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 这个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既可能是溶血性感染，也可能是潜伏期。 HSV潜伏期是病毒最吸引人的生物学特性，也是其最重要的临床特征。 了解HSV潜伏期可能会导致新的治疗方法，甚至治愈这种广泛传播的病原体。第一 这个项目的具体目的是研究潜伏期对裂解基因表达的抑制。至少一个 宿主miR-138的microRNA(MiRNA)抑制裂解基因的表达并促进HSV潜伏期，但 目前尚不清楚这个或其他miRNAs如何影响HSV感染。MiR-138的作用，miRNAs更多 通常，来自潜伏期相关转录本(LAT)基因座的miRNAs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。该等附带条件的影响 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是 在活体中测量，并与项目1和项目3合作，在培养的神经元中进行测量。两个具体的假设 关于LAT基因的产物如何抑制ICP4基因的表达，将进行测试。在项目1中， 关于ICP4基因或相应转录本的转录反义的假说将被测试 使用病毒突变体，在这样的转录中应该表现出减少。MiR-H6抑制的假说 ICP4的表达将使用miR-H6表达中断或其结合位点被破坏的突变体进行测试。这个 第二个具体目标是转录后--很可能是翻译--的抑制机制 表达病毒蛋白ICP34.5，以对抗宿主免疫。突变如何影响5‘ 将研究ICP34.5 mRNA的非翻译区增加ICP34.5的表达和病毒毒力，以及， 在项目3中，将评估它们对免疫机制的影响。第三个具体目的是评估一个岗位-- 转录机制可能使天平向溶血性感染倾斜。HSV-1如何阻断miRNA 在裂解感染期间阻止miRNAs从细胞核输出的生物发生，这可能克服 潜伏的miRNAs的抑制功能将被研究。负责的病毒基因产物(S)将被确定 通过测试来自项目1的HSV-1开放阅读框和病毒miRNAs来阻断前miRNA到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(S)是如何导致这种情况的 封锁将被调查。第四个目标寻求通过使用以下方法从LAT基因座识别miRNAs的靶标 基于深度测序的方法。将测试候选目标在HSV复制和其他方面的角色 与项目1和项目3合作的生物学活动。在整个项目中，对基因表达的研究 染色质状态将得到核心A的帮助，而使用小鼠进行的研究将得到核心B的帮助。