Title: The Nucleocapsid Protein of Severe Acute Respiratory Syndrome-Coronavirus Inhibits the Activity of Cyclin-Cyclin-dependent Kinase Complex and Blocks S Phase Progression in Mammalian Cells
Abstract: Deregulation of the cell cycle is a common strategy employed by many DNA and RNA viruses to trap and exploit the host cell machinery toward their own benefit. In many coronaviruses, the nucleocapsid protein (N protein) has been shown to inhibit cell cycle progression although the mechanism behind this is poorly understood. The N protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) bears signature motifs for binding to cyclin and phosphorylation by cyclin-dependent kinase (CDK) and has recently been reported by us to get phosphorylated by the cyclin-CDK complex (Surjit, M., Kumar, R., Mishra, R. N., Reddy, M. K., Chow, V. T., and Lal, S. K. (2005) J. Virol. 79, 11476–11486). In the present study, we prove that the N protein of SARS-CoV can inhibit S phase progression in mammalian cell lines. N protein expression was found to directly inhibit the activity of the cyclin-CDK complex, resulting in hypophosphorylation of retinoblastoma protein with a concomitant down-regulation in E2F1-mediated transactivation. Coexpression of E2F1 under such conditions could restore the expression of S phase genes. Analysis of RXL and CDK phosphorylation mutant N protein identified the mechanism of inhibition of CDK4 and CDK2 activity to be different. Whereas N protein could directly bind to cyclin D and inhibit the activity of CDK4-cyclin D complex; inhibition of CDK2 activity appeared to be achieved in two different ways: indirectly by down-regulation of protein levels of CDK2, cyclin E, and cyclin A and by direct binding of N protein to CDK2-cyclin complex. Down-regulation of E2F1 targets was also observed in SARS-CoV-infected VeroE6 cells. These data suggest that the S phase inhibitory activity of the N protein may have major significance during viral pathogenesis. Deregulation of the cell cycle is a common strategy employed by many DNA and RNA viruses to trap and exploit the host cell machinery toward their own benefit. In many coronaviruses, the nucleocapsid protein (N protein) has been shown to inhibit cell cycle progression although the mechanism behind this is poorly understood. The N protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) bears signature motifs for binding to cyclin and phosphorylation by cyclin-dependent kinase (CDK) and has recently been reported by us to get phosphorylated by the cyclin-CDK complex (Surjit, M., Kumar, R., Mishra, R. N., Reddy, M. K., Chow, V. T., and Lal, S. K. (2005) J. Virol. 79, 11476–11486). In the present study, we prove that the N protein of SARS-CoV can inhibit S phase progression in mammalian cell lines. N protein expression was found to directly inhibit the activity of the cyclin-CDK complex, resulting in hypophosphorylation of retinoblastoma protein with a concomitant down-regulation in E2F1-mediated transactivation. Coexpression of E2F1 under such conditions could restore the expression of S phase genes. Analysis of RXL and CDK phosphorylation mutant N protein identified the mechanism of inhibition of CDK4 and CDK2 activity to be different. Whereas N protein could directly bind to cyclin D and inhibit the activity of CDK4-cyclin D complex; inhibition of CDK2 activity appeared to be achieved in two different ways: indirectly by down-regulation of protein levels of CDK2, cyclin E, and cyclin A and by direct binding of N protein to CDK2-cyclin complex. Down-regulation of E2F1 targets was also observed in SARS-CoV-infected VeroE6 cells. These data suggest that the S phase inhibitory activity of the N protein may have major significance during viral pathogenesis. Mitotic cells undergo repeated cycles of division in order to produce daughter cells, a process essential for the maintenance of tissue homeostasis. Different steps of a cell division event typically include the G1 phase (preparation for DNA synthesis), followed by the S phase (genome replication), the G2 phase (preparation for cell division), and the M phase (mitosis). One of the most critical phases during cell cycle progression is the S phase, since it involves precise duplication of the