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

描述（由申请方提供）：爱泼斯坦巴尔病毒（EBV）是一种致癌性γ-疱疹病毒，与霍奇金病和无浆性鼻咽癌相关。EBV感染对于晚期HIV疾病或移植特别危险，其中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。主要的EBV癌基因，潜伏膜蛋白1（LMP 1），通过模拟激活的免疫受体促进细胞存活和增殖。通过不完全确定的途径，LMP 1有效地刺激核因子κ B（NF-κ B），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性NF-kB活化，并且在NF-kB阻断后迅速经历细胞凋亡。副作用排除了目前可用的NF-κ B抑制剂的临床使用，尽管LMP 1选择性药物靶点可以提供实质上更少的毒性。因此，确定LMP 1如何激活NF-κ B是很重要的。我已经进行了人类全基因组siRNA筛选LMP 1典型NF-κ B激活的细胞调节剂。抑制或增强NF-κ B的LMP 1活化的命中已经用二次筛选验证。这些筛选涉及LMP 1功能中的许多蛋白质，包括以前与NF-kB无关的新因子。我将进行基于假设和更大规模的二次检测，以确定上皮细胞和B淋巴母细胞中LMP 1 NF-κ B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，包括潜在的药物酶和功能性聚集的命中。二次筛选将根据命中是否影响LMP 1表达、亚细胞运输以及它们在LMP 1/NF-kB通路中的功能进一步将命中分层为功能组。LMP 1独特使用的细胞因子，而不是免疫受体途径，可能作为治疗EBV驱动的恶性肿瘤的重要治疗靶点。同样地，这些研究可能揭示NF-κ B活化的重要一般机制，涉及过敏、自身免疫和宿主防御。 公共卫生相关性：持续性爱泼斯坦巴尔病毒感染是某些淋巴瘤和喉癌的重要原因。该项目将更好地定义EBV如何颠覆细胞机制以驱动受感染细胞的癌性生长。最终，希望从这些研究中获得的知识可以开发特异性阻断EBV功能的化疗药物。

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.