Temporal regulation of the essential Epstein-Barr virus oncoprotein LMP1

描述（由申请人提供）：长期以来已知EB病毒通过其潜伏相关膜蛋白LMP 1诱导NF κ B信号传导。最近，我们的实验室通过证明LMP 1蛋白和信号传导直到感染后大约两周才积累到有意义的水平，挑战了NFkB信号传导在整个感染过程中始终是组成性的这一教条。感染后早期EBV感染的细胞系和EBV感染的B母细胞的转录谱显示，与感染后早期相比，LMP 1基因的转录增加15倍，mRNA半衰期增加2倍，总mRNA增加50倍。LMP 1启动子由病毒基因如EB核抗原（EBNA）2以及EBNA-3C和宿主转录因子PU.1、ATF 4和IRF 7控制。由于PU.1、ATF 4和IRF 7的转录活性在感染后早期被抑制，因此这些或其他反式作用因子可能不存在以在感染后早期将LMP 1转录激活至高水平。为了解决LMP 1 mRNA稳定性的变化，已发表的工作表明，Myc诱导的miRNA，miR 17，对EBV感染的淋巴母细胞系中的LMP 1表达具有负面影响。此外，当LMP 1表达差时，miR 17在感染后早期以较高水平表达。为了研究这些假设，我建议研究在没有miR 17负调控的情况下EBV对B细胞的转化，以及进一步表征感染后早期LMP 1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物的表达以及LMP 1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.