MuLV p12 function in tethering & integration

对感染细胞进行有丝分裂的要求以及对整合的偏好 在转录起始点(TSS)和CpG岛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。本应用研究 MuLV Gag p12蛋白与整合前信号转导相关的新功能 有丝分裂染色体的复合体(PIC)。通过产生嵌合的p12蛋白，它 研究发现，p12的拴系特性可以影响整合靶位，在 特别是远离转录起始点和CpG岛。这具有深远的影响 关于MuLV的致病机制，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。我们的实验方法测试了 来自三种不同病毒的已知系链结构域，以补充p12突变体(PM14)，在 哪种病毒感染在核进入或滞留时被阻止。令人惊讶的是，人们发现 病毒选择与浓缩的有丝分裂染色体结合较弱。插入 泡沫状病毒染色体结合结构域原型，与有丝分裂染色体结合更紧密 比WT p12显示对TSS和CpG岛的偏向减少。我们的工作模式是 P12系留的强度将影响整合站点的偏好。一个特定的 AIM通过绘制嵌合p12病毒的整合位点利用图来直接测试这一点 使用下一代测序技术。第二个具体目标是定义和扩展系绳 用遗传学和生物化学方法研究MuLV p12蛋白的性质。电池组的能力 对补充p12突变体的新的、可供选择的系链结构域进行研究。这个 检测了p12磷酸化的影响。WT p12使用的绑定机制 对有丝分裂染色体的影响尚不清楚。第三个具体目标是利用质谱学来 定义PIC中p12的化学计量比，并确定相互作用的宿主因素 WT p12蛋白。 了解MULV的PIC绑定机制有两个显著特点 即细胞有丝分裂的要求及其发病机制 与启动子插入相关。这项研究的总体目标是扩大这些证据 针对更安全的基因传递载体的改进设计的概念性实验。

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 CpG 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.