Regulation of HSV-1 Gene Expression and Replication by Nuclear Hormone Receptors

描述（由申请人提供）：HSV-1是影响人类的最常见的病毒感染之一，从轻微的唇疱疹到严重的眼角膜炎和致命的脑炎。该病毒在三叉神经节（TG）的神经元内建立终身潜伏感染。文献显示疱疹病毒再激活与激素失衡有关。因此，我们预测激素的改变可能调控HSV-1基因在再激活过程中的表达。为了在HSV-1基因组中寻找激素应答元件，我们在HSV-1胸苷激酶（TK）、潜伏期相关转录物（LAT）和感染细胞蛋白0 （ICP0）的调控区域发现了甲状腺激素受体应答元件（TREs）。TREs是甲状腺激素受体（TR）结合/作为转录因子的结合位点。我们的中心假设是，甲状腺激素（TH或T3）及其受体（TR）可以通过调节HSV-1关键基因的表达来控制HSV-1的潜伏期和再激活，从而影响潜伏感染神经元内病毒的转录和复制。这一假设得到了文献和我们的观察结果的支持：首先，HSV-1 TK和ICP0对感染神经元内病毒DNA的有效复制和转录很重要。其次，TR存在于感觉神经节。第三，我们的研究结果表明，TR在体外调节TK、LAT和ICP0的表达。第四，我们的文章表明T3水平控制着病毒的复制和感染性病毒的体外释放。第五，用于诱导HSV-1再激活的标准程序，如热疗和地塞米松治疗，也可降低TH水平。总之，我们发表的研究结果表明，配体受体（T3/TR，激素受体复合物）控制HSV-1关键基因的表达和复制。N2aTR¿细胞被用于中试研究的优点是：1)通过加入培养基可以方便地控制T3的水平，2)N2aTR¿细胞在处理3天后可以被T3分化，模拟分化神经元的情况。在本项目中，我们将通过以下具体目的进一步研究T3/TR调控HSV-1复制/基因表达的机制。具体目标为了验证T3/TR通过核孤儿受体（Nuclear Orphan receptor, NORs）募集的类视黄醇和甲状腺受体（retinoid and thyroid receptor， SMRT）的沉默介质（silencing mediator of retinoid and thyroid receptors， SMRT）通过TR调控胸苷激酶（TK） T3应答元件（trei）的转录，我们的研究结果表明，配体的TR被募集到TK的trei上，而NORs在T3处理的N2aTR¿细胞中上调。NOR被证明与SMRT相互作用并抑制启动子活性。在Aim 1中，我们将制作一系列TRE突变体，并使用我们的T3去除N2aTR¿细胞培养模型对它们进行测试，以研究：1)TR结合，2)NOR/SMRT募集，以及3)它们在神经元和非神经元细胞中通过瞬时转染和病毒感染对病毒复制/释放的功能。具体目标2。验证T3 /TR通过将多功能转录因子Egr-1和染色质绝缘子CTCF募集到HSV-1的关键调控区域，从而调控LAT和¿基因的转录。在不同的研究中，我们发现T3 /TR在神经细胞感染后诱导Egr-1和CTCF。它们都对HSV-1基因有抑制作用。在Aim 2中，我们将生成一系列部分缺失HSV-1 Repeat Element-1 （RE-1）的突变质粒，以研究CTCF对LAT和¿基因的边界效应。Egr-1介导的调节将通过转染试验以及在神经元和非神经元细胞中使用过表达Egr-1的重组病毒感染进一步研究。特异性目的3：验证染色体凝聚1调控因子（RCC1）和RAs相关核蛋白（Ran）复合体通过维持HSV-1基因组上的抑制染色质结构，通过T3/TR进行基因沉默，从而参与HSV-1转录/复制的全局表观遗传控制的假设。RCC1/Ran复合体通过对染色质和核小体的调节作用参与RNA易位、DNA合成和细胞周期进程的控制。RCC1在溶解性感染中部分降解，但在感染后通过T3/TR在神经元细胞中诱导（C.12，图7）。在Aim 3中，我们将研究RCC1/Ran复合物对HSV-1基因表达、复制和病毒释放的影响。在NCRR/NIH的支持下，我们的实验室一直在研究HSV-1生物学，我们有三份出版物支持这些假设。根据我们未发表的结果，在具体目标中提出了其他研究。我们的短期任务是在马里兰大学东岸分校药学院建立一个活跃的研究项目，这样学生就可以研究病毒学、分子生物学、细胞生物学、神经科学和内分泌学的最新进展。长期目标是确定调节机制，以协助开发新的治疗方案，以获得更好的治疗。

DESCRIPTION (provided by applicant): HSV-1 is one of the most common viral infections affecting humans from mild cold sores to severe ocular keratitis and deadly encephalitis. The virus establishes a life-long latent infection within neurons of the trigeminal ganglia (TG). Literature showed that herpesvirus reactivation is correlated to hormone imbalance. Therefore we predict that hormone alteration may regulate the HSV-1 gene expression during reactivation. In searching for hormone responsive elements in HSV-1 genome, we identified thyroid hormone receptor responsive elements (TREs) in the regulatory regions of HSV-1 thymidine kinase (TK), latency-associated transcript (LAT), and infected cell protein 0 (ICP0). TREs are the binding sites where the thyroid hormone receptor (TR) binds/acts as a transcription factor. Our central hypothesis is that thyroid hormone (TH or T3) and its receptor (TR) can control HSV-1 latency and reactivation by regulating the HSV-1 key gene expression and thus influence the transcription and replication of virus within the latently-infected neurons. This hypothesis is supported by the literature and our observations: First, HSV-1 TK and ICP0 are important for efficient viral DNA replication and transcription within the infected neurons. Second, TR is present in sensory ganglia. Third, our results have shown that TR regulates expression of TK, LAT, and ICP0 in vitro. Fourth, our publication indicated that the level of T3 controls viral replication and the release of infectious viruses in vitro. Fifth, standard procedures used to induce HSV-1 reactivation, such as hyperthermia and dexamethasone treatment, also decrease