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

核激素受体对 HSV-1 基因表达和复制的调节

• 批准号: 8915764
• 负责人: Shaochung Victor Hsia
• 金额: $ 14.98万
• 依托单位: UNIVERSITY OF MARYLAND EASTERN SHORE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8915764
• 关键词: Affect; Animal Model; Antiviral Agents; Beryllium; Binding Sites; Biological Assay; Biology; Cell Culture Techniques; Cell Cycle Progression; Cells; Cellular biology; Chromatin; Chromatin Structure; Chromosome Condensation; Chronic; Complex; Complication; Corticotropin; DNA Viruses; DNA biosynthesis; Data; Development; Dexamethasone; Disease; Disease Outbreaks; Elements; Encephalitis; Endocrinology; Epigenetic Process; Epithelial Cells; Euchromatin; Excision; Exhibits; Fever; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genome; Goals; Grant; Growth; Herpes Labialis; Herpesviridae; Herpesvirus 1; Heterochromatin; Historically Black Colleges and Universities; Hormone Receptor; Hormone Responsive; Hormones; Human; Hydrocortisone; Hyperthermia; Hypothyroidism; In Vitro; Indium; Infection; Keratitis; Knowledge; Laboratories; Lead; Life; Ligands; Literature; Lytic Phase; Maintenance; Maryland; Mediating; Medicine; Mission; Modeling; Molecular; Molecular Biology; National Center for Research Resources; Neuroglia; Neurons; Neurosciences; Neurosecretory Systems; Nuclear Hormone Receptors; Nuclear Orphan Receptor; Nuclear Protein; Nucleic Acid Regulatory Sequences; Nucleosomes; Operative Surgical Procedures; Pharmacy Schools; Pilot Projects; Plasmids; Play; Procedures; Process; Proteins; Protocols documentation; Psychological Stress; Publications; Publishing; RNA; Recruitment Activity; Recurrence; Regulation; Research; Response Elements; Role; Sensory Ganglia; Series; Silencing Mediator of Retinoid Thyroid Receptor; Structure of trigeminal ganglion; Students; Testing; Therapeutic Agents; Thymidine Kinase; Thyroid Hormone Receptor; Thyroid Hormones; Time; Transfection; Trauma; Triiodothyronine; UV Radiation Exposure; United States National Institutes of Health; Universities; Viral; Virus; Virus Diseases; Virus Latency; Virus Replication; Work; base; biological adaptation to stress; data mining; histone modification; latency associated transcript; latent infection; mutant; novel; novel therapeutics; programs; promoter; reactivation from latency; receptor; receptor binding; recombinant virus; research study; response; transcription factor; treatment duration; viral DNA; virology

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.

描述(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)的神经元内建立了终身潜伏感染。文献表明，疱疹病毒的重新激活与激素失衡有关。因此，我们预测，激素变化可能调节HSV-1基因在重新激活过程中的表达。在寻找HSV-1基因组中的激素反应元件时，我们在HSV-1胸苷激酶(TK)、潜伏期相关转录本(LAT)和感染细胞蛋白0(ICP0)的调节区中发现了甲状腺激素受体反应元件(Tres)。TRES是甲状腺激素受体(TR)结合/作为转录因子的结合部位。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的潜伏和重新激活，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对于感染神经元内有效的病毒DNA复制和转录是重要的。第二，受体存在于感觉神经节中。第三，我们的研究结果表明，在体外，tr可以调节TK、LAT和ICP0的表达。第四，我们的出版物指出，在体外，T3水平控制着病毒的复制和传染性病毒的释放。第五，用于诱导HSV-1重新激活的标准程序，如热疗和地塞米松治疗，也会降低TH水平。综上所述，我们发表的结果表明，连接受体(T_3/T_R，激素受体复合体)控制HSV-1关键基因的表达和复制。N2aTR�细胞用于中试研究具有以下优点：1)在培养液中加入T_3可以方便地控制T_3的水平；2)处理3天后，T_3可以向�细胞分化，模拟分化后神经元的状态。在本项目中，我们将通过以下特定目的进一步研究T3/TR调节HSV-1复制/基因表达的机制。具体目的1.通过核孤儿受体(NORs)向TK T_3反应元件(TRE)募集维甲酸受体和甲状腺受体(SMRT)的沉默介质，通过染色质调节T_3/T_r调控胸苷激酶(TK)的转录。我们的结果表明，在经T3处理的N2aTR�细胞中，连接的TRR被募集到TK tre，并且NORs上调。也没有显示与SMRT相互作用并抑制启动子活性。在目标1中，我们将制造一系列TrE突变体，并利用我们的去除T3的N2aTR�细胞培养模型对它们进行测试，以研究：1)TRR结合，2)NOR/SMRT募集，3)它们通过瞬时转染和病毒感染在神经元和非神经元细胞中对病毒复制/释放的作用。具体目的2.通过将多功能转录因子早期生长反应基因(Egr-1)和染色质绝缘体CTcf招募到单纯疱疹病毒1型的关键调控区，验证T3/TR通过招募多功能转录因子早期生长反应基因(Egr-1)和染色质绝缘体CTcf来调控LAT和�基因转录的假说。在不同的研究中，我们发现在感染的神经细胞中，T_3/T_R诱导了Egr-1和CTCF的表达。它们对单纯疱疹病毒1型�基因均表现出抑制作用。在目标2中，我们将构建一系列部分缺失单纯疱疹病毒1重复元件-1(RE-1)的突变体，以研究CTCFs对LAT和�基因的边界效应。EGR-1介导的调控将通过转基因试验以及在神经细胞和非神经细胞中使用过表达Egr-1的重组病毒感染来进一步研究。具体目的3：验证染色体凝聚调节因子1(RCC1)和RAS相关核蛋白(RAN)复合体通过维持HSV-1基因组的抑制性染色质结构，通过T3/tR抑制基因沉默，参与HSV-1转录/复制的全球表观遗传控制的假说。RCC1/RAN复合体通过对染色质和核小体的调节作用，参与调控RNA易位、DNA合成和细胞周期进程。RCC1在裂解感染中部分降解，但在感染时由T3/TR诱导在神经细胞中(C.12，图7)。在目标3，我们将研究RCC1/RAN复合体对HSV-1基因表达、复制和病毒释放的影响。我们的实验室在NCRR/NIH的支持下一直致力于HSV-1生物学的研究，我们有三份出版物支持这一假说。根据我们未发表的结果，在具体目标中提出了更多的研究。我们的短期任务是在马里兰东岸大学药学院(UMES)建立一个积极的研究项目，这是一个土地赠款、历史上一直是黑人的学院和大学(HBCU)，这样学生们就可以学习病毒学、分子生物学、细胞生物学、神经科学和内分泌学的当前进展。长期目标是确定调节机制，以帮助开发新的治疗方案，以实现更好的治疗。