Functional contribution and regulation of EBNA1 and EBP2 in Epstein-Barr virus plasmid segregation.
Published: 2005
Language: eng
Number of pages: 276
Description: Epstein-Barr virus persists indefinitely in human B lymphocytes due to its latent infection cycle. The viral genomes persist as DNA episomes that replicate once per cell cycle and efficiently segregate during host mitotic division. Episomal segregation requires interaction of the EBNA1 protein with the FR element on the episomes and is dependent on the association of the episomes with host mitotic chromosomes through EBNA1. In an EBV-based plasmid partitioning system in S. cerevisiae, segregation of a FR-containing plasmid by EBNA1 requires the EBNA1-interacting human cellular protein hEBP2. To gain a better understanding of the mechanism governing EBV segregation, hEBP2 was characterized, its functional role in EBNA 1-mediated segregation was determined and its regulation was explored. EBNA1 sequences and post-translational modifications involved in EBNA1-mediated segregation were also studied.The EBNA1 residues 325-376 were previously shown to be essential for its segregation activity. In this study, it was shown that EBNA1 N-terminal residues 8-67 also contribute to, but are not required for, plasmid segregation by EBNA1. Serine phosphorylation in the EBNA1 325-376 region was found to promote segregation by EBNA1 but was not required for this activity.Previous work and studies described here provided evidence that the requirements for EBNA1-mediated segregation are the same in S. cerevisiae and human cells. Using the yeast segregation system, it was demonstrated that hEBP2 binds mitotic chromosomes in a cell cycle regulated manner, as it does in human cells, and is required for the association of EBNA1 with mitotic chromosomes. hEBP2 regions that associate with the mitotic chromosomes and EBNA1, which were mapped to amino acids 95-220 and 220-306, respectively, are both required for plasmid partitioning by EBNA1. Using RNA silencing, it was shown that hEBP2 is important for EBV segregation in human cells. Down-regulation of hEBP2 decreased the association of EBNA1 and oriP plasmids with mitotic chromosomes. Factors affecting hEBP2 localization during mitosis were assessed and it was found that the interaction of hEBP2 with mitotic chromosomes is regulated by Aurora kinases in yeast and humans, implicating the involvement of these kinases in EBV persistence.
Notes: Source: Dissertation Abstracts International, Volume: 66-10, Section: B, page: 5240.

Thesis (Ph.D.)--University of Toronto, 2005.

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