MuLV p12 function in tethering & integration

描述(由申请人提供)：要求感染细胞进行有丝分裂，以及倾向于在转录起始点(TSS)和CpG岛整合，这是伽马逆转录病毒的两个特征，极大地影响了它们的致病机制和作为基因治疗载体的用途。这项应用研究了MuLV的Gag p12蛋白的一个新功能，它与前整合复合体(PIC)连接到有丝分裂染色体上有关。通过产生嵌合的p12蛋白，发现p12的拴系特性可以影响整合靶点，特别是远离转录起始点和CP岛。这对MuLV的发病机制有深远的影响，已知启动子/增强子插入会导致癌基因激活。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系链结构域补充p12突变体(PM14)的能力，在该突变体中，病毒感染被阻止在核进入或滞留。令人惊讶的是，人们发现该病毒选择与浓缩的有丝分裂染色体的关联较弱。插入原型泡沫病毒染色体结合域，它与有丝分裂染色体的结合比WT p12更紧密，显示出对TSS和CpG岛的偏见减少。我们的工作模型是，p12系留的强度将影响整合站点的偏好。一个特定的目的是通过使用下一代测序绘制嵌合p12病毒的整合位点利用图来直接测试这一点。第二个特定目的是通过遗传和生物化学研究来定义和扩展MuLV p12蛋白的系留特性。将检验一组新的、可供选择的系链结构域来补充p12突变体的能力。对p12的磷酸化作用进行了研究。WT p12与有丝分裂染色体结合的机制尚不清楚。第三个特定目的是利用质谱学来定义PIC中p12的化学计量比，以及确定与WT p12蛋白相互作用的宿主因素。了解针对MuLV的PIC拴系机制解决了伽马逆转录病毒的两个显著特征，即要求细胞经历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.