

EXPERIMENTAL PROCEDURES
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 g/ml each of penicillin G and streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 .
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 g), pTet-tTAK (5 g), and pCI-neo (0.1 g) 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 MgCl 2 ) and N buffer (50 mM Hepes-KOH, pH 7.5, 10% sucrose, 5 mM KCl, 0.5 mM MgCl 2 , 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 p21 Waf1/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 g 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 32 P-labeled probe using the Quick-Hyb hybridization solution (Stratagene). Full-length HCV NS5A cDNA (1.3 kilobase pairs), human p53 cDNA, mouse p21 Waf1/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 ϫ 10 4 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 (A 260 ) (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 g 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-p21 Waf1/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 (A 600 ϭ 0.6), followed by 1 mM isopropyl-1-thio-␤-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 l 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 g 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.
Histone H1 Kinase Assay-Total cell lysates (50 g of protein from each sample) from Chang liver neo r 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 l reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 10 Ci of [␥-32 P]ATP, and 50 g/ml of histone H1 and incubated at 30°C for 30 min. The reaction was terminated by the addition of 6ϫ SDS-PAGE sample loading buffer. The phosphorylated products were separated by SDS-PAGE and electrotransferred onto nitrocellulose membrane, followed by autoradiography.
Cell Cycle Analysis-Cells in 50 -80% confluency were trypsinized 48 h after seeding and then fixed with 67% cold methanol in phosphatebuffered saline. Before flow cytometric analysis, cells were washed and resuspended in phosphate-buffered saline containing 20 g/ml of RNase A and 50 g/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 fi- 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 g) 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 (ϩ) or lacking (Ϫ) tetracycline for 24 h. Induction of tTA mRNA was analyzed by Northern blot using 32 P-labeled tTA cDNA probe. 28 and 18 S rRNA stained by methylene blue is shown.

Inhibition of Cell Growth by HCV NS5A
broblasts 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 neo r 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 NS5Aexpressing cell lines.
We determined the cell growth rates of these stable cell lines. For these experiments, triplicate pairs of control versus NS5Aexpressing cells (1 ϫ 10 4 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 inhi-bition 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 NS5Aexpressing 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 G 0 /G 1 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 G 2 /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 G 2 arrest. In HeLa cells, however, the Vpr protein caused growth retardation and G 2 arrest.
Expression of Cell Cycle Regulatory Genes in NS5A-expressing Cells-Overexpression of p21 Waf1/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 p21 Waf1/Cip1 protein was significantly increased in the constitutive NS5A-expressing cells from all three different cell types to different extents (Chang liver Ͼ HeLa Ͼ NIH3T3). Comparing the two different NS5A-expressing clones of Chang liver cells, we found that the p21 Waf1/Cip1 protein level of clone 1 was much higher than that of clone 3. The mRNA levels of the p21 Waf1/Cip1 gene were also elevated in these cells (Fig. 7A). Thus, the increased p21 Waf1/Cip1 expression level seems to closely correlate with their cell cycle distribution in this stable human cell line.
Since the p21 Waf1/Cip1 gene expression is regulated by p53, we examined the p53 protein and mRNA levels in these NS5Aexpressing 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 p21 Waf1/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 p21 Waf1/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 G 1 3 S phase and is primarily required for G 2 3 M phase transition (see Refs. 52-54; for reviews, see Refs. [55][56][57]. Cdk1 also plays a role in phosphorylation of key substrates involved in G 1 3 S phase transition (for reviews, see Refs. 28 and 58). Since the cell cycle distribution of human cells expressing NS5A showed an increase in G 2 /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 Total cellular RNA (25 g) 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 32 P-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.
of any effect of NS5A expression on the cell cycle distribution of NIH3T3 cells (Table I, bottom).
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 G 2 3 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 5Ј-leader sequence for capindependent 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, p21 Waf1/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 FIG. 5. NS5A inhibits cell growth and colony formation efficiency in different cell types constitutively expressing NS5A. A, cell growth analysis. neo r control (Ⅺ) or NS5A-expressing cells (q or E). 1 ϫ 10 4 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 CO 2 (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 Ϯ SD of triplicate dishes. B, colony formation efficiency assay. Cells of neo r control or NS5A-expressing cells (5 ϫ 10 2 ) 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.

