Cellular Cofactors of Murine Leukemia Virus Integrase

鼠白血病病毒整合酶的细胞辅因子

• 批准号: 8709737
• 负责人: Mamuka Kvaratskhelia
• 金额: $ 23.07万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-10 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8709737
• 关键词: Adverse event; Affinity; Amino Acids; BMI1 gene; Binding; Binding Sites; Biochemical; Bioinformatics; Bromodomain; CCND2 gene; ChIP-seq; Chromatin; Chromosomes; Clinical Trials; Communities; CpG Islands; Data; Development; Enzymes; Foundations; Genes; Genetic Materials; Genetic Transcription; Genomics; Goals; HIV-1; Histones; Human; Infection; Insertional Activations; Integrase; Lead; Link; Lysine; Maps; Mass Spectrum Analysis; Mediating; Molecular; Murine leukemia virus; Patients; Protein Footprinting; Proteins; Proto-Oncogenes; Publishing; Recombinants; Reporting; Retroviral Vector; Retroviridae; Role; Route; Site; Site-Directed Mutagenesis; Subfamily lentivirinae; System; Tertiary Protein Structure; Testing; Transcription Initiation Site; Virus Integration; adverse outcome; base; biophysical techniques; cellular transduction; cofactor; congenital immunodeficiency; gene therapy; gene therapy clinical trial; insight; interest; lens epithelium-derived growth factor; mutant; novel; public health relevance; research study; transcriptional coactivator p75; vector; viral DNA

DESCRIPTION (provided by applicant): The overarching goal of the present proposal is to elucidate the molecular mechanisms by which cellular cofactors control the distribution of murine leukemia virus (MLV) integration sites in chromatin. The selection of chromosomal targets for retroviral integration is not random and varies markedly for different retroviral gener. For example, the gamma-retroviruses including MLV favor integration near transcription start sites and CpG islands, whereas lentiviruses including HIV-1 preferentially integrate within active genes. These observations have suggested that different cellular binding partners of retroviral integrases could be responsible for distinct integration site selectivity. However, until very recently, only one example has been reported: lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether that engages HIV-1 intasomes and navigates them to active genes. The significance of exploring the molecular mechanisms of gamma-retroviral MLV integration site selectivity is exemplified by the development of MLV-based vectors for human gene-therapy. In clinical trials, the use of gamma-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertional activation of protooncogenes by MLV-based vectors. We have recently discovered that the bromodomain and extra terminal domain (BET) proteins (Brd2, 3, 4) are the principal cellular binding partners of MLV IN and demonstrated their significance for targeting MLV integration at transcription start sites. The present application aims to extend these important initial findings to better understand the underlying mechanism for how BET proteins selectively recognize MLV IN and navigate the gamma-retroviral integration to specific sites in chromatin. In particular, aim 1 will study structural and mechanistic foundations for how MLV IN recognizes BET proteins selectively and with high affinity; and aim 2 will examine a proposed bimodal mechanism for BET proteins-mediated link between MLV integration and select chromatin sites. Our experiments are expected to yield important novel findings, which will benefit a wide scientific community interested in understanding molecular mechanisms of retroviral integration. Since BET proteins-MLV IN interactions are only the second system, besides HIV-1 IN-LEDGF/p75 interactions, our findings will elucidate important mechanistic similarities and differences between MLV and HIV-1 integration site selectivity. Thus, the proposed studies will lead to better understanding of how different retroviruses have evolved to utilize distinct cellular chromatin binding partners to insert their genetic material at select sits in the host chromosome. Additionally, our findings will lead to better understanding of molecular mechanisms for integrations of MLV-based vectors near proto-oncogenes during human gene-therapy trails, which have been linked to significant adverse outcomes in patients.

描述（由申请人提供）：本建议的总体目标是阐明细胞辅助因子控制染色质中鼠白血病病毒（MLV）整合位点的分布的分子机制。逆转录病毒整合染色体靶标的选择不是随机的，对于不同的逆转录病毒生成而有很大变化。例如，包括MLV的伽马病毒在转录起始站点和CPG岛附近有利积分，而包括HIV-1在内的慢病毒优先整合在活性基因中。这些观察结果表明，逆转录病毒整合酶的不同细胞结合伴侣可能导致不同的整合位点选择性。但是，直到最近，只有一个例子报道了：镜头上皮衍生的生长因子（LEDGF/p75），该因子充当双峰束缚，可与HIV-1插入式组合并将其导航到活性基因。探索γ-逆转录病毒MLV整合位点的分子机制的意义通过用于人类基因疗法的基于MLV的载体的发展来体现。在临床试验中，使用γ-逆转录病毒载体纠正原发性免疫缺陷是有效的，但是发生了不良事件，与基于MLV的载体插入原子基因有关。我们最近发现，溴结构域和额外末端结构域（BET）蛋白（BRD2、3、4）是MLV IN的主要细胞结合伙伴，并且证明了它们在转录起始位点靶向MLV整合的重要性。本应用程序旨在扩展这些重要的初始发现，以更好地了解BET蛋白如何选择性地识别MLV并导航γ-逆转录病毒整合到染色质质中特定位点的基本机制。特别是，AIM 1将研究MLV如何选择性地识别蛋白质和具有高亲和力的结构和机械基础。 AIM 2将检查提出的双峰机制，用于BET蛋白介导的MLV整合与选择染色质位点之间的联系。我们的实验有望产生重要的新发现，这将使人们有益于了解逆转录病毒整合的分子机制的广泛科学界。由于相互作用中的BET蛋白-MLV仅仅是第二个系统，除了HIV-1 IN-LEDGF/P75相互作用之外，我们的发现将阐明MLV和HIV-1积分位点选择性之间的重要机理相似性和差异。因此，提出的研究将使人们更好地了解不同的逆转录病毒如何利用不同的细胞染色质结合伴侣在宿主染色体中插入其遗传材料。此外，我们的发现将使人们更好地理解人类基因疗法过程中原始基因基因基于MLV基于MLV的载体的整合，这与患者的严重不良后果有关。