Mechanisms of Silencing of Retroviral DNAs in Embryonic Cell Lines

本文描述了调节Moloney小鼠白血病病毒（简单哺乳动物逆转录病毒的原型）表达的宿主因子的遗传和生化分析。我们特别侧重于描述我们已经确定的限制或限制病毒在胚胎细胞类型中的表达的特定因子的作用机制。我们将描述小鼠胚胎干（ES）细胞通过转录沉默前病毒dna并维持种系完整性的机制。我们将研究三种平行途径——一种快速高效的机制，靶向Moloney原病毒的特定DNA元件，tRNApro引物结合位点（PBS）；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）；以及一种新发现的也广泛作用于许多逆转录病毒的病毒。我们将对介导沉默的DNA结合宿主蛋白（ZFP809、YY1和NP220）进行表征，并确定这些沉默机制是如何被调节的，从而在胚胎干细胞中具有特异性活性。这项研究将包括检查泛素连接酶、SUMO转移酶和蛋白质相互作用，这些蛋白质相互作用需要形成与病毒DNA结合的大复合体，并通过抑制组蛋白修饰诱导沉默。我们还将研究其中一个因子如何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，因此这些实验将提供有关胚胎干细胞的“干性”（即多能性）特性的重要新信息。宿主因子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，这些实验将大大扩展我们对逆转录病毒复制的基本方面的理解，以及对这些重要病毒产生影响的新细胞生物学过程的理解。

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.