Genetic and Proteomic Analysis of Epstein-Barr Virus LMP1 Activation of NF-kB

描述（由申请人提供）：eb病毒（EBV）是一种与霍奇金病和无浆性鼻咽癌相关的致癌γ疱疹病毒。EBV感染在晚期HIV疾病或移植中尤其危险，EBV编码的蛋白质在缺乏免疫监视的情况下驱动异常细胞生长。EBV的主要致癌基因潜伏膜蛋白1 （Latent Membrane Protein 1, LMP1）通过模拟激活的免疫受体来促进细胞存活和增殖。通过不完全确定的途径，LMP1有效刺激核因子κ B (NF-kB)，这是控制炎症、细胞存活和生长的转录因子。EBV转化的细胞依赖于组成型NF-kB激活，并在NF-kB阻断后迅速发生凋亡。尽管lmp1选择性药物靶点的毒性可能会大大降低，但目前可用的NF-kB抑制剂的副作用阻碍了其临床应用。因此，确定LMP1如何激活NF-kB是很重要的。我对LMP1典型NF-kB激活的细胞调节剂进行了人类全基因组siRNA筛选。抑制或增强NF-kB的LMP1激活已经通过二次筛选得到验证。筛选涉及LMP1功能中的许多蛋白质，包括以前未与NF-kB相关的新因子。我将进行基于假设和更大规模的二次分析，以确定上皮细胞和B淋巴细胞中LMP1 NF-kB激活的关键缺失成分。我将对几个特别感兴趣的目标进行详细的生化分析，包括潜在的可药物酶和功能聚集点。二级筛选将进一步根据它们是否影响LMP1表达、亚细胞运输以及它们在LMP1/NF-kB通路中的作用，将击中物分层为功能组。LMP1独特利用的细胞因子，而不是免疫受体途径，可能是治疗ebv驱动的恶性肿瘤的重要治疗靶点。同样，这些研究可能揭示NF-kB活化的重要一般机制，对过敏、自身免疫和宿主防御有影响。

DESCRIPTION (provided by applicant): The Epstein Barr Virus (EBV) is an oncogenic gamma-herpesvirus that is associated with Hodgkin disease and anaplasmic nasopharyngeal carcinoma. EBV infection is particularly hazardous with advanced HIV disease or transplant, where EBV-encoded proteins drive aberrant cell growth in the absence of immune surveillance. The principal EBV oncogene, Latent Membrane Protein 1 (LMP1), promotes cell survival and proliferation by mimicking activated immune receptors. Through incompletely defined pathways, LMP1 potently stimulates Nuclear Factor Kappa B (NF-kB), transcription factors that control inflammation, cell survival and growth. EBV- transformed cells rely on constitutive NF-kB activation, and rapidly undergo apoptosis upon NF-kB blockade. Side-effects preclude the clinical use of currently available NF-kB inhibitors, though LMP1-selective drug targets may afford substantially less toxicity. It is therefore important to define how LMP1 activates NF-kB. I have carried out a human genome-wide siRNA screen for cellular modulators of LMP1 canonical NF-kB activation. Hits that either suppress or enhance LMP1 activation of NF-kB have been validated with secondary screens. The screens have implicated numerous proteins in LMP1 function, including novel factors not previously associated with NF-kB. I will carry out hypothesis-based and larger-scale secondary assays to identify critical missing components of LMP1 NF-kB activation in both epithelial cells and B lymphoblasts. I will pursue detailed biochemical analysis of several targets of particular interest, including potentially druggable enzymes and functionally clustered hits. Secondary screens will further stratify hits into functional groups based on whether they affect LMP1 expression, subcellular trafficking, and where they function within the LMP1/NF-kB pathway. Cellular factors uniquely employed by LMP1, but not by immune receptor pathways, may serve as important therapeutic targets for treatment of EBV-driven malignancies. Likewise, these studies may reveal important general mechanisms of NF-kB activation, with implications for allergy, autoimmunity, and host-defense. PUBLIC HEALTH RELEVANCE: Persistent Epstein Barr virus infection is an important cause of certain lymphoma and throat cancers. This project will better define how EBV subverts cellular machinery to drive cancerous growth of infected cells. Ultimately, it is hoped that knowledge gained from these studies may enable the development of chemotherapies that specifically block EBV function.