Temporal regulation of the essential Epstein-Barr virus oncoprotein LMP1

描述(由申请人提供)：EB病毒通过其潜伏期相关的膜蛋白LMP1诱导NFkB信号转导。最近，我们的实验室通过证明LMP1蛋白和信号直到感染后大约两周才积累到有意义的水平，挑战了NFkB信号在整个感染过程中始终是结构性的这一教条。EBV感染的细胞株和EBV感染的B细胞感染后早期的转录图谱显示，与感染早期相比，LMP1基因的转录增加了15倍，信使核糖核酸半衰期增加了2倍，总信使RNA增加了50倍。LMP1启动子受病毒基因控制，如EB病毒核抗原(EBNA)2、EBNA-3C和宿主转录因子PU.1、ATF4和IRF7。由于PU.1、ATF4和IRF7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在感染后早期转录激活LMP1到高水平。为了解决LMP1 mRNA稳定性的变化，已发表的工作表明，Myc诱导的miR17对EBV感染的淋巴母细胞系中LMP1的表达具有负面影响。此外，当LMP1表达较低时，miR17在感染后早期表达水平较高。为了研究这些假设，我建议在没有miR17负调控的情况下研究EBV对B细胞的转化，并进一步表征感染后早期LMP1的启动子活性。这个项目的目标是表征EBV如何使用宿主因素动态控制自己转录本的表达，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.