FUCTIONS OF EBV PROTEINS IN B CELL IMMORTALIZATION

本研究的长期目标是通过以下方式来识别机制： EB病毒诱导B细胞增殖和永生化 细胞; EBV诱导的增殖可导致免疫抑制的淋巴瘤 个体 这些信息对于我们理解 EB病毒的发病机制和持久性，以及我们的理解， B细胞增殖的过程，是免疫调节的中心事件， 功能 EB病毒潜伏感染导致6种病毒核表达 蛋白（EBNAs）和3种病毒膜蛋白， 以及感染的B细胞的永生化。 我们建议确定 潜伏感染蛋白和细胞途径的功能 他们通过它行动。 具体而言，我们建议：1）确定一个 EBNA 2诱导的B细胞永生化的生化基础，2）阐明 通过表征相关细胞蛋白的EBNA功能和3） 建立个体EBV潜伏感染的生化效应 原代B细胞中的蛋白质。 我们的初步研究表明， 这些核蛋白之一EBNA 2的相互作用结合到 视网膜母细胞瘤蛋白Rb，并且EBNA 2含有 与SV 40大T抗原的Rb结合结构域同源。 我们建议 确定相互作用是否是通过这些序列介导的， 特异性肽来抑制相互作用，并通过检查 EBNA 2缺失突变体结合Rb。 EBNA 2序列在一些实施方案中不同。 残基被认为是不变的，因此这些结果 将提供关于这些残基的相对重要性的信息。 我们最近已经证明，其他细胞蛋白质结合到 EBNAs;这些细胞蛋白质可能不仅包括蛋白质 已经证明其与DNA肿瘤病毒转化有关 蛋白质，如Rb，但也有蛋白质参与了精致的 控制B细胞增殖以及参与其他免疫调节的蛋白质。 细胞过程如转录因子。 使用EBNA融合 蛋白质筛选cDNA表达文库，我们建议分离 编码与EBNA结合的细胞蛋白质的cDNA。 的作用 将通过转染病毒来研究单个病毒蛋白 基因进入原代B细胞;我们将检查激活标志物， 放射性标记的核苷酸掺入，以确定对 B细胞的活化和增殖。 这些研究将使我们能够 定义EBV潜伏蛋白的生化功能。

The long term objective of this research is to discern the mechanism by which Epstein-Barr virus induces proliferation and immortalization of B cells; EBV-induced proliferation can lead to lymphomas in immunosuppressed individuals. This information is essential to our understanding of the pathogenesis and persistence of EBV as well as our comprehension of the process of B cell proliferation, a highly regulated event central to immune function. Latent infection by EBV results in expression of 6 viral nuclear proteins (EBNAs) and 3 viral membrane proteins followed by proliferation and immortalization of the infected B cells. We propose to identify the functions of the latent infection proteins and the cellular pathways through which they act. Specifically, we propose to: 1) determine a biochemical basis of EBNA2-induced immortalization of B cells, 2) elucidate EBNA function by characterization of associated cellular proteins and 3) establish the biochemical effects of individual EBV latent infection proteins in primary B cells. Our preliminary studies have indicated that interaction of one of these nuclear proteins, EBNA2, binds to the retinoblastoma protein, Rb, and that EBNA2 contains sequences which are homologous to the Rb-binding domain of SV40 large T antigen. We propose to determine whether the interaction is mediated via these sequences by using specific peptides to inhibit the interaction and by examining the ability of EBNA2 deletion mutants to bind Rb. The EBNA2 sequence differs in some residues which are believed to be invariant and therefore these results will provide information about the relative importance of these residues. We have recently demonstrated that other cellular proteins bind to the EBNAs; these cellular proteins are likely to include not only proteins which have been demonstrated to associate with DNA tumor virus transforming proteins, such as Rb, but also proteins which are involved in the exquisite control of B cell proliferation as well as proteins involved in other cellular processes such as transcription factors. Using EBNA fusion proteins to screen a cDNA expression library, we propose to isolate the cDNAs encoding cellular proteins which bind to the EBNAs. The role of individual viral proteins will be investigated by transfection of viral genes into primary B cells; we will examine activation markers and radiolabelled nucleotide incorporation to determine the effects on activation and proliferation of B cells. These studies will allow us to define the biochemical function of the EBV latent proteins.