Mechanisms of Silencing of Retroviral DNAs in Embryonic Cell Lines

这项建议描述了调节Moloney鼠白血病病毒表达的宿主因素的遗传和生化分析，Moloney鼠白血病病毒是简单哺乳动物逆转录病毒的原型。我们特别关注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转录沉默前病毒DNA和维持生殖系完整性的机制。我们将研究三条平行的途径--一种针对Moloney前病毒特定DNA元件的快速而高效的机制，一种作用于保守位点的较弱的tRNAproPrimer结合位点;，存在于许多逆转录病毒前病毒上的负控制区；;和一种新发现的也广泛作用于许多逆转录病毒的途径。我们将鉴定介导沉默的DNA结合宿主蛋白(ZFP809、YY1和NP220)，并确定这些沉默机制是如何调节的，以便在ES细胞中具有特异性的活性。这项研究将涉及泛素连接酶、相扑转移酶和蛋白质-蛋白质相互作用的检查，这些酶是形成与病毒DNA结合的大型复合体所需的，并通过进行抑制性的组蛋白修饰来诱导沉默。我们还将研究这些因素中的一个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。由于在胚胎发育过程中，逆转录病毒DNA的表达与宿主基因的表达密切相关，这些实验将提供有关定义“干性”的属性的重要新信息--ES细胞的多能性状态。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的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.