whole genome, which carries all genetic messages for the next generation. Any abnormality during the replication step or thereafter would be disastrous for the organism. Hence, cells employ multiple strategies to ensure accurate and error-free genome replication. First, cells synthesize adequate amounts of raw materials that would be utilized during the S phase; second, cells strategically employ multiple check points (in the form of inhibitory factors) to block the S phase progression should there be a hostile environment arising due to any intracellular or extracellular factors. The majority of the events during cell cycle progression are driven by enzymes called cyclin-dependent kinases (CDKs), 2The abbreviations used are: CDK, cyclin-dependent kinase; N protein, nucleocapsid protein; SARS, severe acute respiratory syndrome; CoV, coronavirus; BrdUrd, bromodeoxyuridine, Rb, retinoblastoma, FBS, fetal bovine serum; HA, hemagglutinin; FACS, fluorescence-activated cell sorting; CAT, chloramphenicol acetyltransferase; CKI, CDK inhibitor; ERK, extracellular signal-regulated kinase. which are dependent on a series of cyclins to remain catalytically active. Regulating the activity of these cyclin-CDK complexes constitutes the crux of cell cycle regulation, which remains under the scrutiny of inhibitors of CDK. On the other hand, this regulatory network has been an attractive target for pathogens to exploit the cellular machinery toward their benefit. For example, herpes simplex virus gene products ICP0 (1Hobbs II, W.E. DeLuca N.A. J. Virol. 1999; 73: 8245-8255Crossref PubMed Google Scholar, 2Lomonte P. Everett R.D. J. Virol. 1999; 73: 9456-9467Crossref PubMed Google Scholar), and ICP27 induce G1 cell cycle arrest and shut off host gene expression during infection (3Song B. Yeh K.C. Liu J. Knipe D.M. Virology. 2001; 290: 320-328Crossref PubMed Scopus (47) Google Scholar). Similarly, IE2 (4Wiebusch L. Hagemeier C. J. Virol. 1999; 73: 9274-9283Crossref PubMed Google Scholar) and UL69 (5Lu M. Shenk T. J. Virol. 1999; 73: 676-683Crossref PubMed Google Scholar) of cytomegalovirus, Zta of Epstein-Barr virus (6Cayrol C. Flemington E.K. EMBO J. 1996; 15: 2748-2759Crossref PubMed Scopus (160) Google Scholar), p28 of mouse hepatitis virus (7Chen C.J. Sugiyama K. Kubo H. Huang C. Makino S. J. Virol. 2004; 78: 10410-10419Crossref PubMed Scopus (61) Google Scholar), and K-bZIP of Kaposi sarcoma-associated herpes virus (8Izumiya Y. Lin S.F. Ellison T.J. Levy A.M. Mayeur G.L. Izumiya C. Kung H.J. J. Virol. 2003; 77: 9652-9661Crossref PubMed Scopus (55) Google Scholar) have been shown to arrest cell cycle progression at the G1 phase. It has been postulated that imposing a G1 block may help these pathogens in utilizing the cellular raw materials to replicate their own genome or provide them shelter for a longer duration to complete their life cycle and bud off. The mechanisms of virus-induced cell cycle arrest differ from one virus to the other. Both Epstein-Barr virus Zta and IE2 of cytomegalovirus bind to and stabilize p53 (3Song B. Yeh K.C. Liu J. Knipe D.M. Virology. 2001; 290: 320-328Crossref PubMed Scopus (47) Google Scholar, 9Flemington E.K. J. Virol. 2001; 75: 4475-4481Crossref PubMed Scopus (141) Google Scholar, 10Zhang Q. Gutsch D. Kenney S. Mol. Cell. Biol. 1994; 14: 1929-1938Crossref PubMed Scopus (53) Google Scholar), which in turn up-regulates the expression of p21, leading to the inhibition of CDK2 and CDK4 activity, whereas the K-bZIP gene product of Kaposi sarcoma-associated herpes virus directly associates with and inhibits the activity of the cyclin-CDK2 complex (8Izumiya Y. Lin S.F. Ellison T.J. Levy A.M. Mayeur G.L. Izumiya C. Kung H.J. J. Virol. 2003; 77: 9652-9661Crossref PubMed Scopus (55) Google Scholar). Among the coronaviruses, the p28 protein of mouse hepatitis virus has been shown to accumulate hypophosphorylated retinoblastoma (Rb), stabilize p53, and up-regulate the levels of p21, thus inducing cell cycle arrest at the G1 phase (7Chen C.J. Sugiyama K. Kubo H. Huang C. Makino S. J. Virol. 