Epigenetic control of cytomegalovirus latency and reactivation

描述(申请人提供)：人巨细胞病毒是一种普遍存在的疱疹病毒，具有建立终身潜伏感染的能力。在实体器官和骨髓移植的接受者中经常观察到潜伏病毒的重新激活，这是新生儿和免疫低下宿主发病率和死亡率的重要原因。潜伏期在操作上被定义为即使存在病毒DNA也无法检测到传染性病毒。然而，CMV建立潜伏感染并从潜伏感染中重新激活的分子机制尚不清楚。由于缺乏研究体内感染巨细胞病毒的动物模型，我们等人利用巨细胞病毒作为模型来研究巨细胞病毒的潜伏期和再激活。我们已经开发了一种用于转录重新激活IE基因表达的移植模型。在这些研究的基础上，我们假设CMV建立了一种真正的潜伏感染，其中与生产性感染相关的基因，包括IE基因，不表达，转录重新激活是由于信号通路的激活，导致转录因子与其在增强子中的同源位置结合。在这里，我们假设潜伏感染中的转录沉默是由于主要的直接早期启动子(MIEP)与维持染色质处于浓缩和转录不活跃状态的蛋白质的相互作用。病毒将能够保持这种状态，因为潜伏感染的细胞对宿主免疫反应是看不见的。我们进一步假设，转录重新激活是由炎症刺激诱导的，炎症刺激导致与MIEP相关的组蛋白发生变化，从而使它们获得与转录活性染色质相关的修饰。这些修饰导致染色质的开放，从而允许NFkB等转录因子访问MIEP。我们假设，IE基因表达的转录重新激活伴随着染色质重塑和NFkB与MIEP的结合。我们建议通过使用染色质免疫沉淀试验来验证这一假设，以表征在急性和潜伏感染期间以及移植诱导的重新激活后与MIEP相关的共激活因子、转录因子、转录抑制因子和组蛋白的修饰。这些研究将为未来的R01研究染色质重塑蛋白被招募到MIEP的机制奠定基础。这些研究将促进我们对CMV潜伏期和再激活机制的理解，并可能导致预防CMV疾病的新疗法的开发。 公共卫生相关性：巨细胞病毒(CMV)是一种普遍存在的疱疹病毒，它建立了终生潜伏感染，其中存在病毒DNA，但不产生病毒。妊娠期间感染巨细胞病毒可导致胎儿死亡或严重的神经系统并发症。潜伏病毒的重新激活可导致免疫功能低下患者的显著发病率或死亡率。这项建议的目标是了解病毒建立潜伏感染的分子机制，以及触发潜伏病毒重新激活的分子途径。这些研究将促进我们对CMV潜伏期和再激活机制的理解，并可能导致预防CMV疾病的新疗法的开发。

DESCRIPTION (provided by applicant): HCMV is a ubiquitous herpesvirus which has the ability to establish a lifelong latent infection. Reactivation of latent virus is frequently observed in recipients of solid organs and bone marrow transplants and is a significant cause of morbidity and mortality in newborns and in immunocompromised hosts. Latency is operationally defined as the inability to detect infectious virus despite the presence of viral DNA. However, the molecular mechanisms by which CMV establishes a latent infection and reactivates from latency are not well understood. Because of the lack of animal models to study HCMV infection in vivo, we and others have used MCMV as a model to study CMV latency and reactivation. We have developed a transplant model for transcriptional reactivation of IE gene expression. On the basis of these studies, we have hypothesized that CMV establishes a true latent infection in which genes associated with productive infection, including the IE genes, are not expressed, and that transcriptional reactivation is due to activation of signaling pathways which lead to binding of transcription factors to their cognate sites in the enhancer. Here we hypothesize that transcriptional silencing in latent infection is due to interaction of the major immediate early promoter (MIEP) with proteins which maintain the chromatin in a condensed and transcriptionally inactive state. The virus would be able to persist in this state because latently infected cells would be invisible to the host immune response. We further hypothesize that transcriptional reactivation is induced by inflammatory stimuli which result in changes in the histones associated with the MIEP such that they acquire modifications associated with transcriptionally active chromatin. These modifications lead to opening of the chromatin which allows transcription factors such as NFkB access to the MIEP. We hypothesize that transcriptional reactivation of IE gene expression is accompanied both by chromatin remodeling and by binding of NFkB to the MIEP. We propose to test this hypothesis by using chromatin immunoprecipitation assays to characterize coactivators, transcription factors, transcriptional repressors and modification of histones associated with the MIEP during acute and latent infection and after reactivation induced by transplantation. These studies will form the basis for a future R01 investigating mechanisms by which chromatin remodeling proteins are recruited to the MIEP. These studies will advance our understanding of the mechanisms of CMV latency and reactivation and may lead to development of new therapies to prevent CMV disease. PUBLIC HEALTH RELEVANCE: Cytomegalovirus (CMV) is a ubiquitous herpesvirus which establishes a lifelong latent infection in which the viral DNA is present, but no virus is produced. Primary infection with CMV during pregnancy can cause death or serious neurological complications in the fetus. Reactivation of latent virus can cause significant morbidity or mortality in immunocompromised patients. The goal of this proposal is to understand the molecular mechanisms by which the virus establishes latent infection and the molecular pathways that trigger reactivation of latent virus. These studies will advance our understanding of the mechanisms of CMV latency and reactivation and may lead to development of new therapies to prevent CMV disease.