Epstein-Barr Virus Nuclear Antigen 3C Facilitates G1-S Transition by Stabilizing and Enhancing the Function of Cyclin D1

Abstract

EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130–160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1–50), and C-terminal domain (residues 171–240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.

EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

“…Aurora kinase B promoter-driven luciferase plasmids pGL3B-AK-B-FL (Ϫ1879), pGL3B-AK-B (Ϫ337), and pGL3B-AK-B (Ϫ74) were provided by Yukio Okano (Gifu University School of Medicine, Tsukasamachi, Japan). The lymphoblastoid cell lines (LCL1 and -2), BL41-B95.8, Mutu I, Mutu III, BJAB stably expressing EBNA3C clones (BJAB7 and BJAB10) and DG75, BJAB, and BL41 were cultured as described earlier (10). pEGFP-EBNA3C fulllength EBNA3C (residues 1 to 992), full-length EBNA3C-dsRed (residues 1 to 992), full-length Flag-tagged EBNA3C (residues 1 to 992, 1 to 365, 366 to 620, and 621 to 992), and glutathione S-transferase (GST)-tagged EBNA3C construct (residues 90 to 365, 366 to 581, 582 to 792, 900 to 992, 90 to 129, 130 to 159, and 160 to 190) in pGEX2TK vector have been previously described (6,17).…”

“…Biological Corp. (Swampscott, MA). Myc epitope (9E10), Flag epitope (M2), and EBNA3C (A10) were described earlier (29). Adherent cells and B cells were transfected as described earlier (30,31).…”

“…Short hairpin oligonucleotides for EBNA3C, control short hairpin RNA (shRNA), and p53 were described earlier (10,32). The sequence for the AK-B shRNA sense strand was 5=-CGAGACCTAT CGCCGCATC, and that for the antisense strand was GCTCTGGATAGC GGCGTAG-3= (33).…”

“…Expression of these latent transcripts results in upregulation of various cellular genes important for transitioning resting B cells into the cell cycle (5). One of these nuclear antigens, EBNA3C, has cell cycle regulatory functions (6)(7)(8), and earlier studies have shown that EBV affects expression of regulatory genes, in particular those for cyclin A, p27, cdc2, cyclin E, and cyclin D1, in infected B cells (7)(8)(9)(10).…”

NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, enhances the radiosensitivity of human glioma stem cells in vitro

“…Cyclin A is involved in meiotic progression and has markedly lower expression in cells arrested in the G 1 phase [33][34][35] . Cyclin D1 is a key regulator of the G 1 to S phase progression and is aberrantly expressed in numerous human cancers [36] . The inhibition of cyclin D1 function results in G 1 phase arrest, whereas the regulation of G 2 /M phases primarily depends on cyclin B1 function [37] .…”