TABLE I
Effect of NS5A expression on cell cycle NS5A-expressing cell lines in a tetracycline-regulated system (top) and a constitutive system (bottom) were analyzed by flow cytometry. The values represent the percentage of the total cell population in each phase of the cell cycle. Chang cells (NS5A-1 clone) were analyzed at two population densities (1, 80%; 2, 50%). The cell cycle analysis shown at the top was done using the CellQuest program by manual setting of regions M1, M2, and M3 for G 0 /G 1 , S, and G 2 /M, respectively, and the data shown at the bottom were obtained using curve-fitting analysis with the ModFit program. 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. It has been suggested that the increased expression of p21 Waf1/Cip1 may target the cyclin A-Cdk2 complex to attenuate mitosis (60). We sought to determine whether the significantly increased expression of p21 Waf1/Cip1 accompanied by the prolonged G 2 /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-p21 Waf1/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 p21 Waf1/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 p21 Waf1/Cip1 . We next sought to determine whether the interaction between p21 Waf1/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 p21 Waf1/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 G 2 3 M transition through a two-pronged mechanism: a p53-dependent p21 Waf1/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 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 G 2 /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 Total cellular RNAs (25 g) 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 32 P-labeled mouse p21 Waf1/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.
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 G 0 /G 1 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 G 2 arrest (48). In HeLa cells, Vpr caused growth retardation and G 2 arrest 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 g 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 g 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 g) from control neo r 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 g/ml) as substrate. The phosphorylated products were separated by SDS-PAGE (10%) and electrotransferred onto nitrocellulose membrane, followed by autoradiography.
FIG. 9. p21 Waf1/Cip1 is bound to Cdk2 in NS5A-expressing Chang liver cells and inhibits histone H1 kinase activity. A, total cell lysates (200 g of protein) from control neo r Chang liver cells or NS5A-expressing 5A-1 and 5A-3 cloned cells were immunoprecipitated with anti-p21 Waf1/ 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 g 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-l reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 10 mCi of [␥-32 P]ATP, and 50 g/ml histone H1 and incubated at 30°C for 30 min. The reaction was terminated by the addition of 6ϫ SDS-PAGE sample-loading buffer. Phosphorylated products were separated by SDS-PAGE (10%), electrotransferred to nitrocellulose membrane, and subjected to autoradiography. (49). Our results indicate that NS5A caused growth retardation and prolongation of G 2 /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 p21 Waf1/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 p21 Waf1/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 G 1 upon exposure to ionizing radiation due to inefficient induction of p21 Waf1/Cip1 (63). A higher number of mitotic cells in G 2 phase was also observed, and cells entered mitosis more rapidly in E6-expressing cells (63). p21 Waf1/Cip1 is induced in a p53-dependent pathway in response to a variety of DNA-damaging agents. The expression of p21 Waf1/Cip1 is also enhanced when cells undergo senescence or differentiation (64,65). In some mammalian cells, p21 Waf1/Cip1 predominantly causes G 2 arrest (35)(36)(37), suggesting that p21 Waf1/Cip1 is a negative regulator of the G 2 /M transition. In Xenopus egg extracts, it has been shown that Cdk2 functions as a positive activator of the cyclin B1-Cdk1 complex, whereas p21 Waf1/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 p21 Waf1/Cip1 in the premitotic G 2 /M phase either by inhibition of its kinase activity or activation of the Cdk-activating kinase. Induction of p21 Waf1/Cip1 expression in NS5A-expressing Chang liver cells may also target the cyclin A-Cdk2 complexes as shown by increased association of Cdk2 with p21 Waf1/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 NS5Aexpressing 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 p21 Waf1/Cip1 levels in HeLa cells, the levels of cyclin B1-Cdk1 kinase activity are sufficiently high due to incomplete inhibition of Cdk2 by p21 Waf1/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 p21 Waf1/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 p21 Waf1/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.