Project 2 - Post-transcriptional mechanisms and the HSV lytic/latent balance

项目 2 - 转录后机制和 HSV 裂解/潜伏平衡

• 批准号: 10226131
• 负责人: DONALD M COEN
• 金额: $ 56.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-02 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226131
• 关键词: 5' Untranslated Regions; Affect; Afferent Neurons; Animal Experiments; Antiviral Agents; Binding Sites; Biogenesis; Biological; Biological Process; Biology; Cell Culture Techniques; Cell Nucleus; Cells; Chromatin; Clinical; Collaborations; DICER1 gene; Equilibrium; Exhibits; Gene Expression; Genes; Genetic Transcription; Herpesvirus 1; Immune; Immunity; Impairment; Infection; Integration Host Factors; Knock-out; Lead; Life; Lytic; Lytic Phase; Lytic Virus; Maintenance; Measures; Messenger RNA; Methods; MicroRNAs; Mus; Mutation; Neurons; Nuclear Export; Open Reading Frames; Organ Culture Techniques; Property; Proteins; Regulator Genes; Repression; Role; Simplexvirus; Testing; Transcript; Viral; Viral Genes; Viral Proteins; Viral Regulatory Proteins; Virulence; Virus; Virus Diseases; Virus Latency; Virus Replication; base; cellular transduction; conditional knockout; deep sequencing; fascinate; gene product; human disease; in vivo; latency associated transcript; latent infection; lytic gene expression; lytic replication; mutant; novel therapeutics; pathogen; prevent; reactivation from latency; statistics; transcription factor; transcriptome sequencing; transmission process

Summary/Abstract – Project 2 The long-term objective of this project is to investigate how post-transcriptional gene regulatory mechanisms tilt the interaction of herpes simplex virus (HSV) with neurons either towards lytic infection or towards latency. HSV latency is the most fascinating biological property of the virus and its most important clinical feature. Understanding HSV latency may lead to new therapies or even a cure for this widespread pathogen. The first specific aim of this project is to investigate repression of lytic gene expression during latency. At least one microRNA (miRNA), host miR-138, represses lytic gene expression and promotes HSV latency, but much remains unknown about how this or other miRNAs impact HSV infections. The roles of miR-138, miRNAs more generally, and miRNAs from the latency associated transcript (LAT) locus will be investigated using mice whose miR-138 or Dicer genes can be inducibly excised in sensory neurons. Effects of such conditional knockouts on viral replication and reactivation, viral gene expression, chromatin status, and latency will be measured in vivo and, in collaboration with Projects 1 and 3, in cultured neurons. Two specific hypotheses regarding how products of the LAT locus repress ICP4 gene expression will be tested. With Project 1, a hypothesis regarding transcription antisense to the ICP4 gene or the corresponding transcripts will be tested using viral mutants that should exhibit decreases in such transcription. The hypothesis that miR-H6 represses ICP4 expression will be tested using mutants with disrupted miR-H6 expression or binding sites for it. The second specific aim focuses on post-transcriptional – most likely translational – mechanisms restraining expression of the viral protein ICP34.5 that counteracts host immunity. How mutations affecting the 5' untranslated region of ICP34.5 mRNA increase ICP34.5 expression and viral virulence will be studied, and, with Project 3, their effects on immune mechanisms will be assessed. The third specific aim assesses a post- transcriptional mechanism that may tilt the balance towards lytic infection. How HSV-1 blocks miRNA biogenesis by preventing export of miRNAs from the nucleus during lytic infection, which may overcome repressive functions of latent miRNAs, will be studied. The viral gene product(s) responsible will be identified by testing HSV-1 open reading frames from Project 1 and viral miRNAs for blocking pre-miRNA to miRNA conversion in miRNA-transduced cells, and by testing viral mutants. How the gene product(s) cause this blockade will be investigated. The fourth aim seeks to identify targets for miRNAs from the LAT locus by using deep sequencing based methods. Candidate targets will be tested for their roles in HSV replication and other biological activities in collaboration with Projects 1 and 3. Throughout this project, studies of gene expression and chromatin status will be assisted by Core A, and studies using mice will be assisted by Core B.

摘要/摘要 - 项目2 该项目的长期目标是研究转录后基因调节机制如何倾斜 单纯疱疹病毒（HSV）与神经元对裂解感染或潜伏期的相互作用。 HSV潜伏期是该病毒及其最重要的临床特征最迷人的生物学特性。 了解HSV潜伏期可能会导致新的疗法，甚至可以治愈这种宽度的病原体。第一个 该项目的具体目的是研究潜伏期期间裂解基因表达的表达。至少一个 microRNA（miRNA），宿主miR-138，反映裂解基因的表达并促进HSV潜伏期，但很多 对于这种或其他miRNA如何影响HSV感染仍不清楚。 mir-138的角色，mirnas更多 通常，将使用小鼠研究潜伏相关笔录（LAT）基因座的miRNA 其miR-138或DICER基因在感觉神经元中可能非常出色。这种条件的影响 敲除病毒复制和重新激活，病毒基因表达，染色质状态和潜伏期的敲除 在体内测量，并与培养的神经元中的项目1和3合作测量。两个特定的假设 考虑如何测试LAT基因座的抑制ICP4基因表达的产品。与项目1一起 关于ICP4基因或相应转录本的转录反义的假设将测试 使用应在这种转录中表现出下降的病毒突变体。 mir-h6复制品的假设 ICP4表达将使用中断的miR-H6表达或结合位点的突变体进行测试。这 第二个特定目的重点是转录后（很可能是翻译 - 限制机制） 病毒蛋白ICP34.5的表达抵消了宿主免疫。突变如何影响5' ICP34.5 mRNA的未翻译区域增加了ICP34.5表达和病毒病毒，将被研究，并且 通过项目3，将评估它们对免疫机制的影响。第三个特定目的评估后 转录机制可能将平衡倾向于裂解感染。 HSV-1如何阻止miRNA 生物发生通过防止在裂解感染期间从细胞核中输出miRNA，这可能会克服 潜在miRNA的压抑功能将研究。将确定负责的病毒基因产物 通过测试项目1和病毒miRNA的HSV-1开放式阅读框，以阻止MiRNA到miRNA 通过miRNA转导细胞和通过测试病毒突变体的转化。基因产品如何引起此 封锁将被调查。第四目标旨在通过使用LAT基因座的miRNA目标 基于深度测序的方法。候选目标将测试其在HSV复制中的作用和其他 与项目1和3合作的生物活动。通过该项目，基因表达的研究 核心A将协助染色质状态，使用小鼠的研究将由核心B进行协助。