2004; 78: 10410-10419Crossref PubMed Scopus (61) Google Scholar). The virus responsible for severe acute respiratory syndrome (SARS) is a recently discovered coronavirus, which shares significant homology with the mouse hepatitis virus. However, no information exists regarding the involvement of this virus in host cell cycle modulation. We have recently shown that the nucleocapsid protein (N protein) of the SARS-CoV localizes to the nucleus as well as cytoplasm and bears signature motifs for binding to the cyclin box and phosphorylation by the CDK, and we have experimentally demonstrated it to be a substrate of the cyclin-CDK complex (11Surjit M. Kumar R. Mishra R.N. Reddy M.K. Chow V.T. Lal S.K. J. Virol. 2005; 79: 11476-11486Crossref PubMed Scopus (129) Google Scholar). However, its involvement in modulation of the host cell cycle remains unknown. In this report, we provide substantial evidence that the N protein of the SARS-CoV binds to and inhibits the activity of the cyclin-CDK complex, resulting in down-regulation of the S phase gene products and subsequent inhibition of S phase progression. Mutational analysis identified the mechanism of inhibition to be different for G1 and late G1/S phase cyclins. In addition, expression of the S phase gene products were found to be down-regulated in SARS-CoV-infected cells, further supporting the above data. The possible significance of this phenomenon during the natural course of SARS-CoV infection is discussed. Plasmids and Reagents—pCDNA3.1N has been described earlier (12Surjit M. Liu B. Jameel S. Chow V.T. Lal S.K. Biochem. J. 2004; 383: 13-28Crossref PubMed Scopus (131) Google Scholar). RXL and RGNSPAR mutants (denoted as C and K, respectively) were constructed on pCR-XL-TOPO-N backbone by site-directed mutagenesis at Bangalore Genei Corp. (Bangalore, India). Mutants were confirmed by sequencing the entire gene. C and K mutants were cloned into pCDNA3.1 Myc vector at BamHI and ApaI restriction sites. The CK dual mutant was created by cloning the HindIII fragment from pCR-XL-TOPO-K into the HindIII restriction site of the pCR-XL-TOPO-C vector construct. The orientation of this insert was checked by restriction enzyme mapping. The pCDNA3.1-CK dual mutant was created by cloning a BamHI-ApaI fragment from pCR-XL-CK into BamHI and ApaI-digested pCDNA3.1 vector backbone. Final clone was again verified by sequencing. pRC/cytomegalovirus cyclin D1-HA plasmid that expresses HA-tagged cyclin D1 protein was a gift from Dr. Mark Ewen (13Lamb J. Ramaswamy S. Ford H.L. Contreras B. Martinez R.V. Kittrell F.S. Zahnow C.A. Patterson N. Golub T.R. Ewen M.E. Cell. 2003; 114: 323-334Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). pHisTrx-cyclin A plasmid and the pMM vector bearing coding sequences for CDK2 and CIV1 was a gift from Dr. Anindya Dutta (14Wohlschlegel J.A. Dwyer B.T. Takeda D.Y. Dutta A. Mol. Cell. Biol. 2001; 21: 4868-4874Crossref PubMed Scopus (67) Google Scholar). Wild-type and mutant cyclin E reporter constructs were obtained from Dr. J. R. Nevins (15Ohtani K. DeGregori J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12146-12150Crossref PubMed Scopus (535) Google Scholar). pSGI-E2F1 plasmid was a gift from Dr. Vijay Kumar (International Centre for Genetic Engineering and Biotechnology, New Delhi, India). Histone H1 and acetyl-CoA were purchased from Calbiochem. All antibodies and glutathione S-transferase Rb protein were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rabbit polyclonal anti-N antibody was used to detect N protein expression. [14C]chloramphenicol, [γ-32P]ATP, [35S]cysteine/methionine promix was obtained from PerkinElmer Life Sciences. The rabbit reticulocyte coupled transcription-translation (TNT) kit was obtained from Promega Corp. (Madison, WI). Bromodeoxyuridine and other biochemicals were obtained from Sigma. Cell Culture and Transfection—COS-7 and Huh7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with penicillin, streptomycin, and 10% fetal bovine serum. Cells were transfected with Lipofectamine or Fugene 6 reagent (Invitrogen or Roche Applied Science, respectively) as per the manufacturer's instructions. Mock-transfected cells were transfected with the empty vector. For synchronization experiments, 24 h postseeding, cells were starved for 34 h in serum-free medium followed by stimulation with 10% serum-containing medium for the indicated time periods. Fluorescence-activated Cell Sorting (FACS)—FACS