FUNCTION OF EBV EBNA-3C IN B CELL IMMORTALIZATION

描述（由研究者提供）：拟议研究的长期目标是确定EBV潜伏核蛋白EBNA-3C对EBV介导的B淋巴细胞永生化的重要贡献。EBV转化细胞的能力和随之而来的EBV诱导的增殖被认为是建立长期潜伏期的核心，因此对于任何EBV相关恶性肿瘤的发展至关重要，这些恶性肿瘤通常发生在感染后数年至数十年。为了确定EBNA-3C在这一过程中的作用，我们一直在研究与其相互作用的细胞途径。EBNA-3C最具特征的功能是它能够通过与J？和Spi蛋白。由于LMP-1在EBV介导的永生化和增殖中起关键作用，EBNA-3C调节LMP-1表达的能力可能在EBV生物学和发病机制中起重要作用。提出了三个具体目标。Aim 1下提出的实验将鉴定潜伏感染细胞中介导内源性LMP-1启动子激活的EBNA-3C结构域，在病毒蛋白全补体存在的情况下。这些实验将确定EBNA-2和EBNA-3C是否与细胞内的Spi蛋白结合或修改Spi的生化特性以影响其调节转录的能力。Aim 2提出的研究将使用NMR在体外研究这些蛋白的相互作用，确定参与结合的特定氨基酸，并解决EBNA-3C的功能域结构。这些研究将使我们能够解释该领域以前的工作，并更合理地产生在给定功能中被破坏的突变蛋白。这一信息不仅对未来实验的规划至关重要，而且可能为未来的研究提供刺激，以确定可能破坏这些相互作用并具有抗病毒价值的试剂。最后，介导EBNA-3C调控作用的两个转录因子Spi和J？，在许多细胞基因中都有结合位点，提示EBNA-3C可以调节细胞基因的表达。利用转录谱分析，我们已经确定了几个EBNA-3C应答基因，这些基因可能与EBV发病机制具有生物学相关性。在Aim 3中，我们将确认这些是EBNA-3C调节基因，并确定它们对潜伏感染的贡献。通过这些分析，我们不仅有可能深入了解EBV使细胞永生的机制，而且有可能利用这些知识开发抗病毒策略，有朝一日可能预防EBV感染和相关疾病。

DESCRIPTION (provided by investigator): 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.