Identification of cellular protein interactions with the EBNA1 protein of Epstein-Barr virus and characterization of the interaction with USP7.
Published: 2004
Language: eng
Number of pages: 210
Description: Epstein-Barr virus is a gamma herpesvirus that infects human B lymphocytes. The viral protein essential for maintaining latent infection within the host is EBNA1, (Epstein-Barr Nuclear Antigen 1). To better understand how EBNA1 mediates its many functions during viral latency, we set out to identify human cellular proteins that interact with EBNA1 and contribute to its various roles within the cell. By coupling EBNA1 affinity chromatography and EBNA1 TAP-tagging with identification of bound proteins by mass spectrometry, previously unknown interactions of EBNA1 with NAP1, TAF-Ibeta, casein kinase II, PRMT5, and USP7 were observed. The significance of the EBNA1 interaction with USP7, a protein formerly found to associate with the Herpes Simplex protein, ICP0, was further investigated.The USP7 protein was purified, and used to characterize the protein  in vitro. The structural organization of USP7 was determined and showed that USP7 is comprised of four structural domains; an N-terminal domain known to bind p53, a catalytic domain, and two C-terminal domains. We showed that the p53 binding domain was also responsible for the EBNA1 interaction, while the ICP0 binding domain mapped to a C-terminal domain between amino acids 599--801. Binding assays indicated that EBNA1 peptide 395--450 bound with 10 fold higher affinity to USP7 than did p53 peptides, suggesting EBNA1 may sequester USP7 in order to moderate p53 levels. EBNA1 was also found to efficiently compete for USP7 from p53 in gel filtration analyses.The region of EBNA1 mediating the interaction with USP7 was mapped. An EBNA1 mutant (Delta395--450) lacking this region was used to investigate the possible role of USP7 interaction on EBNA1 functions. Deletion of the USP7 binding residues from EBNA1 increased the efficiency with which EBNA1 initiated DNA replication, and slightly decreased the transcriptional activation function of EBNA1.Preliminary data suggests that removal of amino acids 395--450 from EBNA1 results in a deregulation of p53 levels, as cells expressing Delta395--450 demonstrate higher steady state levels of p53 as compared to cells expressing wild-type EBNA1. Current evidence also suggests that cells expressing Delta395--450 exhibit a decreased capacity to avoid apoptosis, as compared to cells expressing wild-type EBNA1.
Notes: Adviser:  Lori D. Frappier.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-10, Section: B, page: 4990.