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: 9791977
• 负责人: DONALD M COEN
• 金额: $ 54.96万
• 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/9791977
• 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; Latent Virus; 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; combat; 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 本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，要么走向裂解感染，要么走向潜伏期。 HSV潜伏期是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潜伏期可能会导致新的疗法，甚至治愈这种广泛传播的病原体。第一 本项目的具体目的是研究在潜伏期期间裂解基因表达的抑制。至少一 microRNA（miRNA），宿主miR-138，抑制裂解基因表达并促进HSV潜伏期，但 目前还不清楚这种或其他miRNAs如何影响HSV感染。miR-138、miRNAs的作用更多 一般来说，来自潜伏相关转录物（LAT）基因座的miRNA将使用小鼠进行研究。 其miR-138或Dicer基因可在感觉神经元中被诱导切除。这种条件的影响 对病毒复制和再活化、病毒基因表达、染色质状态和潜伏期的敲除将被 与项目1和3合作，在培养的神经元中进行体内测量。两个具体假设 关于LAT基因座的产物如何抑制ICP 4基因表达，将进行测试。在项目1中， 将检验关于ICP 4基因或相应转录物的反义转录的假设 使用应该表现出这种转录减少的病毒突变体。miR-H6抑制 将使用具有破坏的miR-H6表达或其结合位点的突变体来测试ICP 4表达。 第二个具体的目标集中在转录后-最有可能的翻译-机制抑制 表达抵抗宿主免疫的病毒蛋白ICP34.5。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。第三个具体目标是评估一个职位， 转录机制，可能会倾斜平衡裂解感染。HSV-1如何阻断miRNA 通过阻止裂解感染期间miRNAs从细胞核输出，这可能克服 潜在的miRNA的抑制功能，将被研究。将确定相关的病毒基因产物 通过测试来自项目1的HSV-1开放阅读框架和病毒miRNA， 在miRNA转导的细胞中的转化，以及通过测试病毒突变体。基因产物是如何导致这种情况的 封锁将被调查。第四个目标是通过使用基因组学方法， 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和项目3合作开展生物活动。在整个项目中， 和染色质状态将由核心A辅助，使用小鼠的研究将由核心B辅助。