analysis of cell cycle progression was done as described by Krishan (16Krishan A. J. Cell Biol. 1975; 66: 188-193Crossref PubMed Scopus (1499) Google Scholar) using the propidium iodide staining method. Metabolic Labeling and Immunoprecipitation—Forty hours post-transfection, cells were starved for 1 h in cysteine/methionine-deficient medium and then labeled with 100 μCi of [35S]Cys/Met promix for 4 h. After labeling, cells were washed once in phosphate-buffered saline and lysed in immunoprecipitation buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm β-glycerolphosphate, 1 mm Na3VO4) with protease inhibitor mixture. For immunoprecipitation, equal amounts of protein were incubated overnight with 1 μg of the corresponding antibody. This was followed by a 1-h incubation with 100 μl of 10% protein A-Sepharose suspension. The beads were washed four times with lysis buffer, and the protein was eluted by boiling the samples in 2× SDS dye. Proteins were then resolved by SDS-PAGE. Data obtained are representative of three independent sets of experiments conducted. Results were quantified, and normalized values were calculated using the NIH Image version 1.32 program. The graphs represent ± S.E. of three independent sets of experiments. Nuclear extracts were prepared as described earlier (11Surjit M. Kumar R. Mishra R.N. Reddy M.K. Chow V.T. Lal S.K. J. Virol. 2005; 79: 11476-11486Crossref PubMed Scopus (129) Google Scholar). Protein Expression by Coupled in Vitro Transcription-Translation—DNA was isolated using a miniscale DNA isolation kit (Qiagen Corp.). Transcription of the plasmid DNA was initiated from the T7 promoter, and the resultant transcript was translated by the translation machinery present in the rabbit reticulocyte lysate. The reaction was conducted using a commercially available TNT kit (Promega Corp.), following the manufacturer's protocol. The resultant protein was stored at –20 °C. An aliquot of the lysate was mixed with equivalent amounts of 2× SDS-loading dye and boiled for 5 min, and protein bands were visualized by SDS-PAGE followed by staining in Coomassie Brilliant Blue or by autoradiography. In Vitro Phosphorylation Assay—Immunoprecipitated cell lysates were washed twice with kinase buffer (25 mm Tris, pH 7.5, 5 mm β-glycerolphosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, 10 mm MgCl2) and then incubated with the indicated substrate along with 100 μm ATP and 10 μCi of [γ-32P]ATP for 45 min at 30 °C. Samples were subsequently boiled for 5 min in 10 μl of 4× SDS dye. Protein bands were resolved on 12% SDS-PAGE and detected by autoradiography. Chloramphenicol Acetyltransferase (CAT) Assay—Cells cultured in 60-mm dishes were transfected with respective plasmids. The total amount of transfected DNA was kept equal for each sample by adjusting the amount with respective empty vectors. 34 h poststarvation, cells were stimulated with 10% fetal bovine serum (FBS) for the indicated time period and harvested in phosphate-buffered saline. The CAT assay was done as described by Kalra et al. (16Krishan A. J. Cell Biol. 1975; 66: 188-193Crossref PubMed Scopus (1499) Google Scholar). Cell Lysate Preparation and Immunoblotting—Cells were washed once in phosphate-buffered saline and harvested in 1× SDS dye followed by vigorous vortexing and incubation in a boiling water bath for 5 min. Protein amount was equalized using the Bio-Rad protein assay kit. Samples were resolved by SDS-PAGE, followed by electrotransfer into nitrocellulose membrane (Amersham Biosciences) and incubated with respective antibodies. Protein bands were developed by the enhanced chemiluminiscence method using a commercially available kit (Cell Signaling Technology). Virus Infection and Lysate Preparation—Vero E6 cells (ATCC number CRL-1586) were cultured in M199 medium (Invitrogen) supplemented with 10% fetal calf serum, 2.2 g/liter sodium bicarbonate, 10 mm HEPES, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate at 37 °C under 5% CO2 in a humidified incubator. When cells reached ∼90% confluence, one batch of Vero E6 cells that served as the control was mock-infected with sterile medium (18Kramer M.F. Cook W.J. Roth F.P. Zhu J. Holman H. Knipe D.M. Coen D.M. J. Virol. 