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: 10545182
• 负责人: STEPHEN Paine GOFF
• 金额: $ 59.03万
• 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/10545182
• 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-1; Haploid Cells; Histone Deacetylase; Human; Human T-lymphotropic virus 1; Infection; Integration Host Factors; Knock-out; Maintenance; Mediating; Methylation; Modeling; Modification; Moloney Leukemia Virus; Monitor; Mus; Pathway interactions; Phosphorylation; Positioning Attribute; Proline-Specific tRNA; Property; Protein Family; Proteins; Provirus Integration; Proviruses; RNA; RNA Binding; Regulation; Regulatory Pathway; Repression; Retroviridae; Role; SETDB1 gene; SUMO1 gene; Signal Pathway; Signaling Molecule; Site; Specificity; Structure; TRIM Motif; Testing; Transferase; Viral; Virus; Work; Yin-Yang; Zinc Fingers; blastomere structure; cell type; cofactor; embryo cell; 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.

该提案描述了调节莫洛尼鼠白血病病毒（简单哺乳动物逆转录病毒的原型）表达的宿主因素的遗传和生化分析。我们特别关注于表征我们已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 (ES) 细胞转录沉默前病毒 DNA 并维持种系完整性的机制。我们将研究三种平行途径——一种针对莫洛尼原病毒特定 DNA 元件（tRNApro 引物结合位点 (PBS)）的快速高效机制；一种作用于保守位点（许多逆转录病毒原病毒 DNA 上的阴性控制区 (NCR)）的效力较弱的机制；以及一种新发现的也广泛作用于许多逆转录病毒的机制。我们将表征介导沉默的 DNA 结合宿主蛋白（ZFP809、YY1 和 NP220），并确定如何调节这些沉默机制以便在 ES 细胞中特异性活跃。该研究将涉及检查泛素连接酶、SUMO 转移酶以及形成大型复合物所需的蛋白质-蛋白质相互作用，该复合物与病毒 DNA 结合并通过抑制性组蛋白修饰诱导沉默。我们还将研究这些因素之一独特地激活而不是沉默逆转录病毒家族（HTLV-1）成员之一的机制。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，因此这些实验将提供有关定义“干性”（ES 细胞的多能状态）特性的重要新信息。宿主因素控制逆转录病毒的这些方面将为抗病毒治疗提供新的靶点。最重要的是，这些实验将极大地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。