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控机制如何倾斜 单纯疱疹病毒（HSV）与神经元的相互作用，要么导致裂解性感染，要么导致潜伏期。 HSV潜伏期是该病毒最迷人的生物学特性，也是其最重要的临床特征。 了解 HSV 潜伏期可能会带来新的疗法，甚至治愈这种广泛传播的病原体。第一个 该项目的具体目的是研究潜伏期裂解基因表达的抑制。至少一个 microRNA (miRNA)，宿主 miR-138，抑制裂解基因表达并促进 HSV 潜伏期，但 目前尚不清楚该 miRNA 或其他 miRNA 如何影响 HSV 感染。 miR-138、miRNA 的作用更多 一般来说，来自潜伏相关转录本 (LAT) 位点的 miRNA 将使用小鼠进行研究 其 miR-138 或 Dicer 基因可以在感觉神经元中被诱导切除。这种有条件的效果 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将 在体内进行测量，并与项目 1 和 3 合作在培养的神经元中进行测量。两个具体假设 关于如何测试 LAT 基因座的产物抑制 ICP4 基因表达。对于项目 1， 将测试有关 ICP4 基因或相应转录本的反义转录的假设 使用应该表现出这种转录减少的病毒突变体。 miR-H6 抑制的假设 将使用 miR-H6 表达或其结合位点被破坏的突变体来测试 ICP4 表达。这 第二个具体目标侧重于转录后（最有可能是翻译）抑制机制 病毒蛋白 ICP34.5 的表达会抵消宿主免疫。突变如何影响 5' 将研究 ICP34.5 mRNA 的非翻译区增加 ICP34.5 表达和病毒毒力，并且， 在项目 3 中，将评估它们对免疫机制的影响。第三个具体目标评估后 转录机制可能使平衡向裂解性感染倾斜。 HSV-1 如何阻断 miRNA 通过在裂解感染期间阻止 miRNA 从细胞核输出来进行生物发生，这可能会克服 将研究潜在 miRNA 的抑制功能。将确定负责的病毒基因产物 通过测试项目 1 中的 HSV-1 开放阅读框和病毒 miRNA 来阻断 pre-miRNA 到 miRNA miRNA 转导细胞中的转化，以及通过测试病毒突变体。基因产物如何导致这种情况 封锁将受到调查。第四个目标是通过使用从 LAT 基因座识别 miRNA 的靶标 基于深度测序的方法。将测试候选靶点在 HSV 复制和其他方面的作用 与项目 1 和 3 合作进行生物活动。在整个项目中，基因表达的研究 染色质状态将由 Core A 协助，而使用小鼠的研究将由 Core B 协助。