Project 1 - Chromatin and the lytic/latent balance

项目摘要/摘要-项目1 本项目研究的长期目标是：1)确定疱疹病毒 单纯疱疹病毒1型(HSV-1)基因产物在裂解感染过程中促进病毒裂解基因上的常染色质 异染色质对这些基因在感觉神经元潜伏感染过程中的切换确定 裂解性和潜伏性感染，以及2)应用这些知识开发治疗HSV潜伏性感染的药物。 抑制HSV裂解感染的抗病毒药物已经开发出来，但还没有抗病毒治疗方法。 潜伏感染。以前，我们已经定义了HSV潜伏感染感觉神经元的许多方面 小鼠三叉神经节，特别是潜伏的HSV-1染色质的结构和病毒基因产物 调节病毒染色质结构。在拟议的研究中，我们将测试潜伏感染的机制 小鼠三叉神经节模型，在培养的小鼠神经元中，在更大的医学意义上，在人类中 可诱导的多能干细胞来源的神经元。我们将重点研究病毒基因产品的功能，促进 一种稳定的潜伏感染，准备重新激活。所有AIMS都与项目2和3以及核心集成在一起 A和B。 特定目标1将测试关于潜伏期相关记录(LAT)如何的各种假设 促进潜伏的HSV-1 DNA上的总组蛋白和兼性异染色质负载。我们将测试LAT是否 在LAT中插入转录终止子，通过反义转录促进表观遗传沉默 HSV-1基因组中的转录单位，并检测其对染色质的影响，转录ICP4反义片段， 以及下游反义基因的表达。我们将测试LAT是否招募了组蛋白加载者和表观遗传学 对HSV-1基因组促进异染色质装载到病毒基因组并稳定的因素 保持稳定的延迟。 具体目标2将探索ICP0促进潜伏期稳定维持的机制 单纯疱疹病毒1型基因组。我们将确定ICP0在维持病毒基因组、异染色质和LAT中的作用 在人类神经元、小鼠神经元和小鼠体内系统中的表达。一部小说的效果，令人兴奋 将在小鼠和人类神经潜伏期系统中定义ICP0特异性抑制小分子。 具体目标3将研究CCCTC结合因子(CTCF)结合位点的作用机制 (CTRL2)位于LAT和ICP0基因启动子之间，作为染色质绝缘体，促进 重新激活。我们将定义CTRL2站点的CTCF绑定在建立和 潜伏感染的维持。我们将定义CTRL2在病毒三维结构中的作用 人类和小鼠神经元的染色质与项目3。 这些研究将为了解HSV潜伏感染机制提供重要的新的基础知识 并可能为HSV潜伏感染提供新的治疗方法。

Project Summary/Abstract – Project 1 The long-term goals of the research of this project are 1) to define the mechanisms by which herpes simplex virus 1 (HSV-1) gene products promote euchromatin on viral lytic genes during lytic infection and heterochromatin on these genes during latent infection of sensory neurons to determine the switch between lytic and latent infection, and 2) to apply this knowledge to develop therapeutics for HSV latent infection. Antiviral drugs that inhibit HSV lytic infection have been developed, but there is no antiviral therapeutic for latent infection. Previously, we have defined many aspects of HSV latent infection of sensory neurons in murine trigeminal ganglia, in particular the structure of latent HSV-1 chromatin and the viral gene products that regulate viral chromatin structure. In the proposed research, we will test mechanisms of latent infection in the murine trigeminal ganglion model, in cultured mouse neurons and, for greater medical relevance, in human inducible pluripotent stem cell-derived neurons. We will focus on functions of viral gene products that promote a stable latent infection that is poised for reactivation. All aims are integrated with Projects 2 and 3 and Cores A and B. Specific Aim 1 will test various hypotheses about how the latency-associated transcript (LAT) promotes total histone and facultative heterochromatin loading on latent HSV-1 DNA. We will test whether LAT promotes epigenetic silencing by antisense transcription by inserting transcriptional terminators in the LAT transcriptional unit in the HSV-1 genome, and testing the effects on chromatin, transcripts antisense to ICP4, and expression of downstream antisense genes. We will test if LAT recruits histone loaders and epigenetic factors to the HSV-1 genome to promote loading of heterochromatin to the viral genome and stable maintenance of poised latency. Specific Aim 2 will explore mechanisms by which ICP0 promotes stable maintenance of the latent HSV-1 genome. We will determine the role of ICP0 in maintaining the viral genome, heterochromatin, and LAT expression in human neurons, murine neurons, and murine in vivo systems. The effects of a novel, exciting ICP0-specific inhibitory small molecule will be defined in murine and human neuronal latency systems. Specific Aim 3 will study the mechanism of action of the CCCTC-binding factor (CTCF) binding site (CTRL2) between the LAT and ICP0 gene promoters, which serves as a chromatin insulator and promotes reactivation. We will define the role of CTCF binding at the at the CTRL2 site on establishment and maintenance of latent infection. We will define the role of CTRL2 in the 3-dimensional structure of viral chromatin in human and murine neurons with Project 3. These studies will provide important new basic knowledge of the mechanisms of HSV latent infection and may enable new therapeutics for the HSV latent infection.