2003; 77: 9533-9541Crossref PubMed Scopus (56) Google Scholar, 19Munir S. Kapur V. J. Virol. 2003; 77: 4899-4910Crossref PubMed Scopus (21) Google Scholar). Another batch was infected with SARS-CoV (strain 2003VA2774 isolated from a SARS patient in Singapore) at a multiplicity of infection of 1 (20Ng M.L. Tan S.H. See E.E. Ooi E.E. Ling A.E. J. Gen. Virol. 2003; 84: 3291-3303Crossref PubMed Scopus (96) Google Scholar), with a virus inoculum volume of 0.75 ml diluted with 1.25 ml of maintenance medium with only 3% fetal calf serum. After adsorption for 1 h, the inoculum was removed, and 20 ml of maintenance medium was added. At 12 h postinfection, cytopathic effects were observed in infected cells. Following incubation at 37 °C for 12 h, both uninfected and infected Vero E6 cells were treated with 0.2 ml of 5% formalin (Merck) and then incubated at 4 °C for 24 h. The formalin-treated cells were further subjected to heat at 60 °C for 30 min and UV for 5 min to ensure complete inactivation. Finally, 2 ml of 10% Triton X-100 was added to both mock-infected and SARS-CoV infected Vero E6 cells and stored at –80 °C until further use. Lysate was prepared from these samples as described by Ikeda et al. (21Ikeda K. Monden T. Kanoh T. Tsujie M. Izawa H. Haba A. Ohnishi T. Sekimoto M. Tomita N. Shiozaki H. Monden M. J. Histochem. Cytochem. 1998; 46: 397-403Crossref PubMed Scopus (191) Google Scholar) with minor modifications. Briefly 200 μl of sample was mixed with 50 μl of 5× sample buffer (1× sample buffer: 1 m sodium dihydrogen phosphate, 10 mm disodium hydrogen phosphate, 154 mm sodium chloride, 1% Triton X-100, 12 mm sodium deoxycholate, 0.2% sodium azide, 0.95 mm fluoride, 2 mm phenylmethylsulfonyl fluoride, 50 mg/ml aprotinin, 50 mm leupeptin, 2% SDS, pH 7.6), and the contents were incubated under different conditions as follows: at 0 °C for 2 h, at 37 °C for 2 h, at 60 °C for 2 h, and at 100 °C for 20 min, followed by incubation at 60 °C for 2 h. After incubation, the tissue lysates were centrifuged at 15,000 × g for 20 min at 4 °C. The supernatants were equalized for protein content, boiled for 5 min in 6× SDS dye, and loaded onto SDS-polyacrylamide gel, followed by Western immunoblotting. Bacterial Expression and Purification of Cyclin-CDK Complex—Bacterial expressed cyclin-CDK complex was reconstituted as described earlier by Wohlschlegel et al. (14Wohlschlegel J.A. Dwyer B.T. Takeda D.Y. Dutta A. Mol. Cell. Biol. 2001; 21: 4868-4874Crossref PubMed Scopus (67) Google Scholar). Bromodeoxyuridine (BrdUrd) Incorporation Assay—Cells seeded on the coverslip were transfected with respective pCDNA3.1N plasmid. 34 h post-transfection, cells were stimulated with 10% FBS for 14 h and further incubated for 1 h in the presence of 10 mm BrdUrd. The medium was then aspirated, and cells were fixed with ice-cold fix (70 ml of ethanol plus 30 ml of glycine, pH 2.0) for 30 min at –20 °C. The cells were then washed in phosphate-buffered saline, and an immunofluorescence assay was conducted as described earlier (12Surjit M. Liu B. Jameel S. Chow V.T. Lal S.K. Biochem. J. 2004; 383: 13-28Crossref PubMed Scopus (131) Google Scholar). BrdUrd staining was observed using anti-BrdUrd antibody (1:100 dilution), and N protein expression was checked using anti-Myc antibody (1:100 dilution). Nucleus was stained with 4′,6-diamidino-2-phenylindole. BrdUrd-positive cells were counted by using a Nikon TE 2000U immunofluorescence microscope. Exogenous Expression of the N Protein Inhibits the Activity of Cyclin-CDK Complex—Earlier experiments done in our laboratory have demonstrated that the N protein bears the structural motif necessary for cyclin binding and phosphorylation by cyclin-dependent kinases. Accordingly, the N protein was found to be a substrate of the cyclin-CDK complex (11Surjit M. Kumar R. Mishra R.N. Reddy M.K. Chow V.T. Lal S.K. J. Virol. 