TH levels. Together our published results demonstrate that the liganded receptor (T3/TR, the hormone-receptor complex) controls the expression of HSV-1 key genes and replication. N2aTR¿ cell has been used for pilot study because of the advantages that 1) it is convenient to control the level of T3 by adding it into media, and 2) N2aTR¿ cells can be differentiated by T3 after 3-days of treatment, mimicking the condition of differentiated neurons. In this project, we will further study the mechanisms by which T3/TR regulates HSV-1 replication/gene expression via the following Specific Aims. Specific Aim 1. To test the hypothesis that T3/TR control the transcription of thymidine kinase (TK) by chromatin regulation using silencing mediator of retinoid and thyroid receptors (SMRT) recruited by Nuclear Orphan Receptors (NORs) to the TK T3 Response Element (TRE) via TR. Our results indicated that liganded TR was recruited to the TK TRE, and NORs were up-regulated in T3-treated N2aTR¿ cells. NOR is shown to interact with SMRT and repress promoter activity. In Aim 1, we will make a series of TRE mutants and test them using our T3 removal N2aTR¿ cell culture model to investigate: 1) TR binding, 2) NOR/SMRT recruitment, and 3) their functions on viral replication/release by transient transfection and viral infection in neuronal and non-neuronal cells. Specific Aim 2. To test the hypothesis that T3 /TR modulate transcription of LAT and ¿ genes by recruiting multifunctional transcription factor Early Growth Response gene (Egr-1) and chromatin insulator CTCF to the key regulatory regions of HSV-1. In different studies we showed that Egr-1 and CTCF were induced by T3 /TR in neuronal cells upon infection. They both exhibit repressive effects on HSV-1 ¿ genes. In Aim 2, we will generate a series of mutant plasmids with partial deletion of HSV-1 Repeat Element-1 (RE-1) to study the boundary effect of CTCF on LAT and ¿ genes. Egr-1-mediated regulation will be further investigated by transfection assays as well as infections using recombinant virus over-expressing Egr-1 in neuronal and non-neuronal cells. Specific Aim 3: To test the hypothesis that Regulator of Chromosome Condensation 1 (RCC1) and RAs- related Nuclear protein (Ran) complex participated in the global epigenetic control of HSV-1 transcription/ replication by maintaining repressive chromatin structure on HSV-1 genome for gene silencing via T3/TR. RCC1/Ran complex is involved in the control of RNA translocation, DNA synthesis, and cell cycle progression via modulating effects on chromatin and nucleosomes. RCC1 is partially degraded in lytic infection but induced in neuronal cells by T3/TR upon infection (C.12, Fig. 7). In Aim 3, we will study the effects of RCC1/Ran complex on HSV-1 gene expression, replication, and virus release. Our laboratory has been working on HSV-1 biology through the support from NCRR/NIH, and we have three publications supporting the hypotheses. Additional studies are proposed in the Specific Aims based on our unpublished results. Our short-term mission is to establish an active research program at the School of Pharmacy at the University of Maryland Eastern Shore (UMES), a land-grant, historically black college and university (HBCU) so the students can study the current progress of virology, molecular biology, cell biology, neuroscience, and endocrinology. The long-term goal is to identify the regulatory mechanisms to assist in the development of novel therapeutic protocols for better treatments.