MuLV p12 function in tethering & integration

描述（由申请人提供）：受感染细胞进行有丝分裂的要求以及在转录起始位点（TSS）和CpG岛整合的偏好是γ -逆转录病毒的两个特征，这极大地影响了它们的发病机制和作为基因治疗载体的效用。本应用研究了MuLV Gag - p12蛋白在有丝分裂染色体上拴住预整合复合体（pre-integrative complex， PIC）的新功能。通过嵌合蛋白p12的生成，发现p12的拴系特性可以影响整合靶位点，特别是远离转录起始位点和Cp岛。这对MuLV的发病机制具有深远的意义，其中启动子/增强子插入已知会导致癌基因激活。提出了三个具体目标。我们的实验方法检测了来自三种不同病毒的已知栓系结构域补充p12突变体（PM14）的能力，其中病毒感染在核进入或保留时被阻断。令人惊讶的是，发现病毒选择与浓缩的有丝分裂染色体弱关联。插入的原型泡沫病毒染色体结合域比WT p12更紧密地结合有丝分裂染色体，显示出对TSS和CpG岛的偏好降低。我们的工作模型是，聚合的强度将影响整合站点的偏好。一个具体的目标是通过使用下一代测序绘制嵌合病毒的整合位点利用来直接测试这一点。第二个具体目标是利用遗传和生化研究来定义和扩展MuLV p12蛋白的系留特性。我们将研究一组新的、可选择的拴系结构域来补充p12突变体的能力。研究了p12磷酸化的影响。WT p12与有丝分裂染色体结合的机制尚不清楚。第三个具体目的是利用质谱法确定PIC内p12的化学计量，并确定与WT p12蛋白相互作用的宿主因子。了解PIC系住MuLV的机制，可以解决伽马逆转录病毒的两个标志性特征，即细胞需要进行有丝分裂及其与启动子插入相关的发病机制。这项研究的总体目标是将这些概念验证实验扩展到更安全的基因传递载体的改进设计。

DESCRIPTION (provided by applicant): The requirement for infected cells to undergo mitosis as well as the preference to integrate at transcriptional start sites (TSS) and CpG islands are two features of gammaretroviruses that greatly influence their pathogenesis and utility as gene therapy vectors. This application studies a new function of the Gag p12 protein of MuLV associated with tethering the pre-integrative complex (PIC) to the mitotic chromosomes. Through the generation of chimeric p12 proteins, it was found that the tethering property of p12 can influence the integration target-site, in particular away from transcription start sites and Cp islands. This has profound implications with respect to MuLV pathogenesis, where promoter/enhancer insertions are known to cause oncogene activation. Three specific aims are proposed. Our experimental approach examined the ability of known tethering domains from three different viruses to complement a p12 mutant (PM14), in which viral infection was blocked at nuclear entry or retention. Surprisingly, it was found that the virus selects for weak association to the condensed mitotic chromosomes. Insertion of the prototype foamy virus chromosome-binding domain, which binds to mitotic chromosomes tighter than the WT p12, displayed the decreased bias to TSS and CpG islands. Our working model is that the strength of the p12 tethering will influence the integration site preferences. One specific aim directly tests this through mapping the integration site utilization of the chimeric p12 viruses using next-generation sequencing. A second specific aim defines and expands the tethering property of the MuLV p12 protein using genetic and biochemical studies. The ability of a panel of novel, alternative tethering domains to complement p12 mutants will be examined. The effects of phosphorylation of p12 are examined. The mechanism utilized by the WT p12 to bind to the mitotic chromosomes is not known. The third specific aim utilizes mass spectrometry to define the stoichiometry of p12 within the PIC as well as to identify the host factors that interact with the WT p12 protein. Understanding the mechanism of PIC tethering for MuLV addresses two hallmark features of gammaretroviruses, namely the requirement for cells to undergo mitosis and its pathogenesis associated with promoter insertions. The overall goal of the research is to extend these proof-of- concept experiments towards the improved design of safer gene delivery vectors.