Interactions of MuLV IN with host proteins and DNA

 描述（由申请人提供）：鼠白血病病毒优先整合在活性启动子区域内，靠近转录起始位点（TSS）和CpG岛。整合位点与病毒的致癌潜力相关，因此与其发病机制和作为基因递送载体的用途相关。我们的合作研究最近确定了宿主BET蛋白家族，特别是Brd 2、3和4蛋白作为与MLV IN的相互作用物，这种相互作用影响了整合到TSS和CpG岛中。这为我们理解MLV靶位点选择机制提供了第一个窗口。 本申请研究了MLV IN和BET家族成员之间新定义的病毒-宿主相互作用。四个具体目标是建立在两个关键的观察。第一个链接Brd ET结构域与MLV IN C-末端结构域的结合。第二个指示缺乏C-末端23个氨基酸的IN蛋白不再偏向于整合到TSS和CpG岛。基于我们实验室的MLV IN CTD的溶液结构，第一个具体目标是定义MLV IN CTD：Brd 3 ET复合物的NMR结构。将对CTD与交叉骨DNA整合中间体的复合物进行额外的结构分析。 第二个具体目标是应用突变和生化测定来确定主要和潜在的次要Brd结合位点。将检查IN CCD在结合BET蛋白中的有争议的作用。将研究MLV IN蛋白与已知ET结构域相互作用物竞争的能力。第三个具体目标是利用下一代测序和生物信息学来探测一组IN蛋白的靶位点偏好，包括在整合位点的局部以及在人类基因组内的全局。检查核小体结构内的靶DNA的识别。最终的具体目标是将这些信息应用于开发靶向整合载体。 这项研究将我们对MLV IN的丰富知识转化为快速理解与宿主靶细胞的相互作用。这项研究解决了一个悬而未决的问题，有关的机制，针对MLV整合到启动子区。这些研究有可能适用于多种γ逆转录病毒。该研究的目标是将这些知识应用于减少基因递送系统中启动子/增强子插入对癌基因激活的影响。

 DESCRIPTION (provided by applicant): Murine leukemia virus preferentially integrates within active promoters regions, proximal to transcriptional start sites (TSS) and CpG islands. The site of integration is linked to the oncogenic potential of the virus and thus its pathogenesis and use as a gene delivery vector. Our collaborative research has recently identified the host BET family of proteins, specifically the Brd 2, 3, and 4 proteins as interactors with the MLV IN and this interaction influences the integration into TSS and CpG islands. This provides the first window into our understanding of the mechanism of MLV target-site selection. This application studies the newly defined viral-host interaction between the MLV IN and BET family members. Four specific aims are built on two key observations. The first links the binding of Brd ET domains to the MLV IN C-terminal domain. The second indicates that IN proteins lacking the C-terminal 23 amino acids are no longer biased towards integration to TSS and CpG islands. Based on the solution structure of the MLV IN CTD from our laboratory, the first specific aims at defining the NMR structure of the MLV IN CTD:Brd3 ET complex. Additional structural analysis of the CTD in complex with the crossbone DNA integration intermediate will be pursued. The second specific aim applies mutational and biochemical assays to define the primary as well as potential secondary Brd binding sites. The controversial role of the IN CCD in binding BET proteins will be examined. The ability of the MLV IN protein to compete with known ET domain interactors will be studied. The third specific aim utilizes next-generation sequencing and bioinformatics to probe a panel of IN proteins for their target-site preferences, both locally at the site of integraion as well as globally within the human genome. The recognition of the target DNA within the nucleosome structure is examined. The final specific aim applies this information towards developing targeted integrating vectors. The research translates our strong knowledge of MLV IN towards rapidly understanding this interaction with the host target cell. The research addresses one of the outstanding questions relating to the mechanism of targeting MLV integration into promoter regions. These studies have the potential to be applicable to multiple gammaretroviruses. The goal of the research is to apply this knowledge towards decreasing the effects of promoter/enhancer insertions on oncogene activation in gene delivery systems.