2005; 79: 11476-11486Crossref PubMed Scopus (129) Google Scholar). We thus asked whether the possession of signature motifs and phosphorylation by the CDK is a strategy employed by the N protein to manipulate the host cell cycle machinery toward its benefit by acting as a competitive inhibitor to in vivo substrates of the cyclin-CDK complex. Hence, experiments were designed to check the effect of N protein expression on the kinase activity of different cyclin-CDK complex. Initially, control experiments were done to check the temporal profile of different phases of cell cycle in COS7 (African green monkey kidney) and Huh7 (human hepatoma) cells. For this, cells were maintained in the absence of growth factors for different time periods to arrest majority of the cell population at the G0 phase and then stimulated with 10% FBS to allow reentry into the cell cycle. Poststimulation, cells were harvested at every 3-h interval up to 30 h and the total cell lysate was immunoblotted to check the expression profile of different factors like cyclin D, cyclin E, cyclin A, cyclin B, p27, P-p27, CDK2, and CDK1. Also, aliquots of the lysate were used for FACS analysis to check percentage distribution of different phases of cell cycle and for in vitro phosphorylation assay to check the activity profile of cyclin D, cyclin E, cyclin A, and cyclin B, whose activities start at early G1,G1/S, S, and M phase, respectively. Approximately 30–48-h starvation (without FBS) was sufficient for arresting the majority of the Huh7 cells at the G0 phase, since no cyclin E, cyclin A, or cyclin B activity was observed. However, some level of cyclin D activity was observed during starvation too. In COS7 cells, all cyclins demonstrated basal activity at the same time period, which may be attributed to the presence of large T antigen in those cells. After the addition of 10% FBS, activity of G1 and S phase cyclins peaked at 6–9 and 12–15 h, respectively (data not shown). These experiments gave us a rough idea regarding the cell cycle profile of COS7 and Huh7 cells, and the same experimental condition was followed in all of the subsequent experiments. Based on the above observations, Huh7 cells were transfected with empty vector only (mock) or with pCDNA3.1N (N). Post-transfection, cells were starved for 34 h and then stimulated for different time periods in order to harvest different cyclin-CDK complexes during the peak of their activity. An in vitro phosphorylation assay using histone H1 as a substrate was used to check the activity of the holoenzyme. First we tested the activity of CDK4-cyclin D complex. Its activity was found to be significantly inhibited in N protein-expressing cells (Fig. 1A, first and second panels) at all three time points chosen. To ensure that an equal amount of protein was used in each reaction, aliquots of the lysate were immunoblotted with CDK4 antibody (third panel). The graph represents a quantitative estimation of the normalized band intensities with reference to loading control. As a control, a parallel experiment was conducted to check the CDK4 activity in enhanced green fluorescent protein-expressing cells. There was no difference in CDK4 activity of enhanced green fluorescent protein-transfected cells in comparison with mock-transfected cells (data not shown), thus ruling out the possibility that the observed CDK4 inhibitory activity of the N protein is an artifact of the transient transfection method. Similarly, we checked the activity of CDK6. The inhibitory effect of the N protein on CDK6 kinase activity appeared to be less intense as compared with that of CDK4 (fourth panel). The fifth panel shows the protein level of CDK6 as a loading control. Aliquots of the lysate were immunoblotted with anti-Myc (9E10) antibody to confirm the expression of N protein (sixth panel). The bottom panel shows schematics of cell cycle distribution at the respective time points as judged by subjecting control cells to fluorescence-assisted cell sorting analysis. A similar set of experiment was performed using COS7 cells, which showed similar results (data not shown).FIGURE 1N protein expression down-regulates cyclin-CDK activity. A, Huh7 cells transfected with pCDN3.1 (M) or pCDNA3.1N (N) plasmid were starved for 34 h followed by stimulation with 10% bovine serum for the indicated time periods. Aliquots of the lysate were immunoprecipitated with CDK4 (first panel), cyclin D (second panel), and CDK6 (fourth panel) antibody and used for in vitro phosphorylation assay (IVP). PS, poststimulation. A fraction of the total cell lysate was immunoblotted (WB) with CDK4 (third panel), CDK6 (fifth panel), or anti-Myc (sixth