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.

受感染的细胞需要进行有丝分裂， 转录起始位点（TSS）和CpG岛是γ逆转录病毒的两个特征， 极大地影响它们的发病机理和作为基因治疗载体的效用。本应用研究 MuLV的Gag p12蛋白的一种新功能与束缚前整合 复合体（PIC）的有丝分裂染色体。通过嵌合p12蛋白的产生， 发现p12的束缚性质可以影响整合靶位点， 特别是远离转录起始位点和CpG岛。这有着深远的影响 关于MuLV发病机制，其中已知启动子/增强子插入引起 癌基因激活 提出了三个具体目标。我们的实验方法检查了 来自三种不同病毒的已知系链结构域，以补充p12突变体（PM 14）， 该病毒感染在核进入或保留时被阻断。令人惊讶的是，发现 病毒选择与浓缩的有丝分裂染色体弱联合。插入 原型泡沫病毒染色体结合域，与有丝分裂染色体结合更紧密 与野生型p12相比，对TSS和CpG岛的偏好降低。我们的工作模式是 p12绑定的强度将影响集成站点偏好。一个具体 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定义并扩展了网络共享 使用遗传和生物化学研究MuLV p12蛋白的特性。小组的能力 新的，替代拴系结构域，以补充p12突变体将被检查。的 检测p12磷酸化的作用。WT p12结合的机制 与有丝分裂染色体的关系尚不清楚。第三个具体目标是利用质谱法， 定义PIC中p12的化学计量，并确定相互作用的宿主因子 野生型p12蛋白 了解MuLV的PIC网络共享机制可以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 与启动子插入有关。该研究的总体目标是扩展这些证据 概念实验，以改进更安全的基因传递载体的设计。