Temporal regulation of the essential Epstein-Barr virus oncoprotein LMP1

描述（由申请人提供）：人们早就知道eb病毒通过其潜伏期相关膜蛋白LMP1诱导NFkB信号传导。最近，我们的实验室通过证明LMP1蛋白和信号直到感染后两周左右才积累到有意义的水平，挑战了NFkB信号在整个感染过程中始终构成的教条。对感染ebv的细胞系和感染ebv的B母细胞的转录谱分析显示，与感染后早期相比，LMP1基因的转录增加了15倍，mRNA半衰期增加了2倍，mRNA总量增加了50倍。LMP1启动子受EBNA- 2、EBNA- 3c和宿主转录因子PU.1、ATF4和IRF7等病毒基因控制。由于PU.1、ATF4和IRF7的转录活性在感染后早期被抑制，因此有可能这些或其他反式作用因子在感染后早期不存在以高水平转录激活LMP1。为了解决LMP1 mRNA稳定性的变化，已发表的研究表明，myc诱导的miRNA miR17对ebv感染淋巴母细胞样细胞系中LMP1的表达有负面影响。此外，miR17在感染后早期表达水平较高，而LMP1表达水平较低。为了验证这些假设，我建议在没有miR17负调控的情况下研究EBV对B细胞的转化，并在感染后早期进一步表征LMP1的启动子活性。该项目的目标是表征EBV如何利用宿主因子动态控制其自身转录本的表达，以及LMP1的时间调节如何影响B细胞转化。

DESCRIPTION (provided by applicant): Epstein-Barr virus has long been known to induce NFkB signaling through its latency associated membrane protein, LMP1. Recently our lab has challenged the dogma that NFkB signaling is constitutive at all times throughout infection by demonstrating that LMP1 protein and signaling do not accumulate to meaningful levels until around two weeks after infection. Transcriptional profiling of both EBV-infected cell lines and EBV-infected B blasts early after infection has revealed that transcription of the LMP1 gene increases 15-fold, the mRNA half-life increases 2-fold, and the total mRNA increases 50-fold compared to early times after infection. The LMP1 promoter is controlled by viral genes such as Epstein-Barr Nuclear Antigen (EBNA) 2 as well as EBNA-3C and the host transcription factor PU.1, ATF4, and IRF7. Since the transcriptional activity of PU.1, ATF4, and IRF7 is repressed early after infection, it is possible that these or other trans-acting factors are not present to transcriptionally activate LMP1 to high levels early after infection. To address the change in LMP1 mRNA stability, published work has demonstrated that a Myc-induced miRNA, miR17, has a negative effect on LMP1 expression in EBV-infected lymphoblastoid cell lines. Furthermore, miR17 is expressed at higher levels early after infection when LMP1 is expressed poorly. To investigate these hypotheses I propose to study the transformation of B cells by EBV in the absence of negative regulation by miR17 as well as further characterize the promoter activity of LMP1 early after infection. The goal of this project is to characterize how EBV dynamically controls the expression of its own transcripts using host factors and how temporal regulation of LMP1 effects B cell transformation.