panel) antibody. The graph represents mean ± S.D. relative band intensity from three independent experiments. In the graph, each set of bars represents the corresponding lane in the gel above. Numbers 1–6 represent mock- and pCDNA3.1N-transfected sample at the 3, 6, and 9 h time point, respectively. Dark gray, light gray, and black bars represent CDK4, cyclin D, and CDK6 band intensity, respectively. The seventh panel represents FACS analysis of cell cycle status at 3, 6, and 9 h after the stimulation period. Numbers represent the percentage of cells in that particular phase. B, cells maintained and harvested as described in A were immunoprecipitated with cyclin D antibody, and aliquots of the lysate were immunoblotted with CDK4 (second panel) or p27 (third panel) antibody. The p27 blot was stripped and reprobed with cyclin D antibody (first panel). A fraction of the total cell lysate was immunoblotted with total ERK antibody to check equal loading (fourth panel). C, cells maintained as described above were harvested at the indicated time periods, and in vitro phosphorylation was done using CDK2 (first panel), cyclin E (second panel), and cyclin A (third panel) antibody. Aliquots of the total cell lysate were immunoblotted with CDK4 (fourth panel) or anti-Myc (sixth panel) antibody. In the graph, dark gray, light gray, and black bars represent CDK2, cyclin E, and cyclin A activity, respectively. Numbers 1–6 represent mock- and pCDNA3.1N-transfected sample at the 9, 12, and 15 h time point, respectively. The bottom panel represents FACS analysis of cell cycle status at 9, 12, and 15 h after the stimulation period. Numbers represent percentage of cells in that particular phase. D, cells maintained and harvested as described in C were immunoprecipitated with CDK2 antibody, and aliquots of the lysate were immunoblotted with cyclin E (first panel) and cyclin A (second panel) antibody. Cyclin A and cyclin E blot were stripped and reprobed with CDK2 (third panel) and total p38 (fourth panel) antibody, respectively. In the graph, the dark gray, light gray, and black bars represent cyclin E, cyclin A, and CDK2 protein level, respectively. Numbers 1–6 represent mock- and pCDNA3.1N-transfected sample at the 9, 12, and 15 h time point, respectively. E, Huh7 cells were transfected with the indicated amounts of respective plasmids and harvested 4 h (lanes 1–3) or 12 h (lanes 4–6) poststimulation with 10% bovine serum. An in vitro phosphorylation assay was done from an equal amount of lysate using CDK4 (lanes 1–3) or CDK2 (lanes 4–6) antibody. F, CDK4 in vitro phosphorylation as described in A using Rb as a substrate (first panel). Aliquots of the total cell lysate were immunoblotted with CDK4 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since down-regulation of the CDK4-cyclin D activity may be a result of decreased association of CDK4 with cyclin D or p27 with the CDK4-cyclin D complex, we checked these associations in N protein-expressing cells. As shown in Fig. 1B, immunoprecipitation of mock- or N protein-expressing cell lysate with cyclin D antibody and immunoblotting with CDK4 or p27 antibody revealed that there was no interference in the assembly of cyclin D-CDK4-p27 complex in N protein-expressing cells as compared with mock-transfected cells. Aliquots of total cell lysate were immunoblotted with total ERK antibody to ensure that equal amounts of lysate were used for each immunoprecipitation reaction (Fig. 1B, bottom panel). Having observed that N protein expression could down-regulate CDK4 activity without destabilizing the formation of the CDK4-cyclin D complex, we next investigated whether N protein expression inhibited CDK2 activity as well. An in vitro phosphorylation assay using CDK2, cyclin E, and cyclin A antibodies clearly indicated that the N protein could effectively inhibit CDK2 activity as well (Fig. 1C, first, second, and third panels and accompanying graph). The fourth panel shows an immunoblot of the total CDK4 protein, used as a loading control for this set of experiments. Aliquots of the lysate were also immunoblotted with anti-Myc antibody to check the expression of N protein (fifth panel). The bottom panel shows schematics of cell cycle distribution at the respective time p