MuLV p12 function in tethering & integration

MuLV p12 在网络共享中的功能

• 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
• 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

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.

受感染细胞经历有丝分裂的要求以及整合的偏好 在转录起始站点（TSS）和CpG岛是伽马环病毒的两个特征，它们是这些特征 作为基因治疗载体，极大地影响了他们的发病机理和效用。该应用研究 MULV的GAG p12蛋白的新功能与束缚前整合性有关 与有丝分裂染色体的复合物（PIC）。通过嵌合p12蛋白的产生， 发现p12的束缚特性会影响集成目标位点， 特别远离转录起始站点和CPG岛。这具有深远的含义 关于MULV发病机理，已知启动子/增强子插入引起 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以补充P12突变体（PM14），在 哪些病毒感染在核进入或保留时被阻塞。令人惊讶的是，发现 病毒选择与冷凝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 比WT p12表现出对TSS和CPG岛的偏差减少。我们的工作模型是 p12束缚的强度将影响整合位点的偏好。一个具体 瞄准通过映射嵌合p12病毒的集成位点利用来直接测试这一点 使用下一代测序。第二个特定目标定义并扩展了束缚 使用遗传和生化研究的MULV p12蛋白的特性。面板的能力 将检查新颖的，替代的绑扎域以补充P12突变体。这 检查了p12磷酸化的影响。 WT P12使用的机制结合 尚不清楚有丝分裂染色体。第三个特定目的利用质谱法 定义PIC中p12的化学计量法以及识别相互作用的宿主因子 与WT P12蛋白。 了解MULV的PIC绑定机理解决了两个标志性功能 伽马肌病毒的含有有丝分裂的要求及其发病机理的要求 与启动子插入相关。该研究的总体目标是扩展这些证明 概念实验，以改善更安全的基因递送向量的设计。