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 J. Virol. 1999; 73: PubMed and G1 cell cycle and host gene expression during J. PubMed J. Virol. 1999; 73: PubMed and M. J. Virol. 1999; 73: PubMed of of virus J. PubMed of virus K. S. J. Virol. PubMed and of herpes virus J. Virol. PubMed have been shown to cell cycle progression the G1 has been that a G1 block may these pathogens in the cellular raw materials to their own genome or for a to their cycle and The of cell cycle virus to the virus and of bind to and J. PubMed J. Virol. PubMed S. PubMed which in the expression of to the inhibition of CDK2 and CDK4 the gene of herpes virus directly with and the activity of the complex J. Virol. PubMed the coronaviruses, the protein of virus has been shown to retinoblastoma and the levels of cell cycle the G1 phase K. S. J. Virol. PubMed The virus for severe acute respiratory is a recently which with the the of this virus in host cell cycle have recently shown that the nucleocapsid protein (N protein) of the SARS-CoV to the and bears signature motifs for binding to the cyclin and phosphorylation by the CDK, and we have it to be a of the cyclin-CDK complex M. R. J. Virol. PubMed in of the host cell cycle remains In this we that the N protein of the SARS-CoV to and the activity of the cyclin-CDK complex, resulting in down-regulation of the S phase gene products and inhibition of S phase identified the mechanism of inhibition to be different for G1 and phase In expression of the S phase gene products found to be in SARS-CoV-infected cells, the The significance of this during the of SARS-CoV is and has been M. S. J. PubMed RXL and and on by by the and and The mutant was by the the of the The of this was by The mutant was by a and was by cyclin that cyclin protein was a J. S. PubMed A and the for CDK2 and was a PubMed and mutant cyclin J. R. K. J. S. PubMed was a for and and and protein A was used to N protein was The was and other and and cells in with and fetal bovine with or or the cells with the For cells for in followed by with for the of cell cycle progression was by J. PubMed the and cells for in and with of for cells in and in with For amounts of protein with of the was followed by a with of protein The with and the protein was by the in by are of of and the The of of M. R. J. Virol. PubMed by in was a DNA of the DNA was the and the was by the machinery present in the The was a the The protein was of the was with amounts of and for and protein by followed by in or by In cell with kinase and with the with and of for for in of on and by in with The of DNA was for by the with cells with fetal bovine for the and in The was by J. PubMed and in and in followed by and in a for was the protein by followed by and with by the a and cells in with fetal under in a cells of cells that the was with J. J. Virol. PubMed S. V. J. Virol. PubMed was with SARS-CoV a in a of of J. Virol. PubMed with a virus of with of maintenance with fetal for the was and of maintenance was observed in cells. for and cells with of and for The cells to for and for to ensure of was to and SARS-CoV cells and was these by K. M. M. M. J. PubMed with of was with of and the under different conditions for for for and for followed by for the tissue for The for protein for in and followed by and of cyclin-CDK complex was by PubMed on the with cells with for and for in the of The was and cells with of of for The cells in and an was M. S. J. PubMed was observed and N protein expression was was with cells by a of the N the of in have that the N protein bears the for cyclin binding and phosphorylation by cyclin-dependent the N protein was found to be a of the cyclin-CDK complex M. R. J. Virol. PubMed the of signature motifs and phosphorylation by the CDK is a strategy employed by the N protein to the host cell cycle machinery toward by a to in of the cyclin-CDK complex. Hence, to check the of N protein expression on the kinase activity of different cyclin-CDK complex. to check the of different phases of cell cycle in and cells. For cells in the of for different to majority of the cell the phase and with to the cell cells to and the cell was to check the expression of different cyclin cyclin E, cyclin cyclin CDK2, and of the used for to check of different phases of cell cycle and for in phosphorylation to check the activity of cyclin cyclin E, cy

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