Mechanisms of Silencing of Retroviral DNAs in Embryonic Cell Lines

胚胎细胞系中逆转录病毒 DNA 沉默的机制

• 批准号: 10083189
• 负责人: STEPHEN Paine GOFF
• 金额: $ 46.65万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10083189
• 关键词: Acetylation; Acquired Immunodeficiency Syndrome; Affect; Antiviral Therapy; Binding; Binding Sites; Biochemical; Biological Process; Cell Differentiation process; Cell Line; Cells; Characteristics; Chromatin; Complex; Cullin Proteins; DNA; DNA Binding; DNA-Binding Proteins; Development; Elements; Embryo; Embryonic Development; Epigenetic Process; Event; Family; Funding; Gene Expression; Gene Silencing; Genes; Genetic; Genome; Germ Lines; Goals; HIV; Haploid Cells; Histone Deacetylase; Human; Human T-lymphotropic virus 1; Infection; Integration Host Factors; Knock-out; Maintenance; Mediating; Modeling; Modification; Moloney Leukemia Virus; Monitor; Mus; Pathway interactions; Phosphorylation; Positioning Attribute; Proline-Specific tRNA; Property; Protein Family; Proteins; Proviruses; RNA; RNA Binding; Regulation; Regulatory Pathway; Repression; Retroviridae; Role; SETDB1 gene; Signal Pathway; Signaling Molecule; Site; Specificity; Structure; TRIM Motif; Testing; Transferase; Virus; Work; Yin-Yang; Zinc Fingers; blastomere structure; cell type; cofactor; embryonic stem cell; experimental study; histone modification; integration site; interest; knock-down; member; novel; novel strategies; peripherin; primitive cell; protein complex; protein protein interaction; prototype; stemness; ubiquitin ligase; viral DNA; viral leukemia

This proposal describes genetic and biochemical analyses of host factors regulating the expression of the Moloney murine leukemia virus, the prototype of the simple mammalian retroviruses. We are especially focused on characterizing the mechanism of action of specific factors that we have identified that limit or restrict virus expression in embryonic cell types. We will characterize the mechanisms by which murine embryonic stem (ES) cells transcriptionally silence proviral DNAs and maintain the integrity of the germ line. We will study three parallel pathways – a rapid and highly efficient mechanism targeting a specific DNA element of the Moloney provirus, the tRNApro Primer Binding Site (PBS); a less potent one acting at a conserved site, the negative control region (NCR) present on the proviral DNA of many retroviruses;; and a newly-­identified one also acting broadly on many retroviruses. We will characterize the DNA-­binding host proteins that mediate the silencing (ZFP809, YY1, and NP220) and determine how these silencing mechanisms are regulated so as to be specifically active in ES cells. The study will involve examination of ubiquitin ligases, SUMO transferases, and protein-­protein interactions needed to form the large complex that binds to the viral DNA and induces silencing by making repressive histone modifications. We will also examine the mechanism by which one of these factors uniquely activates, rather than silences, one member of the retrovirus family (HTLV-1). Because the expression of retroviral DNAs is so closely correlated with expression of host genes during embryonic development, these experiments will provide important new information about the properties that define “stemness” – the pluripotent state of ES cells. These aspects of control of retroviruses by host factors will provide new targets for antiviral therapy. Most importantly, these experiments will significantly extend our understanding of fundamental aspects of retrovirus replication, and of new cell biological processes that impact on these important viruses.

该提案描述了宿主因子的遗传和生化分析，调节表达的莫洛尼鼠白血病病毒，原型的简单的哺乳动物逆转录病毒。我们特别关注表征我们已经确定的限制或限制病毒在胚胎细胞类型中表达的特定因素的作用机制。我们将描述小鼠胚胎干细胞（ES）转录沉默前病毒DNA和保持生殖系完整性的机制。我们将研究三个平行的途径-一个快速和高效的机制，靶向特定的DNA元件的莫洛尼前病毒，tRNApro引物结合位点（PBS）-一个较弱的一个作用于保守位点，负控制区（NCR）上存在于许多逆转录病毒的前病毒DNA;和一个新发现的一个也广泛地作用于许多逆转录病毒。我们将表征介导沉默的DNA结合宿主蛋白（ZFP 809、YY 1和NP 220），并确定这些沉默机制如何被调节，以便在ES细胞中特异性地活化。该研究将涉及检查泛素连接酶、SUMO转移酶和蛋白质-泛素蛋白相互作用，这些蛋白质-泛素蛋白相互作用需要形成与病毒DNA结合的大复合物，并通过进行抑制性组蛋白修饰来诱导沉默。我们还将研究这些因子之一独特地激活而不是沉默逆转录病毒家族（HTLV-1）的一个成员的机制。由于逆转录病毒DNA的表达与胚胎发育过程中宿主基因的表达密切相关，这些实验将提供有关定义“干细胞”--ES细胞多能状态--特性的重要新信息。宿主因素对逆转录病毒的这些控制将为抗病毒治疗提供新的靶点。最重要的是，这些实验将大大扩展我们对逆转录病毒复制的基本方面以及影响这些重要病毒的新细胞生物学过程的理解。