Nuclear Sensing of Herpesviral DNA

疱疹病毒 DNA 的核传感

• 批准号: 9751707
• 负责人: DAVID M. KNIPE
• 金额: $ 52.21万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751707
• 关键词: ATRX gene; Affect; Biological; Cell Nucleus; Cells; Chromatin; DNA; DNA Virus Infections; Detection; Enzymes; Epigenetic Process; Funding; Gene Delivery; Gene Expression; Genes; Genetic Transcription; Genome; Genomic DNA; Goals; Herpesviridae; Heterochromatin; Histones; Hour; Image Analysis; Integration Host Factors; Kinetics; Knowledge; Lytic Phase; Maintenance; Mammalian Cell; Molecular; Nuclear; Pathway interactions; Plasmids; Property; Proteins; Reporting; Research; Role; Simplexvirus; Study models; Technology; Testing; Transactivation; Transfection; Viral; Viral Genome; Viral Load result; Viral Vector; Virion; Virus; Virus Replication; base; chromatin immunoprecipitation; design; gene therapy; genome integrity; histone modification; improved; latent infection; mutant; novel; novel therapeutics; plasmid DNA; promoter; quantitative imaging; recruit; response; sensor; small molecule; tool; viral DNA

Project Summary The long-term goals of this research are to define the mechanisms by which foreign viral or plasmid DNA is recognized or sensed in mammalian cells and then epigenetically silenced. Herpes simplex virus (HSV) genomic DNA in the virion is not associated with histones, but the host cell rapidly adds heterochromatin to the HSV genome upon entry into the nucleus. The cellular factors that sense the foreign viral DNA are poorly characterized. We had shown previously that the host IFI16 protein senses HSV DNA and stimulates an innate response, and during the previous funding period we showed that IFI16 promotes epigenetic silencing of ICP0-null HSV. IFI16 is the only host protein known to increase heterochromatin marks on HSV chromatin. The ND10 proteins PML, Sp100, Daxx and ATRX have all been reported to restrict ICP0-null mutant virus replication and gene expression, but no information is available about how they affect HSV chromatin. We have designed a novel quantitative imaging analysis tool for detection of input viral DNA and host factors, and we have exciting new results showing the sequential association of IFI16 with viral input DNA at 30 minutes postinfection (pi) and ATRX with viral input DNA at 0.5-2 hour pi. The associations of IFI16 and ATRX with input viral DNA appear to be independent and their effect on viral replication is additive. Because we have shown that the viral genome is loaded with heterochromatin by 1-2 hours pi, IFI16 and/or ATRX are strong candidates for a role in the initial sensing of viral DNA and loading of heterochromatin. We also have used a novel small molecule screen to identify molecules that inhibit ICP0-specific transactivation that will provide important probes of ICP0 function. In this application, our specific aims are to 1. Determine the role and mechanisms involving IFI16 in early HSV DNA sensing and chromatinization. 2. Determine the role and mechanisms involving ATRX and other ND10 proteins in early HSV DNA chromatinization. 3. Define the functional mechanisms of ICP0 action on host DNA sensors by determining the mechanisms by which ICP0 promotes degradation of IFI16 and by studies of the mechanism of action of small molecules inhibiting ICP0-dependent gene expression. These studies will provide important new basic knowledge of the mechanisms of sensing of foreign DNA and enable new therapeutics for the herpesviruses and improved gene delivery mechanisms using viral and DNA-based technology.

项目摘要 这项研究的长期目标是确定外源病毒或质粒DNA 在哺乳动物细胞中被识别或感知，然后表观遗传沉默。单纯疱疹病毒 (HSV)病毒体中的基因组DNA与组蛋白不相关，但宿主细胞迅速添加 异染色质进入细胞核后与单纯疱疹病毒基因组结合。细胞因子感受到 外来病毒DNA的特征很差。我们之前已经证明，宿主IFI 16蛋白感知 HSV DNA并刺激先天反应，在上一个资助期间，我们表明， IFI 16促进ICP 0无效HSV的表观遗传沉默。IFI 16是已知的唯一一种宿主蛋白， HSV染色质上的异染色质标记。ND 10蛋白PML、Sp100、Daxx和ATRX都具有 据报道，限制ICP 0无效突变病毒复制和基因表达，但没有信息是 关于它们如何影响HSV染色质。我们设计了一种新的定量成像分析方法， 检测输入病毒DNA和宿主因子的工具，我们有令人兴奋的新结果显示， 在感染后30分钟（pi），IFI 16与病毒输入DNA的顺序结合，以及ATRX与病毒输入DNA的顺序结合。 在感染后0.5-2小时输入DNA。IFI 16和ATRX与输入病毒DNA的关联似乎是 它们对病毒复制的作用是相加的。因为我们已经证明， 基因组在pi后1-2小时加载异染色质，IFI 16和/或ATRX是异染色质的强候选者。 在病毒DNA的初始感应和异染色质的装载中起作用。我们还使用了一种新颖的小 分子筛选以鉴定抑制ICP 0特异性反式激活的分子， ICP 0功能的探针。 在此应用程序中，我们的具体目标是1。确定国际金融机构16在以下方面的作用和机制： 早期HSV DNA传感和染色质化。2.确定涉及ATRX的作用和机制 和其他ND 10蛋白在早期HSV DNA染色质化中的作用。3.定义的功能机制 通过确定ICP 0促进降解的机制来确定ICP 0对宿主DNA传感器的作用 通过对抑制ICP 0依赖性基因的小分子的作用机制的研究， 表情 这些研究将为外源DNA的传感机制提供重要的新的基础知识 并使疱疹病毒的新疗法和使用病毒的改进的基因递送机制成为可能 和基于DNA的技术