Epstein-Barr Nuclear Antigen 1 (EBNA1) is the only viral latent protein continuously expressed in all Epstein-Barr Virus (EBV)-associated tumors, including nasopharyngeal carcinoma (NPC), Hodgkin’s lymphoma and different B-cell and epithelial cell malignancies. EBNA1 maintains and replicates EBV’s genome by recruiting human cellular replication proteins and binding to the viral origin of replication, oriP. It is also important for EBV-infected cell survival. Its DNA-binding and dimerization domain (DBD, amino acid 459-607) is responsible for formation of EBNA1 homodimer and binding to the dyad symmetry (DS) element in oriP, facilitating DNA replication. However, EBNA1 mechanisms for EBV latent infection have not been clearly revealed. The aims of this thesis study were to ident...[ Read more ]

Epstein-Barr Nuclear Antigen 1 (EBNA1) is the only viral latent protein continuously expressed in all Epstein-Barr Virus (EBV)-associated tumors, including nasopharyngeal carcinoma (NPC), Hodgkin’s lymphoma and different B-cell and epithelial cell malignancies. EBNA1 maintains and replicates EBV’s genome by recruiting human cellular replication proteins and binding to the viral origin of replication, oriP. It is also important for EBV-infected cell survival. Its DNA-binding and dimerization domain (DBD, amino acid 459-607) is responsible for formation of EBNA1 homodimer and binding to the dyad symmetry (DS) element in oriP, facilitating DNA replication. However, EBNA1 mechanisms for EBV latent infection have not been clearly revealed. The aims of this thesis study were to identify possible EBNA1-DBD interacting genes and to investigate their functions and novel mechanisms contributing to EBV latent infection.

In biochemical studies, multiple EBNA1-DBD interacting proteins were identified by yeast two-hybrid screening. I showed that an E3 ubiquitin ligase RNF216 could interact with EBNA1 DNA-binding and dimerization domain by different in vitro and in vivo approaches. To further study this interaction, different fragments of RNF216 were designed to identify the binding region(s) or motif(s) involved. Results indicated that TRIAD domain of RNF216 was not involved in EBNA1-DBD binding, while positively charged KRKKRK motif on RNF216 was important for EBNA1-DBD binding. Furthermore, the full-length interaction was also confirmed in HEK293T cells.

To hypothesize the interacting model, we propose that RNF216 carrying KRKKRK motif could physically bind to EBNA1-DBD on the opposite site of DNA-binding region. The interaction was neither DNA- nor RNA-mediated.