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Functional analysis of the Epstein Barr virus nuclear antigen leader protein (EBNA-LP) in a viral context.

Robert White

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Medical Research Council (MRC)
Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is involved in 1% of cancer incidence worldwide. In vitro EBV transforms resting B cells into lymphocyte cell lines. EBNA-LP is one of the EBV nuclear antigens (EBNAs) important for this process. Exploration of its role in EBV biology has been neglected because of its complex repetitive nature and the technical challenge of making mutants of EBNA-LP in EBV. I propose to take a genetic approach to clarify the role of EBNA-LP in B cell transformation and identify the mechanisms central to its key biological properties. In vitro EBNA-LP is able to enhance the ability of EBNA2 to activate gene transcription. Sp100, NCoR and HDAC4 have variously been implicated as mediators of this effect. Using an EBNA-LP-knockout EBV (LPKO) and its revertant, we will identify the functions of EBNA-LP in B cell transformation. We will analyse the phenotype of LPKO-infected B cells, and undertake a microarray analysis of LPKO-infected BL31 cells to allow us to identify genes that are co-regulated with EBNA2 and with the EBNA3s, as well as those altered independently by EBNA-LP. In parallel we will map the interaction domains of 10 known EBNA-LP-binding partners. Having identified EBNA-LP mutants that lack defined interactions, we will complement LPKO EBV to establish which mutants restore which phenotypes. This will identify candidate mediators of those phenotypes, which can be tested by knockdown or overexpression. We will establish whether enhancement of EBNA2 function plays a significant role in gene regulation, by altering expression of Sp100, NCoR and HDAC4, and by analysing a recombinant EBV encoding S36 mutants of EBNA-LP. Finally we will use chromatin immunoprecipitation to reveal mechanisms of gene regulation by EBNA-LP and the proteins that mediate its effects. This program will integrate existing knowledge of EBNA-LP interactions with its biological role, providing mechanistic insights for future application.

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