MuLV p12 function in tethering & integration

MuLV p12 在网络共享中的功能

基本信息

批准号：
9193098
负责人：
MONICA J ROTH
金额：
$ 29.57万
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 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9193098
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 Gammaretrovirus Gene Delivery Gene Transduction Agent Generations Genetic Goals Herpesviridae Human Human Papillomavirus Integrase Integration Host Factors Investigation Island 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 Testing Transcription Initiation Site Universities Viral Virus Virus Assembly Virus Diseases Virus Replication Work base design experimental study gag Gene Products gene therapy histone modification improved integration site mutant next generation sequencing novel particle predictive modeling preference pressure promoter prototype public health relevance research study restoration stoichiometry vector

项目摘要

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.

描述（由申请人提供）：受感染细胞经历有丝分裂的要求，以及在转录起始位点（TSS）和CPG岛的偏爱是γ-逆转录病毒的两个特征，这些特征极大地影响了其发病机理和作为基因治疗载体的发病机理和效用。该应用研究了与将前整合性复合物（PIC）绑定到有丝分裂染色体相关的MULV的GAG p12蛋白的新功能。通过嵌合p12蛋白的产生，发现p12的束缚特性会影响整合目标位点，尤其是远离转录起始位点和CP岛。这在MULV发病机理上具有深远的影响，在MULV发病机理中，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来自三种不同病毒的已知绑定结构域补充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，发现该病毒选择了与凝结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，该结合结构域与WT P12的结合比有丝分裂的染色体结合，显示出对TSS和CPG岛的偏置降低。我们的工作模型是，p12束缚的强度将影响集成位点的偏好。一个特定的目的是通过使用下一代测序来映射嵌合p12病毒的集成位点利用的直接测试。第二个特定目的是使用遗传和生化研究来定义并扩大MULV p12蛋白的束缚特性。将检查一组新型的，替代的绑带域补充P12突变体的能力。检查了p12磷酸化的作用。 WT P12使用的机制与有丝分裂染色体结合。第三个特定目的利用质谱法来定义PIC中p12的化学计量法，并确定与WT p12蛋白相互作用的宿主因子。了解MULV的PIC束缚的机制解决了伽马甲病毒的两个标志性特征，即细胞经历有丝分裂及其与启动子插入相关的发病机理的要求。该研究的总体目标是将这些概念证明实验扩展到改进的更安全基因递送向量的设计。