Function of Ebv EBNA-3 in B Cell Immortalization

Sample, Clare

Abstract

The long-term objective of the proposed studies is to determine the essential contribution of the EBV latent nuclear protein EBNA-3C to EBV-mediated immortalization of B lymphocytes. The ability of EBV to transform cells and the ensuing EBV-induced proliferation is believed to be central to the establishment of long-term latency, and thus essential to the development of any of EBV-associated malignancy, which typically occur years to decades following infection. To identify the contribution of EBNA-3C to this process, we have been studying cellular pathways with which it interacts. The best-characterized function of EBNA-3C is its ability to regulate expression of the EBV LMP-1 oncoprotein, through interactions that we have identified with the J? and Spi proteins. Due to the critical role that LMP-1 plays in EBV-mediated immortalization and proliferation, the ability of EBNA-3C to regulate LMP-1 expression likely plays an important role in EBV biology and pathogenesis.
Three specific aims are proposed. Experiments proposed under Aim 1 will identify the domains of EBNA-3C that mediate activation of the endogenous LMP-1 promoter within latently infected cells, in the presence of the full complement of viral proteins. These experiments will determine whether EBNA-2 and EBNA-3C bind to Spi proteins within cells or modify biochemical properties of Spi to affect its ability to regulate transcription. Studies proposed under Aim 2 will use NMR to study the interaction of these proteins in vitro, to identify the specific amino acids that are involved in binding, and to solve the structure of functional domains of EBNA-3C. These studies will allow us to interpret previous work in the field, and more rationally generate mutant proteins disrupted in a given function. This information is not only crucial for the planning of future experiments but may also provide a stimulus for future studies to identify reagents that might disrupt these interactions and have value as anti-virals. Lastly, two transcription factors that mediate EBNA-3C's regulatory effects, Spi and J?, have binding sites in numerous cellular genes, suggesting that EBNA-3C will regulate cellular gene expression. Using transcriptional profiling, we have identified several EBNA-3C responsive genes that are likely to be biologically relevant to EBV pathogenesis.
In Aim 3, we will confirm that these are EBNA-3C regulated genes and define their contribution to latent infection. Through these analyses, we are likely to gain significant insight not only into the mechanism by which EBV immortalizes cells, but potentially use this knowledge to develop anti-viral strategies that might one day prevent EBV infection and associated diseases.