Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) interacts with Regulator of Chromosome Condensation (RCC1) dynamically throughout the cell cycle

Thibaut Deschamps (1) , Quentin Bazot (1) , Derek M Leske (2) , Ruth Macleod (2) , Dimitri Mompelat (1) , Lionel Tafforeau (3) , Vincent Lotteau (3) , Vincent Maréchal (4) , George S Baillie (2) , Henri Gruffat (1) , Joanna B Wilson (2) , Evelyne Manet (1)

1 Herpesvirus oncogènes – Oncogenic Herpesviruses [CIRI]
2 College of Medical, Veterinary and Life Sciences [Glasgow]
3 Biologie cellulaire des infections virales – Cell biology of viral infection [CIRI]
4 CIMI - Centre d'Immunologie et des Maladies Infectieuses

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a sequence-specific DNA binding protein which plays an essential role in viral episome replication and segregation, by recruiting the cellular complex of DNA replication onto the origin (oriP) and by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication are well documented, those involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here, we have identified Regulator of Chromosome Condensation 1 (RCC1) as a novel cellular partner for EBNA1. RCC1 is the major nuclear guanine nucleotide exchange factor (RanGEF) for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation, and nuclear envelope reassembly following mitosis. Using several approaches, we have demonstrated a direct interaction between these two proteins and found that the EBNA1 domains responsible for EBNA1 tethering to the mitotic chromosomes are also involved in the interaction with RCC1. The use of an EBNA1 peptide array confirmed the interaction of RCC1 with these regions and also the importance of the N-terminal region of RCC1 in this interaction. Finally, using confocal microscopy and FRET analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase, suggesting an essential role for the interaction during this phase, perhaps in tethering EBNA1 to mitotic chromosomes.

Mots clés: Protein Interaction Domains and Motifs; chromatin; Spindle Apparatus; Metaphase; Flouresence Resonance Energy Transfer; cell cycle proteins; Nuclear Proteins; amino acid motifs; HeLa Cells; Mitosis; human; Protein Array Analysis; HEK293 Cells; Guanine nucleotide Exchange Factor; Confocal; Protein Interaction Mapping; Epstein-Barr Virus Nuclear Antigens; Microscopy

Domaines: Virologie; Biologie moléculaire; Immunologie; Bactériologie

Thibaut Deschamps, Quentin Bazot, Derek M Leske, Ruth Macleod, Dimitri Mompelat, et al.. Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) interacts with Regulator of Chromosome Condensation (RCC1) dynamically throughout the cell cycle. Journal of General Virology, 2017, 98 (2), pp.251-265. ⟨10.1099/jgv.0.000681⟩. ⟨hal-01927928⟩