MuLV p12 function in tethering & integration

描述（由申请人提供）：需要感染细胞进行有丝分裂以及偏好在转录起始位点（TSS）和CpG岛整合是γ逆转录病毒的两个特征，这极大地影响了其发病机制和作为基因治疗载体的效用。本申请研究了MuLV的Gag p12蛋白与将前整合复合物（PIC）拴系到有丝分裂染色体相关的新功能。通过嵌合p12蛋白的产生，发现p12的拴系性质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的发病机制有深远的影响，其中已知启动子/增强子插入会引起癌基因激活。 提出了三个具体目标。我们的实验方法研究了已知的拴系结构域的能力，从三种不同的病毒，以补充p12突变体（PM 14），其中病毒感染被阻止在核进入或保留。令人惊讶的是，发现病毒选择与浓缩的有丝分裂染色体的弱关联。插入原型泡沫病毒染色体结合结构域（其与有丝分裂染色体的结合比WT p12更紧密）显示对TSS和CpG岛的偏倚降低。我们的工作模型是，p12的强度将影响整合网站的偏好。一个特定的目标是通过使用下一代测序绘制嵌合p12病毒的整合位点利用率来直接测试这一点。第二个具体目标是使用遗传和生物化学研究来定义和扩展MuLV p12蛋白的拴系特性。一组新的，替代拴系结构域，以补充p12突变体的能力将被检查。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。第三个具体目标是利用质谱法来确定PIC内p12的化学计量，以及鉴定与WT p12蛋白相互作用的宿主因子。 了解MuLV的PIC系留机制解决了γ逆转录病毒的两个标志性特征，即细胞进行有丝分裂的要求及其与启动子插入相关的发病机制。该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递送载体的改进设计。

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.