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

描述(申请人提供)：爱泼斯坦-巴尔病毒(EBV)是一种致癌的伽玛-疱疹病毒，与霍奇金病和无浆性鼻咽癌有关。在晚期艾滋病毒疾病或移植中，EBV感染尤其危险，在没有免疫监测的情况下，EBV编码的蛋白质会驱动细胞异常生长。EBV的主要癌基因潜伏膜蛋白1(LMP1)通过模仿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，LMP1有效地刺激核因子-kB(NF-kB)，核因子-kB是控制炎症、细胞生存和生长的转录因子。EB病毒转化的细胞依赖于组成性的核因子-kB的激活，并在核因子-kB被阻断后迅速发生凋亡。副作用阻碍了目前可用的核因子-kB抑制剂的临床应用，尽管LMP1选择性药物靶点的毒性可能会大大降低。因此，确定LMP1如何激活核因子-kB是很重要的。我已经进行了一次人类基因组范围的siRNA筛选，寻找LMP1典型的核因子-kB激活的细胞调节器。抑制或增强核因子-kB的LMP1活性的HIT已经通过二次筛选得到验证。这些筛选涉及LMP1功能中的许多蛋白质，包括以前没有与核因子-kB相关的新因子。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LMP1核因子-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，包括潜在的可用药酶和功能聚集的命中。二级筛查将根据它们是否影响LMP1的表达、亚细胞运输以及它们在LMP1/NF-kB途径中的功能位置，将它们进一步划分为功能组。LMP1特有的细胞因子，而不是免疫受体通路，可能成为治疗EBV驱动的恶性肿瘤的重要靶点。同样，这些研究可能揭示核因子-kB激活的重要一般机制，与过敏、自身免疫和宿主防御有关。 公共卫生相关性：持续的Epstein Barr病毒感染是某些淋巴瘤和喉癌的重要原因。这个项目将更好地定义EBV如何颠覆细胞结构，推动受感染细胞的癌细胞生长。最终，人们希望从这些研究中获得的知识可以使专门阻断EBV功能的化疗药物的开发成为可能。

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.