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（由申请人提供）：HSV-1 是影响人类的最常见病毒感染之一，从轻度唇疱疹到严重眼部角膜炎和致命性脑炎，该病毒在三叉神经节 (TG) 神经元内建立终生潜伏感染。文献表明，疱疹病毒的重新激活与激素失衡相关，因此我们预测激素改变可能在重新激活过程中调节HSV-1基因的表达。 HSV-1 基因组中，我们在 HSV-1 胸苷激酶 (TK)、潜伏相关转录本 (LAT) 和感染细胞蛋白 0 (ICP0) 的调节区域中鉴定了甲状腺激素受体反应元件 (TRE)。我们的中心假设是甲状腺激素（TH 或 T3）及其受体 (TR) 可以通过调节来控制 HSV-1 潜伏期和重新激活。 HSV-1 关键基因表达，从而影响病毒在潜伏感染神经元内的转录和复制，这一假设得到了文献和我们的观察的支持：首先，HSV-1 TK 和 ICP0 对于有效的病毒 DNA 复制和复制非常重要。其次，TR 存在于感觉神经节中。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病毒复制。释放的第五，用于诱导 HSV-1 重新激活的标准程序（例如热疗和地塞米松治疗）也可降低 TH 水平，我们的结果表明配体受体（T3/TR，激素受体复合物）进行控制。 HSV-1 关键基因的表达和复制。细胞已被用于试点研究，因为它具有以下优点：1）通过将T3添加到培养基中可以方便地控制T3的水平，2）N2aTR¿治疗 3 天后，T3 可以分化细胞，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标进一步研究 T3/TR 调节 HSV-1 复制/基因表达的机制。目标 1. 验证 T3/TR 使用核募集的类视黄醇和甲状腺受体沉默介体 (SMRT) 通过染色质调节来控制胸苷激酶 (TK) 转录的假设通过 TR 与 TK T3 反应元件 (TRE) 的孤儿受体 (NOR) 我们的结果表明，配体 TR 被招募到 TK TRE，并且 NOR 在 T3 处理的 N2aTR 中上调。 NOR 被证明与 SMRT 相互作用并抑制启动子活性。在目标 1 中，我们将制作一系列 TRE 突变体并使用我们的 T3 去除 N2aTR 进行测试。细胞培养模型研究：1) TR 结合，2) NOR/SMRT 募集，3) 它们通过神经元和非神经元细胞中的瞬时转染和病毒感染对病毒复制/释放的功能。 具体目标 2. 检验假设。 T3 /TR 调节 LAT 和 ¿ 的转录通过将多功能转录因子早期生长反应基因（Egr-1）和染色质绝缘子 CTCF 募集到 HSV-1 的关键调控区域，我们在不同的研究中表明，Egr-1 和 CTCF 在神经元细胞中被 T3 /TR 诱导。它们都对 HSV-1 表现出抑制作用。在目标 2 中，我们将生成一系列部分删除 HSV-1 Repeat Element-1 (RE-1) 的突变质粒，以研究 CTCF 对 LAT 和 ¿将通过转染测定以及使用在神经元和非神经元细胞中过度表达 Egr-1 的重组病毒进行感染来进一步研究 Egr-1 介导的调节。 具体目标 3：检验染色体缩合调节因子 1 的假设。 (RCC1) 和 RA 相关核蛋白 (Ran) 复合物通过维持抑制性染色质参与 HSV-1 转录/复制的全局表观遗传控制RCC1/Ran 复合物通过 T3/TR 抑制 HSV-1 基因组上的结构，通过调节染色质和核小体的作用参与控制 RNA 易位、DNA 合成和细胞周期进程，但 RCC1 在裂解感染中部分降解。感染后 T3/TR 在神经元细胞中诱导（C.12，图 7）。在目标 3 中，我们将研究 RCC1/Ran 复合物对神经元细胞的影响。 HSV-1 基因表达、复制和病毒释放。我们的实验室在 NCRR/NIH 的支持下一直致力于 HSV-1 生物学研究，并且根据我们的具体目标，我们发表了三篇出版物来支持这些假设。我们的短期任务是在马里兰大学东海岸分校 (UMES) 药学院建立一个积极的研究项目，这是一所赠地的历史悠久的黑人学院和大学 (HBCU)，以便学生能够学习病毒学、分子生物学的最新进展生物学、细胞生物学、神经科学和内分泌学的长期目标是确定调节机制，以协助开发新的治疗方案以实现更好的治疗。