A Cyclin-Binding Motif within the Amino-Terminal Homology Domain of EBNA3C Binds Cyclin A and Modulates Cyclin A-Dependent Kinase Activity in Epstein-Barr Virus-Infected Cells

Abstract

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In this report we demonstrate that although the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C. Detailed mapping studies employing both in vitro binding assays and coimmunoprecipitation experiments implicated a small region of EBNA3C, amino acids 130 to 159 within the EBNA3 homology domain, as having the greatest affinity for cyclin A. The EBNA3 homology domain has the highest degree of amino acid similarity (approximately 30%) between the EBNA3 proteins, and, indeed, EBNA3B, but not EBNA3A, showed binding activity with cyclin A. We also show that EBNA3C binds to the ␣1 helix of the highly conserved mammalian cyclin box, with cyclin A amino acids 206 to 226 required for strong binding to EBNA3C amino acids 130 to 159. Interestingly, EBNA3C also bound human cyclins D1 and E in vitro, although the affinity was approximately 30% of that seen for cyclin A. Previously it was demonstrated that full-length EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity (J. S. Knight and E. S. Robertson, J. Virol. 78:1981-1991, 2004). It was also demonstrated that the carboxy terminus of EBNA3C recapitulates this phenotype. Surprisingly, the amino terminus of EBNA3C with the highest affinity for cyclin A was unable to rescue p27 suppression of kinase activity and actually downregulates cyclin A activity when introduced into EBV-infected cells. The data presented here suggests that the amino terminus of EBNA3C may play an important role in recruiting cyclin A complexes, while the carboxy terminus of EBNA3C is necessary for the functional modulation of cyclin A complex kinase activity.

Epstein-Barr Virus Nuclear Antigen 3C Interacts with and Enhances the Stability of the c-Myc Oncoprotein

“…Previous studies have demonstrated the ability of EBNA3C to target cell cycle regulators, resulting in the deregulation of the mammalian cell cycle (21)(22)(23)(24)32). EBNA3C targets the key cell cycle regulators p27 and pRb by recruiting components of the SCF Skp2 ubiquitin ligase and facilitating the degradation of these inhibitors of the mammalian cell cycle (21,23).…”

“…Using pulse-chase experiments, we show that EBNA3C can stabilize c-Myc. This domain of EBNA3C also recruits and modulates the activity of pRb and p27, both major regulators of the mammalian cell cycle (21)(22)(23)(24). The inclusion of c-Myc in the group of proteins modulated by this region further demonstrates the importance of this region of EBNA3C in regulating the cell cycle in EBV infections.…”

“…More recently, it was demonstrated that EBNA3C can target the SCF Skp2 complex, thereby regulating the activity and stability of cyclin A/cdk2 and pRb complexes (21)(22)(23)(24). We further explored the possible regulation of c-Myc, a critical cell cycle modulator and a known substrate of the SCF Skp2 complex (14,15,18,19).…”

SCFSkp2 Complex Targeted by Epstein-Barr Virus Essential Nuclear Antigen

“…Previous studies have demonstrated that EBNA3C targets cell cycle regulators, resulting in deregulation of the mammalian cell cycle (3,20,21,32,33). EBNA3C manipulates the Rb-E2F axis in reporter assays and in rat fibroblast transformation experiments (32).…”

Epstein-Barr virus nuclear antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16 INK4A and p14 ARF expression

“…Indeed, all mutations in EBNA3A or EBNA3C that inhibit association with RBPJ are null for LCL growth (22)(23)(24), whereas EBNA3A or EBNA3C mutations that affect binding to the adenovirus E1a C-terminal binding protein (CtBP) corepressor result in continuous, albeit slower, growth (22)(23)(24)(25)(26). EBNA3C or EBNA3A have many specific and potentially significant interactions with other transcription factors or modifiers, including PU.1, Spi-B, HDAC1, DP103, prothymosin-α, p300, Nm23-H1, SUMO1, and SUMO3, cyclin A, SCF SKP2 ubiquitin ligase, pRb, Chk2, Mdm2, and MRS18-2, and these interactions could be relevant to CDKN2A p16 INK4A or p14 ARF regulation and LCL growth (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). Furthermore, EBNA3C upregulates TCL1A and ITGA4, down-regulates JAG1 and NCALD RNAs, and cooperates with EBNA3A in repressing Bim, a proapoptotic Bcl-2 family protein (24,(39)(40)(41).…”