The Induction of Interferon Regulatory Factor 7 Sumoylation by EBV LMP1

描述(申请人提供)：EB病毒(EBV)是一种与多种癌症相关的致癌病毒，主要的EBV癌蛋白是潜伏膜蛋白(LMP)1，它是一种具有结构性活性的膜蛋白，模拟肿瘤坏死因子受体家族诱导的信号传导。我们已经报道，LMP1诱导干扰素调节因子(IRF)-7的表达，IRF-7是I型干扰素在先天性免疫反应中的关键调节因子。LMP1通过翻译后修饰，包括磷酸化和泛素化，调节IRF7的转录活性。因此，我们研究了LMP1是否诱导IRF7的其他修饰，特别是通过小泛素样蛋白修饰物(SUMO)-1，以调节其功能。我们发现LMP1可能通过未被充分研究的C末端活化区(CTAR3)与Ubc9相互作用，Ubc9是唯一已报道的相扑结合酶。LMP1还诱导了Ubc9与IRF7的相互作用，导致IRF7的总和甲基化，从而导致IRF7周转减少。Sumoylated IRF7聚集在细胞核中，其转录活性略有增加。因此，我们假设LMP1诱导IRF7的相加作用缓和了免疫反应的激活，我们提出了两个目的来检验这一假设。首先，我们将确定IRF7总甲基化的要求。将检查几个IRF7缺失和点突变，其中赖氨酸突变为精氨酸，以确定哪些赖氨酸残基(S)是和基化的。此外，LMP1 CTAR3缺失突变体将被检测其对IRF7求和的能力。最后，我们将研究IRF7苏莫化在诱导先天性免疫反应中的作用。这些结果将为我们深入了解IRF7是如何被求和的，并为未被研究的LMP1 CTAR3提供一个新的功能。其次，我们将研究IRF7的苏莫化和泛素化之间的竞争。每一种翻译后修饰都能以不同的方式改变IRF7的功能，因此我们将研究苏莫化对IRF7泛素化的作用，反之亦然。这些修改对irf7转录活性的影响也将被检测，以破译不同的修改如何改变irf7‘S作为天然免疫反应的中心转录因子的功能。与公众健康相关：调节蛋白质总和甲基化是抗癌治疗的新目标。这项工作将通过定义LMP1 CTAR3的新角色并破译相思如何影响IRF7的功能，进一步加深我们对EBV潜伏期间蛋白质相加作用的理解。综上所述，该项目将深入了解LMP1和IRF7在EBV潜伏期中的新作用，以及抗病毒免疫反应调节的潜在靶点。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is a tumor causing virus that is associated with several types of cancer, and the main EBV oncoprotein is latent membrane protein (LMP) 1, a constitutively active membrane protein that mimics the signaling induced by the tumor necrosis factor receptor family. We have reported that LMP1 induces the expression of interferon regulatory factor (IRF)-7, a transcription factor that is a key regulator of Type I interferons in innate immune responses. LMP1 regulates the transcriptional activity of IRF7 via post-translational modifications, including phosphorylation and ubiquitination. Therefore, we investigated if LMP1 induces other modifications of IRF7, specifically that by the small ubiquitin-like protein modifier (SUMO)-1, in order to regulates its function. We found that LMP1 interacts with Ubc9, the only reported SUMO-conjugating enzyme, possibly via the understudied C-terminal activating region (CTAR3). LMP1 also induced the interaction of Ubc9 with IRF7 leading to the sumoylation of IRF7, which resulted in decreased IRF7 turnover. Sumoylated IRF7 accumulated in the nucleus where its transcriptional activity is modestly increased. Therefore, we hypothesize that the induction of the sumoylation of IRF7 by LMP1 moderates the activation of the immune response, and we propose two aims to test this hypothesis. First, we will determine the requirements for IRF7 sumoylation. Several IRF7 deletion and point mutants, where lysines are mutated to arginines, will be examined to determine which lysine residue(s) is/are sumoylated. In addition, a LMP1 CTAR3 deletion mutant will be examined for its ability to sumoylate IRF7. Finally, we will examine the role IRF7 sumoylation has on the induction of innate immune responses. The results will provide insight into how IRF7 is sumoylated and implicate a new function for understudied LMP1 CTAR3. Second, we will investigate competition between sumoylation and ubiquitination of IRF7. Each post- translational modification can alter IRF7 function in a distinct manner, so we will investigate the role sumoylation has on IRF7 ubiquitination, and vice-versa. The effect these modifications have on IRF7 transcriptional activity will also be examined to decipher how the different modifications alter IRF7's function as a central transcription factor for innate immune responses. Relevance to public health: Regulating protein sumoylation is a new target for anti-cancer therapies. This work will further our understanding of protein sumoylation during EBV latency, by defining a new role for LMP1 CTAR3 and deciphering how sumoylation affected the function of IRF7. Together, this project will provide insight into new role for LMP1 and IRF7 during EBV latency as well as potential targets for the regulation of anti-viral immune responses.