REGULATION OF REPLICATION AND LATENCY BY EBV EBNAS

描述（由申请人提供）：EB病毒（EBV）是一种γ疱疹病毒，可引起传染性单核细胞增多症，并与多种人类淋巴和上皮癌相关。免疫功能低下是EBV相关疾病与恶性肿瘤的风险因素，例如移植患者中出现的移植后淋巴增生性疾病和中枢神经系统淋巴瘤以及AIDS患者中与EBV相关的一部分全身性淋巴瘤。EBV还与伯基特淋巴瘤、鼻咽癌、鼻T细胞淋巴瘤以及霍奇金淋巴瘤和胃癌的子集相关。在初次感染后，个体将保持潜伏感染EBV，并且潜伏感染细胞的这种终身储存库是随后恶性疾病发展的因素。 EBV感染培养物中的B细胞导致永生化B细胞系的生长，EBNA 2是EBV编码的蛋白质之一，对于该过程是必需的。EBNA 2通过靶向DNA结合蛋白CBF 1/RBPJk并将CBF 1抑制的启动子转换为激活状态来改变细胞基因表达。在CBF 1的靶向中，EBNA 2模拟活化的Notch信号传导。最近发现的存在于EBV BARTs中的ORF编码一种蛋白质RPMS，该蛋白质也与Notch途径相互作用，在这种情况下负调节Notch活性。本研究计划的目的是了解EBNA 2对EBV驱动的B细胞永生化的贡献以及RPMS在潜伏期免疫和EBV相关上皮恶性肿瘤中的作用。具体目标是：[1]通过产生Nur 77结合缺陷的EBV突变体并进一步表征EBNA 2-Nur 77相互作用及其后果来检查EBNA 2介导的细胞存活活性。[2]鉴定并比较EBNA 2和NotchIC的下游靶点。原代B细胞和EBV阴性B细胞系将用表达EBNA 2或NotchIC的慢病毒载体和用EBV EBNA 2突变体病毒感染，并且细胞基因表达中诱导的变化将使用基因阵列技术测定。[3]检查RPMS的功能和RPMS稳定性的规定。将检查磷酸化对RPMS蛋白周转和蛋白体靶向的贡献，并探讨RPMS抗Notch活性在上皮肿瘤发展中的潜在作用。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is a gamma herpesvirus that causes infectious mononucleosis and is associated with a variety of human lymphoid and epithelial cancers. Immunocompromise is a risk factor for EBV associated disease with malignancies such as post-transplant lymphoproliferative disease arising in transplant patients and central nervous system lymphoma and a proportion of systemic lymphomas being EBV associated in AIDS patients. EBV is also associated with Burkitt' s lymphoma, nasopharyngeal carcinoma, nasal T cell lymphoma, and a subset of Hodgkin' s lymphomas and gastric carcinomas. After primary infection an individual will remain latently infected with EBV and it is this life-long reservoir of latently infected cells that is a factor in the development of subsequent malignant disease. EBV infection of B cells in culture leads to the outgrowth of immortalized B cell lines and EBNA2 is one of the EBV encoded proteins essential for this process. EBNA2 alters cellular gene expression by targeting a DNA binding protein, CBF1/RBPJk, and switching CBF1 repressed promoters to an activated state. In its targeting of CBF1, EBNA2 mimics activated Notch signaling. A recently recognized ORF present in the EBV BARTs encodes a protein, RPMS, that also interacts with the Notch pathway, in this case negatively regulating Notch activity. The goal of this research program is to understand the contribution of EBNA2 to EBV driven B cell immortalization and the role of RPMS in latency maintainance and in EBV associated epithelial malignancies. The Specific Aims are: [1] To examine EBNA2 mediated cell survival activities by generating an EBV mutant defective for Nur77 binding and further characterizing the EBNA2-Nur77 interaction and its consequences. [2] To identify and compare the downstream targets of EBNA2 and NotchIC. Primary B cells and EBV negative B cell lines will be infected with lentivirus vectors expressing EBNA2 or NotchIC and with EBV EBNA2 mutant viruses and the changes induced in cell gene expression will determined using gene array technologies. [3] To examine RPMS function and the regulation of RPMS stability. The contribution of phosphorylation to RPMS protein turnover and proteosomal targeting will be examined and the potential role of RPMS anti-Notch activity in epithelial tumor development will be probed.