Integration and epigenetic regulation of the HHV-6 genome

 描述（由申请方提供）：在初次感染期间，疱疹病毒建立潜伏感染，使病毒在宿主体内持续存在。最近发现，某些疱疹病毒在潜伏期将其基因组整合到宿主染色体的端粒中，而大多数其他疱疹病毒将其基因组作为细胞核中的附加体。其中一种病毒是人类疱疹病毒6型（HHV-6），它与一系列疾病有关，包括癫痫发作、脑炎和移植患者的移植排斥反应。然而，我们对疱疹病毒感染的分子机制，包括控制病毒基因表达和DNA整合的机制以及病毒再活化所需的事件的认识存在重大差距。为了解决我们知识中的这些关键差距，我们建议使用我们的体外感染系统进行HHV-6整合，潜伏和再激活，并结合功能基因组学来确定病毒基因组在这些过程中的命运。我们将首先通过测量从裂解性感染到潜伏性感染的转变期间的差异基因表达、染色质修饰和整合事件来确定潜伏期和整合建立期间的病毒基因组特征（具体目标1）。接下来，我们将研究病毒和细胞的决定因素参与病毒的再活化。处理后 组蛋白去乙酰化酶抑制剂药物（HDACi）在体外对潜伏感染细胞的作用，只有一部分细胞重新激活。因此，我们假设病毒再活化细胞对药物治疗有特征性的基因表达反应，这将揭示参与潜伏期维持或病毒再活化的细胞和病毒基因。我们将通过用HDACi处理细胞，通过FACS分离再活化和非再活化细胞，并进行目标1中所述的测定以确定再活化的特征来定义这种药物应答（具体目标2）。最后，我们将确定细胞的病毒基因组特征， iciHHV-6患者，以深入了解这些个体中的病毒调节，并了解我们的体外模型（具体目标3）。我们提出的实验将描述，与高分辨率，转录，染色质调节和病毒DNA整合的过程中的初始感染，建立潜伏期和再激活之间的联系。我们的具体目标的成功完成将提供所需的知识，以了解如何从整合的潜伏病毒基因组的转录控制，从而提供可能的治疗目标，以治疗移植过程中的病毒并发症。

 DESCRIPTION (provided by applicant): During primary infection, herpesviruses establish a latent infection that allows the virus to persist in the host for life. It was recently discovered hat certain herpesviruses integrate their genome into the telomeres of host chromosomes during latency, while most others maintain their genome as an episome in the nucleus. One of these viruses is human herpesvirus 6 (HHV-6), which is associated with a spectrum of diseases including seizures, encephalitis, and graft rejection in transplant patients. However, there are critical gaps in our knowledge regarding the molecular mechanisms of herpesvirus infections, including the mechanisms by which virus gene expression and DNA integration is controlled as well as the events required for virus reactivation. To address these critical gaps in our knowledge, we propose to use our in vitro infection system for HHV-6 integration, latency and reactivation coupled with functional genomics to determine the fate of the virus genome in these processes. We will first determine the viral genomic signatures during the establishment of latency and integration by measuring differential gene expression, chromatin modification and integration events during the transition from lytic to latent infection (Specific Aim 1). Next, we ill then investigate the viral and cellular determinants involved in virus reactivation. Upon treatment of latently infected cells with histone deacetylase inhibitor drugs (HDACi) in vitro, only in a fraction of the cells reactivate. We therefore hypothesize that virus-reactivating cells have a characteristic gene expression response to drug treatment, which will reveal cellular and viral genes that participate in either maintenance of latency or virus reactivation. We will define this drug response by treating cells with HDACi, isolation of reactivating and non-reactivating cells by FACS and performing the assays described in Aim 1 to determine the signature of reactivation (Specific Aim 2). Finally, we will determine the viral genomic signature of cells from iciHHV-6 patients to obtain an insight into the viral regulation in these individuals and to validae our in vitro model (Specific Aim 3). Our proposed experiments will describe, with high-resolution, the connections between transcription, chromatin regulation and viral DNA integration over the course of initial infection, establishment of latency and reactivation. Successful completion of our specific aims will provide knowledge required to understand how transcription from the integrated latent virus genome is controlled, thereby providing possible therapeutic targets to treat viral complications during transplantation.