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: 8551783
• 负责人: Shaochung Victor Hsia
• 金额: $ 14.45万
• 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 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8551783
• 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; 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; ultraviolet; 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（由适用提供）：HSV-1是影响人类从轻度唇疱疹到严重的眼部角膜炎和致命脑炎的最常见病毒感染之一。该病毒在三叉神经节（TG）的神经元内建立了终生的潜在感染。文献表明，疱疹病毒的重新激活与马龙失衡有关。因此，我们预测马酮的改变可能会调节重新激活过程中HSV-1基因的表达。在HSV-1基因组中搜索马酮反应性元素，我们确定了HSV-1甲状腺素激酶（TK），延迟相关转录本（LAT）和感染的细胞蛋白0（ICP0）的调节区域中甲状腺激素受体反应元件（TRE）。 TRE是甲状腺激素受体（TR）结合/起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关键基因表达来控制HSV-1潜伏期和重新激活，从而影响纬叶感染神经元内病毒的转录和复制。该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对于感染神经元内的有效病毒DNA复制和转录很重要。其次，TR在感觉神经节中呈现。第三，我们的结果表明，TR在体外调节TK，LAT和ICP0的表达。第四，我们的出版物表明，T3的水平控制病毒复制和体外传染病的释放。第五，用于诱导HSV-1重新激活的标准程序，例如高温和地塞米松治疗，也降低了TH水平。我们发表的结果共同表明，配体受体（T3/TR，马龙受体复合物）控制HSV-1关键基因的表达和复制。 N2ATR¿细胞已被用于试点研究，因为1）通过将其添加到培养基中来控制T3的水平很方便，而2）N2ATR？细胞可以通过治疗3天后通过T3区分，模仿分化神经元的状况。在该项目中，我们将进一步研究T3/TR通过以下特定目的调节HSV-1复制/基因表达的机制。具体目的1。为了测试T3/TR通过染色质调节T3/TR控制胸苷激酶（TK）的转录，使用核孤儿受体（NORS）募集的视视视感素和甲状腺受体和甲状腺受体（SMRT）的沉默介体通过TR募集到TK T3反应元素（TRE）。我们的结果表明，将配体TR募集到TK TRE中，并在T3处理的N2ATR细胞中上调NOR。也没有证明与SMRT相互作用并反映启动子活性。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？细胞培养模型进行测试：1）TR结合，2）NOR/SMRT募集和3）它们在神经元和非神经元细胞中通过短暂转染和病毒感染在病毒复制/释放中的功能。具体目的2。通过募集多功能转录因子早期生长反应基因（EGR-1）和染色质绝缘子CTCF来检验T3 /TR调节LAT和基因的转录的假设。在不同的研究中，我们表明感染后神经元细胞中T3 /TR诱导EGR-1和CTCF。他们都暴露了对HSV-1基因的反射作用。在AIM 2中，我们将生成一系列具有HSV-1重复元素1（RE-1）部分缺失的突变质粒，以研究CTCF对LAT和„基因的边界效应。 EGR-1介导的调节将通过翻译以及使用重组病毒过表达EGR-1在神经元和非神经元细胞中的感染进行进一步研究。具体目的3：测试以下假设：染色体冷凝1（RCC1）和RAS相关的核蛋白（RAN）复合物的调节剂通过在HSV-1基因组上维持HSV-1转录/复制的全球表观遗传控制，以通过T3/ TR维持HSV-1基因组上的抑制性染色质结构。 RCC1/RAN复合物通过调节对染色质和核小体的影响控制RNA易位，DNA合成和细胞周期进展。 RCC1在裂解感染中部分降解，但在感染后通过T3/TR诱导神经元细胞（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。我们的实验室一直在通过NCRR/NIH的支持来研究HSV-1生物学，我们有三本支持假设的出版物。根据我们未发表的结果，在具体目的中提出了其他研究。我们的短期任务是在马里兰州东海岸大学（UMES）的药学院建立一个活跃的研究计划，这是一所土地授予的，历史悠久的黑人学院和大学（HBCU），以便学生可以研究病毒学，分子生物学，细胞生物学，神经科学，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更好地治疗。