MuLV p12 function in tethering & integration

MuLV p12 在网络共享中的功能

基本信息

批准号：
8603648
负责人：
MONICA J ROTH
金额：
$ 28.53万
依托单位：
RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-01-01 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8603648
关键词：
Address Affinity Area Binding Biochemical CD4 Positive T Lymphocytes Cells Chimera organism Chimeric Proteins Chromatin Chromosomes Collaborations Complement Complex CpG Islands Development Engineering Enhancers Epigenetic Process Gagging Gammaretrovirus Gene Delivery Gene Transduction Agent Generations Genetic Goals Herpesviridae Human Human Papillomavirus Integrase Integration Host Factors Investigation Island Maps Mass Spectrum Analysis Mitosis Mitotic Chromosome Mitotic spindle Modeling Modification Molecular Mus Mutation Nuclear Oncogene Activation Pathogenesis Pennsylvania Phosphorylation Property Proteins Research Role Safety Site Spumavirus Staging Testing Transcription Initiation Site Universities Viral Virus Virus Diseases Work base design gag Gene Products gene therapy histone modification improved mutant next generation sequencing novel particle preference pressure promoter prototype public health relevance research study restoration stoichiometry vector

项目摘要

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.

受感染细胞进行有丝分裂的要求以及整合的偏好转录起始位点 (TSS) 和 CpG 岛是伽马逆转录病毒的两个特征极大地影响它们的发病机制和作为基因治疗载体的用途。本应用研究 MuLV Gag p12 蛋白的一个新功能与束缚预整合相关有丝分裂染色体复合物（PIC）。通过嵌合 p12 蛋白的产生，发现 p12 的束缚特性可以影响整合目标位点，特别是远离转录起始位点和 CpG 岛。这具有深远的意义关于 MuLV 发病机制，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。我们的实验方法检验了来自三种不同病毒的已知束缚结构域可补充 p12 突变体 (PM14)，该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的弱关联。插入原型泡沫病毒染色体结合结构域，与有丝分裂染色体结合更紧密与 WT p12 相比，对 TSS 和 CpG 岛的偏向性降低。我们的工作模式是 p12 束缚的强度将影响整合位点偏好。一项具体 Target 通过绘制嵌合 p12 病毒的整合位点利用率来直接测试这一点使用下一代测序。第二个具体目标定义并扩展了网络共享使用遗传和生化研究研究 MuLV p12 蛋白的特性。面板的能力将检查补充 p12 突变体的新颖的替代束缚结构域。这检查了 p12 磷酸化的影响。 WT p12 结合的机制有丝分裂染色体的作用尚不清楚。第三个具体目标是利用质谱法定义 PIC 内 p12 的化学计量并确定相互作用的宿主因子与 WT p12 蛋白。了解 MuLV 的 PIC 网络共享机制可解决两个标志性特征 γ逆转录病毒，即细胞进行有丝分裂的要求及其发病机制与启动子插入相关。该研究的总体目标是扩展这些证明- 旨在改进更安全的基因传递载体设计的概念实验。